Proteinoid carriers and methods for preparation and use thereof

Field of the Invention

This invention relates to proteinoids and proteinoid carriers made from them. The proteinoid carriers releasably encapsulate active agents and have extended longer shelf life and/or photostability. Methods for the preparation of such proteinoid carriers are also disclosed.

Background of the Invention

The available modes of delivery of pharmaceutical and therapeutic agents often are severely limited by chemical or physical barriers or both, which are imposed by the body. For example, oral delivery of many such agents would be the route of choice if not for the presence of chemical and physicochemical barriers such as extreme pH in the gut, exposure to powerful digestive enzymes, and impermeability of gastrointestinal membranes to the active ingredient. Among the numerous pharmacological agents which are known to be unsuitable for oral administration are biologically active peptides and proteins, such as insulin. These agents are rapidly destroyed in the gut

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PCT/US93/05723 by acid hydrolysis and/or by proteolytic enzymes.

A great deal of research has been devoted to developing effective oral drug delivery methods and systems for these vulnerable pharmacological agents. The proposed solutions have included :

(a) co-administration of adjuvants (such as resorcinols and non-ionic surfactants polyoxyethylene oleyl ether and nhexadecyl polyethylene ether to increase the permeability of the intestinal walls; and (b) co-administration of enzymatic inhibitors, such as pancreatic trypsin inhibitor, diisopropylfluorophosphate (DFF) and trasylol to avoid enzymatic degradation.

The use of such substances, in drug delivery systems, is limited however either because of:

(a) their inherent toxicity when employed at effective amounts;

(b) their failure to protect the active ingredient or promote its absorption;

(c) their adverse interaction with the drug.

Liposomes as drug delivery systems have also been described. They provide a layer of lipid around the encapsulated pharmacological agent. The use of liposomes containing heparin is disclosed in U.S. Patent No. 4,239,754 and several studies have been directed to the use cZ liposomes containing insulin;

e.g., Patel et al. (1976) FEBS Letters Vol. 62, page 60 and

Hashimoto et al. (1979) Endocrinol. Janan, Vol. 26, page 337.

The use of liposomes, however, is still in the development stage and there are continuing problems, including:

(a) poor stability;

(b) inadequate shelf life;

(c) limited to low MW (< 30,000) cargoes;

(d) difficulty in manufacturing;

(e) adverse interactions with cargoes.

More recently- synthetic amino acid polymers or proteinoids, forming microspheres, have been described for

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PCT/US93/05723 encapsulating pharmaceuticals. For example, U.S. Patent No. 4,925,673 (the '673 patent), the disclosure which is hereby incorporated by reference in its entirety, describes such microsphere constructs as well as methods for their preparation and use. The '673 patent also describes microspheres which encapsulate pharmaceutical agents for delivery into the gastrointestinal tract or into the blood.

While the proteinoid microspheres described in the '673 patent are useful for their intended purposes, the physicochemi10 cal properties of the proteinoid microspheres, such as light sensitivity, shelf life and the selectivity of their solubility in various portions of the gastrointestinal tract, could be improved. Additionally, there is a need in the art for microspheres that can encapsulate a broader range of active agents such as polar drugs.

The method employed in the '673 patent to prepare proteinoids produces a complex mixture of high molecular weight (MW) (> 1000 daltons) and low MW (< 1000 daltons) peptide-like polymers which are difficult to separate. Moreover, the method produces a small amount of the low MW proteinoids which is the microsphere-forming fraction. Hence, an improved method of preparing of the proteinoids is also desired.

Accordingly, there is a need in the art for improved proteinoid carriers as well as improved methods for their preparation.

Objects of the Invention

It is an object of this invention to provide proteinoids which forms proteinoid carriers as a delivery system with enhanced stability towards at least one of photodegradation and decomposition over time.

It is another object of the invention to provide a proteinoid that forms proteinoid carriers with more selective solubility under various conditions such as pH.

It is yet another object of the invention to provide

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PCT/US93/05723 proteinoid carriers encapsulating biologically active agents which are selectively releasable witnin particular portions of the gastrointestinal tract.

It is a further object of the invention to provide 5 proteinoid carriers which promotes the bioavailability of pharmaceutical agents which otherwise display poor absorption in the gastrointestinal tract.

It is yet a further object of the invention to provide an improved method for producing proteinoid carriers having particular characteristics and for improving yield of the desired proteinoid carriers.

It has been found that these objects and other advantages, which will be apparent from this specification, are achieved by the invention described below. _

Summary of the Invention

The present invention relates to improved proteinoid carriers and methods of making and use thereof.

Proteinoids of a MW ranging between about 250 and about 20 2400 daltons and of defined amino acids are useful in preparing proteinoid carriers with improved stability against photodegrada' tion and/or decomposition. The proteinoids comprise a peptide polymer selected from the group consisting of:

(i) peptide polymers made from at least one first 25 monomer selected from the group consisting of tyrosine and phenylalanine; and from at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid;

(ii) peptide polymers made from at least one first 30 monomer selected from the group consisting of tyrosine and phenylalanine; and from at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid; and from at least one third monomer selected from the group consisting of lysine, arginine and ornithine, the proteinoid being a microsphere- and/or microcapWO 93/25583

PCT/US93/05723 sule-forming proteinoid and being soluble within a selected pH range.

The proteinoid molecules of the invention contain between about 2 and about 20 amino acid residues, preferably between about 2 and about 8 amino acid residues, and has a molecular weight which ranges becween about 250 and about 2400 daltons, preferably between about 250 and about 600, and most preferably between about 250 and 400 daltons.

The proteinoid carriers are useful as delivery systems 10 to releasably encapsulate and carry a broad range of cargoes including pharmaceutical agents, dye reagents and cosmetic ingredients. In particular, the proteinoid carriers are useful as oral delivery systems of sensitive pharmaceutical agents, which normally would not be administrable via the oral route, for selective release at targeted regions of the gastrointestinal tract.

Description of the Drawings ' Figure 1 illustrates the molecular weight distribution as a function of monomer concentration of poly (Asp.Bz-co-Phe) polymer prepared by the NCA method as described in Example 3 .

Figure 2 illustrates the molecular weight distribution of a function of monomer concentration of poly (Asp.Bz) polymer prepared by the DPPA method as described in Example 5.

Figure 3 illustrates the effect of reaction time duration on yields of poly (Asp.Bz) polymer prepared by the DPPA method as described in Example 5. .

Figure 4 illustrates the effect of temperature of the molecular weight of poly (Asp.Bz) polymer prepared by the DPPA method as described in Example 5.

Figure 5 illustrates the effect of changing the molar ratios of [DPPA]/[M] on the molecular weight of poly (Asp.Bz) polymer by the DPPA method as escribed in Example 5.

Figure 6 is a photograph of an x-ray film of the western immunoblot analysis, as described in Example S, of

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PCr/US93/05723 purified murine mAb 9BG5 (2pg, lane 1; lmg, lane 2; and 0.25 pg, lane 3) ; empcy proceinoid carrier supernatant after encapsulating process (no mAb) (lane 4) : empcy proteinoid carrier pellet (lane 5) ; proceinoid carrier encapsulated mAb supernatant after encapsulating process (lane 6); and proteinoid carrier encapsulated mAb pellet. Lane MW contained standard molecular weight markers .

Figure 7 is a photograph of an x-ray film of a western immunoblot analysis of samples described in Example 10.

Figures 8 (a-c) illustrate the levels of serum proteins which bound to immobilized reovirus type 3 and V_LSH under ELISA conditions as described in Example 11. Empty spheres refers to animals orally administered empcy proteinoid carriers (no mAb 9BG5) ; mAb spheres refers co animals orally administered mAb

9BG5 encapsulated proteinoid carriers; IV refers to animals intravenously administered unencapsulated mAb 9BG5; and oral refers to animals orally adminiscered unencapsulated mAb 9BG5.

Figure 9 show mAb binding under conventional ELISA procedures using immobilized reovirus ty_h ~ 3 and V_LSH proteins with serial dilutions of purified mAb in 0.85 N citrate-0.5% gum (Figure 9(a)) or phosphate buffered saline (Figure 9 (b) ) as described in Example 11.

Figure 10 illuscrates levels of erythropoietin (EPO) detecced in rat serum taken from rats administered proteinoid carrier encapsulated EPO (15μg EPO/kg body weight) and encapsulated EPO (15μg EPO/kg body weight) as described in Example 15.

Figure 11 illustrates EPO serum levels in racs thac were administered either erythropoietin (50μg/kg) or encapsulated erythropoietin (50μg/kg) directly into the proximal duodenum as described in Example 15. Serum erythropoietin levels were determined over time with a erythropoietin enzyme immunoassay kit.

Figure 12 illustraces EPO serum levels in rats who were orally gavaged with either encapsulated or unencapsulated erythropoietin (100μg/kg) or received a subcutaneous injection of

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PCT/US93/05723 either 2/zg/kg or 10/zg/kg as described in Example 15. Serum erythropoietin levels were determined over time with an erythropoietin enzyme immunoassay kit.

Figure 13 illustrates serum calcium changes after oral administration of salmon calcitonin proteinoid carriers (0.25 mg calcitonin/kg body weight) in cynomolgus monkeys as described in Example 17. The results are expressed as absolute change in serum calcium from baseline values. The data represents means +/- SEM. ** Serum calcium levels significally different from baseline values.

Figure 14 illustrates serum calcium changes following oral administration of salmon calcitonin proteinoid carriers (0.60 mg/kg body weight) in rats as described in Example 18. The results are expressed as absolute change in serum calcium from baseline values. The data represents means +/- SEM. **Serum calcium levels significantly different compared to the control group at the corresponding time point.

Figure 15 illustrates serum calcium changes after intraduodenal administration of salmon calcitonin or calcitonin proteinoid carriers (3 ug/kg body weight) in rats as described in Example 18. The results are expressed as absolute change in serum calcium from baseline values. The data represents means +/- SEM. ** Significantly different from the unencapsulated control group at the indicated time points.

Figure 16 illustrates clotting times after oral administration of proteinoid carrier encapsulated Factor IX (FIX sph PO) and IV administration of FIX solution (FIX IV) as described in Example 20.

Figure 17 illustrates clotting times after oral administration of proteinoid carrier encapsulated Factor IX (FIX sph PO) and FIX solution (FIX unencap PO) or IV administration of FIX solution (FIX IV) as described in Example 21.

Figure 18 illustrates the percentage of intact alphainterferon (IFN) remaining after incubating IFN and IFN prote35 inoid carriers in simulated gastric fluid (SGF).

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Figure IS illustrates the percentage of intact IFN remaining after incubating IFN and IFN proteinoid carriers in 0.08N HCl.

Figure 20 illustrates the percentage of intact IFN 5 remaining after incubating IFN and IFN proteinoid carriers in simulated intestinal fluid (SIF).

Figure 21 illustrates the clotting times in rats dosed with heparin or proteinoid/heparin, both in water. The data represents an average of 6 rats. The data represents means +/10 SEM.

Figure 22 illustrates clotting times in rats dosed ID with USP heparin or heparin proteinoid carriers, both in citric acid. Each time point is an average of 12 rats. The data represents means +/- SEM.

Figure 23 illustrates clotting times in rats dosed orally with heparin-spiked empty proteinoid carriers or heparin proteinoid carriers. Each time point is an average of 12 rats. The data represents means +/- SEM.

Figure 24 illustrates the average titers of rats immunized orally with Ml proteinoid carriers versus unencapsulated Ml. Only responders in each group were averaged.

Figure 25 illustrates HA-NA titers of rats immunized orally with HA-NA micropspheres versus unencapsulated HA-NA.

Detailed Description of the Invention.

All patents and literature references cited in this specification are hereby incorporated by reference in their entirety. In case of inconsistencies, the present description, including the definitions and interpretations, will prevail.

The instant invention arose from the discovery that proteinoids of a MW of between about 250 and about 2400 daltons and of defined amino acid composition can be obtained by modifying known reactions and selecting starting materials. These proteinoids form proteinoid carriers with surprisingly enhanced stability against at least one of photodegradation and

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PCT/US93/05723 decomposition over time, In addition, proteinoid carriers prepared from such proteinoids carry a broader range of pharmaceutical agents, including labile polypeptides such as insulin, alpha-interferon, calcitonin, antigens, e.g. influenza virus Ml5 protein, and Factor IX and display a selective releasability within various portions of the gastrointestinal tract, relative to prior art proteinoid microspherns.

The proteinoids of the invention comprise a peptide polymer selected from the group consisting of:

(i) peptide polymers made from at least one first monomer selected from the group consisting of tyrosine and phenylalanine; and from at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid;

(ii) peptide polymers made from at least one first monomer selected from the group consisting of tyrosine and phenylalanine; at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid; and from at least one third monomer selected from the group consisting of lysine, arginine and ornithine, the proteinoid being a microsphere- or microcapsule-forming proteinoid and being soluble within a selected pH range.

The proteinoid molecules of the invention contain between about 2 and about 20 amino acid residues, preferably between about 2 and about 8 amino acid residues, and have a molecular weight which ranges between 250 and about 2400 daltons, preferably between about 250 and about 600, and most preferably between about 250 and 400 daltons.

Proteinoid carriers prepared from the proteinoid molecules, in accordance with the present invention, display a selective solubility at specific acidic or basic pH ranges, depending on the choice and amount of the second and third monomers in the proteinoid.

Proteinoid carriers which are selectively soluble under alkaline pH environments, such as those found in the distal

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PCT/US93/05723 portion of the intestine, are prepared from base-soluble proteinoids. These proteinoids contain, as starting monomers in the reaction mixture, at least one second monomer selected from the group consisting of glutamic acid, glutamine, pyroglutamic acid, and aspartic acid. At a pH ranging between about 7.2 and about 11.0, the base-soluble proteinoid exists largely as the anion and is soluble. At a pH below about 7.0, the proteinoid is largely protonated and insoluble in water.

Similarly, proteinoid carriers which are selectively 10 soluble under acidic pH environments, such as the stomach, are prepared from acid-soluble proteinoids. In this case, the proteinoid contain, as starting monomers in the proteinoid reaction mixture, at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid and at least one third monomer selected from the group consisting of lysine, arginine, and ornithine. At a pH ranging between about 1 and about 7, the base-soluble proteinoid exists largely as the cation and is soluble. At a pH above about 7.2, the proteinoid is largely unprotonated and insoluble in water.

The pH and the solubility characteristics of the acidsoluble proteinoid depends largely, but not exclusively, upon the pH and solubilty of the last amino acid added during the synthesis of the proteinoid. For instance, the incorporation of a basic amino acid, e.g., a third monomer, celected from the group consisting of lysine, arginine and ornithine in the acidsoluble proteinoid will result in the elevation of the pi (pH at the isoelectric point) of the proteinoid.

The proteinoids of the present invention are preparable by a thermal condensation reaction by heating mixtures of the appropriate amino acids under conditions described in the '673 patent. In contrast with the '673 patent procedures which use as many as eighteen amino acids, mixtures of two to five specific amino acids with at least one selected from each of the aforemen35 tioned groups yield proteinoids which form proteinoid carriers

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PCT/US93/05723 with selective solubility at particular pH ranges and at high yields .

In carrying out the thermal condensation reaction, it has now been discovered that inclusion of tetramethylene sulfone, an inert, high boiling, polar solvent, maximizes the yield (> 80%) of low MW proteinoids. Omission of solvent does not produce high yields of low MW proteinc ..ds . Presumably this is due to the poor solubility of the amino acid monomers in these solvents and/or unavoidable side reactions between the monomers and the solvent under the reaction conditions.

In general, individual amino acids are added to a reaction flask containing tetramethylene sulfone (sulfolane) which has been heated to a temperature ranging between about 130°C and about 200°C, preferably about 175°C to 195°C, under an inert atmosphere of argon or nitrogen gas. After each addition, the solution is stirred ..for a period of time anging between about 10 minutes and about 5 hours, depending o.. the amino acid type and the order of addition.

Upon heating mixtures of amino acids to temperatures of about 190°C as described above, a reaction takes place and water, ammonia and carbon dioxide are produced as side-products. Water is removed from the reaction as formed and the reaction is terminated when water formation ceases. Thereafter, the proteinoid are precipitated out of the reaction solution by quenching with excess water, under vigorous stirring. After stirring for a period of about 1 hour, the proteinoids are collected by filtration, washed with water and dried under vacuum.

Chemical condensation methods which utilize derivatized amino acids are also useful for making the proteinoids of the present invention as they permit greater control of molecular weight. Such reactions are generally conducted at lower reaction temperature and with initiators. In particular, low MW proteinoids produced by the alpha-amino acid N-carboxyanhydride (NCA) method and the diphenylphosphoryl azide (DPPA) method (N. Nishi

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PCT/US93/05723 et al. (1991) Makromol Chem.. Vol.192, pages 1789-1798) were found to form proteinoid carriers having selected solubility within a particular pH range.

The NCA method involves the preparation of N-carboxyan5 hydrides of alpha-amino acid esters and their subsequent polymerization, using low MW amines as initiators. It has been discovered that non-NCA derived amino esters, e.g., a-methyl tyrosine ester, are effective initiators which are stable and soluble in many organic solvents such as tetrahydrofuran (THF).

The use of amino acids as initiators, presumably due to their poor solubility in organic solvents and their low stability, are not known. The NCA reaction produces a high yield of proteinoids with high purity.

The DPPA method involves the direct condensation of benzyl esters of alpha-amino acids in the presence of DPPA and a low MW amine, followed by removal of the protective benzyl groups, contained in the proteinoid produce, by alkaline hydrolysis. If catalytic hydrogenation is used in place of alkaline hydrolysis, low MW proteinoids of unexpected high purities and yields are obtained.

Proteinoids prepared by any of the above methods can be used immediately to microencapsulate an active pharmacological agent or the proteinoid can be concentrated or dried by conventional means and stored for future use.

The proteinoids of the invention are purified as follows: crude proteinoids are slurried with water at room temperature, e.g. 25°C. While at this temperature, the pH of the slurry is adjusted to about pH 8 using an aqueous alkaline solution, e.g. 40% sodium hydroxide and 10% sodium bicarbonate solutions for an acid-soluble proteinoid. For a base-soluble proteinoid, the slurry is adjusted to an acidic pH with an aqueous acidic solution, e.g. 10% acetic acid solution. The mixture is then filtered and the filter cake washed with a volume of water. The washes and filtrate are then combined and evaporated to dryness in vacuo to afford proteinoids. If

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PCT/US93/05723 necessary, this process can he repeated until proteinoids of a desired purity level are obtained.

If desired, the proteinoid may be further purified by fractionating on a column containing solid supports which include silica gel or alumina, using methanol or propanol as mobile phase; ion exchange resin using water as the mobile phase; reverse phase column supports using trif luoroacetic acid/acetonitrile mixtures as mobile phase. The proteinoids may also be purified by extraction with a lower alcohol such as propanol or butanol to remove low molecular weight contaminants.

Proteinoid carriers are made from purified proteinoids as follows: proteinoids are dissolved in deionized water at a concentration ranging between about 75 and about 200 mg/ml, preferably about 100 mg/ml, at a temperature between about 25 C and about 60^rC, preferably about 40'C. Particulates remaining in the solution may be filtered out by conventional means such as gravity filtration over filter paper.

Thereafter, the proteinoid solution, maintained at a temperature of about 40°C, is mixed with an aqueous acid solution (also at about 40°C) having an acid concentration ranging between about 1 N and about 2 N, preferably about 1.7 N. The resulting mixture is further incubated at 40 °C for a period of time effective for microsphere and microcapsule formation as observed by light microscopy. In practicing this invention, the preferred order of addition is adding the proteinoid solution to the aqueous acid solution.

Suitable acids include any acid which does not (a) ’ adversely effect the proteinoid, e.g., chemical decomposition; (b) interfere with microsphere or microcapsule formation; (c) interfere with microsphero or microcapsule encapsulation of cargo; and (d) adversely interact with the cargo. Preferred acids for use in this invention include acetic acid, citric acid, hydrochloric acid, phosphoric acid, malic acid and maleic acid.

In practicing the invention, a proteinoid carrier stabilizing additives are preferably incorporated into the

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PCT/US93/05723 aqueous acid solution or into the proteinoid solution, prior co che microsphere or microcapsule formation process. The presence of such additives promotes the suability and dispersibility of the proteinoid carriers in solution. 5 The additives may be employed at a concentration ranging between about 0.1 and 5 % (W/V), preferably about 0.5 % (W/V) . Suitable, but non-limiting, examples of stabilizing additives include gum acacia, gelatin, polyethylene glycol, and polylysine.

Thereafter, the proteinoid carriers may be used immediately or may be stored at 4 C or lyophilized and stored under desiccant at room temperature or below.

Under the aforementioned conditions, the proteinoid molecules form spherical proteinoid carriers comprising protein15 oid microcapsules and proteinoid microspheres of less than 10 micron diameter. As defined herein, a microsphere is spherical homogeneous mesh work structure having no discrete inner chamber. A microcapsule refers to a spherical structure having a proteinoid wall which forms a hollow or chamber. If the proteinoid carriers are formed in the presence of a soluble material, e.g., a pharmaceutical agent in the aforementioned aqueous acid solution, this material is believed to be encapsulated within the hollows of the microcapsules and confined within the proteinoid wall defined by the spherical structure or entrapped within the matrix of proteinoid molecules in the microsphere structure. In this way, one can encapsulate or entrap pharmacologically active materials such as. peptides, proteins, and polysaccharides as well as charged organic molecules, e.g., quinolones or antimicrobial agents, having poor bioavailability by the oral route. The amount of pharmaceutical agent which may be encapsulated or entrapped by the proteinoid carrier is dependent on a number of factors which include the concentration of agent in the encapsulating solution.

The proteinoid carriers of the invention are pharmaco35 logically harmless and do not alter the physiological and

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PCT/US93/05723 biological properties of the active agent. Furthermore, the encapsulation process does not alter the pharmacological properties of the active agent. While any suitable pharmacological agent can be encapsulated within proteinoid carriers, it is particularly valuable for delivering agents which otherwise would be destroyed or rendered less effective by conditions encountered in the animal body before it reaches its target zone and which are poorly absorbed in the gastrointestinal tract.

The proteinoid carriers of the invention are particularly useful for the oral administration of certain pharmacological agents, e.g., small peptide hormones, which, by themselves, pass slowly or not at all through the gastro-intestinal mucosa and/or are susceptible to chemical cleavage by acids and enzymes in the gastrointestinal tract. Non-limiting examples of such agents include human or bovine growth hormone, interferon and interleukin-II, calcitonin, atrial naturetic factor, antigens, monoclonal antibodies, and Factor IX, a vitamin K-dependent blood coagulation proenzyme.

The choice of a particular proteinoid for use in encapsulating or entrapping a pharmacological agent depends on a number of factors which include:

(1) the acidity or basicity of the agent;

(2) the targeted area for release in the gastrointestinal tract;

(3) the solubility of the drug at certain pH ranges;

(4) efficiency of encapsulation;

(5) interaction of drug with proteinoid.

For example, proteinoids made from glutamic acid, aspartic acid, tyrosine, and phenylalanine are especially suitable for encapsulating polysaccharides like heparin.

In addition to selective pH solubility, the particle size of the proteinoid carrier plays an important role in determining release of the active agent in the targeted area of the gastrointestinal tract. Proteinoid carriers having diameters between about < 0.1 microns and about 10 microns, preferably

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PCT/US93/05723 between about 5.0 microns- and about 0.1 microns, and containing encapsulated or entrapped active agents are sufficiently small to effectively release the active agent at the targeted area within the gastrointestinal tract. Large proteinoid carriers (>10 microns) tend to be less effective as oral delivery systems.

The size of the proteinoid carriers formed by contacting proteinoids with water or aqueous solution containing active agents can be controlled by manipulating a variety of physical or chemical parameters, such as the pH, osmolarity or salt content of the encapsulating solution, and the choice of acid used in the encapsulating process .

By tailoring both the solubility characteristics of a proteinoid and the particle size of the proteinoid carriers, active agent bearing proteinoid carriers can be produced from 15 base-soluble proteinoids which are stable in the highly acidic stomach (normal pH of from about 2 to about 6) , but which dissolve in the distal portion of the intestines. Such systems are suitable for oral administration of peptide hormones, e.g., insulin, and polysaccharides, e.g., heparin, which otherwise 20 would be quickly destroyed in the GI tract. They also are suitable for protecting the stomach from gastric irritants, such as aspirin. When such aspirin-containing proteinoid carriers are orally administered, they pass through the gastrointestinal mucosa and release the aspirin far more rapidly than conventional 25 enterically coated aspirin, which first must traverse the stomach and then must enter the bloodstream from the intestine after the enteric coating has dissolved.

It also is possible to produce systems from acidsoluble proteinoids which are stable under weakly basic condi3 0 tions (pH of about 8) , but which release active agent under acidic conditions (pH of about 2 to 5) . Such systems are suitable for the intravenous administration of pharmacological agents such as calcium regulators and redox carrier systems for dopamine or gamma-aminobutyric acid.

The proteinoid carriers of the invention may be orally

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PCT/US93/05723 administered alone as solids in the form of tablets, pellets, capsules, and granulates suitable for suspension in liquids such as edible oils. Similarly, the proteinoid carriers can be formulated into an orally administrable composition containing one or more physiologically compatible carriers or excipients. These compositions may contain conventional ingredients such as gelatin, polyvinylpyrrolidone and fillers such as starch and methyl cellulose.

The proteinoid carriers of the invention may also be 10 administered by injection.

The following examples are illustrative of the invention but are not intended to limit the scope of the invention.

Example 1: Preparation of a Base-soluble Proteinoid by a Thermal condensation Reaction

750 ml of tetramethylene sulfone was heated to 190°C in an inert nitrogen atmosphere in a 4 liter flask with stirring. 294 g of glutamic acid was added and the mixture was heated for one-half hour. 266 g of aspartic acid was added and the mixture heated as rapidly as possible to 190°C and held there for 15 minutes. 362 g of tyrosine was added and the mixture heated at 190°C for 3 hours. 330 g of phenylalanine was added and the mixture heated at 190°C for 1.5 hours. The hot melt was then poured into 5 liters of water with vigorous stirring. After stirring for about 1 hour, the mixture was filtered and the filtrate discarded. The cake was reslurried in 5 liters of water, filtered and the cake was again reslurried in 5 liters of water. The pH of the slurry (at 25°C) was adjusted to 8 using

40% sodium hydroxide solution. The mixture was filtered and the cake washed with a small amount of water. The washes and filtrate are combined and evaporated to dryness in vacuo to give Glu/Asp/Tyr/Phe proteinoid.

Appendices A, B, and C describe examples of other proteinoids prepared by the thermocondensation method.

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IS

Example 2: Preparation of an Acid-soluble Proteinoid by a Thermal Condensation Reaction

750 ml of tetramethylene sulfone is heated to 190°C in an inert nitrogen atmosphere in a 4 liner flask with stirring.

294 g of glutamic acid is added and the mixture is heated for one-half hour. 362 g of tyrosine is added and the mixture is heated at 190°C for 3 hours. 330 g of phenylalanine is added and the mixture is heated at 190°C for 1.5 hours. 266 g of arginine is added and the mixture is heated for an additional 1.5 hours.

The hot melt is then poured into 5 liters of water with vigorous stirring. After stirring for about 1 hour, the mixture is filtered and the filtrate is discarded. The cake is reslurried in 5 liters of water, filtered and the cake is again reslurried in 5 liters of water. The pH of the slurry (at 25 °C) was adjusted to 5 using 10% acetic acid solution. The mixture is filtered and the cake is washed with a small amount of water. The washes and filtrate are combined and evaporated to dryness in vacuo to give proteinoid.

Appendices A, B, and C describe examples of other proteinoids prepared by the thermocondensation method.

Example 3: Preparation of Proteinoids by the

NCA Method Using Amine Initiator

This example illustrates the NCA method for preparing copolypeptides consisting of Asp.Bz, Glu.Bz, Phe, and Tyr components. The NCA monomers of these amino acids were prepared according to the reported method.

The reactions were carried out in tetrahydrofuran (THF) or in dichloromethane using benzylamine (BzNH₂) or 4-methylbenzyl amine (MeBzNH₂) as initiator at room temperature ( [M] = 10%) . The characterization of the resulting copolymers was performed by *H NMR and GPC. The results obtained are listed in Table 1.

As shown in Table 1, proteinoids having Asp and/or Glu as the second monomers and Phe and/or Tyr as the first monomers were obtained in high yield from the polymerization initiated with BzNH₂ at the ratio of [M] / [I] = 5 (No. 2-1 to 2-7) .

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The GPC curve (Figure 1) for poly(Asp.Bz-co-Phe), from which a polydispersity of 1.91 was determined. Similar molecular weight distributions were observed for other copolymers.

Polydispersity is defined herein as the molecular 5 weight distribution of a sample. The distribution is assigned a numerical value derived from che molecular weight (MW) divided by the molecular number (Mn) . The polydispersity value for a homopolymer is 1 because the molecular weight is equal to the molecular number. Any polymer with a polydispersity value of 1 is considered to have a very narrow distribution. A polymer with polydispersity value of 1.6 co 1.7 is considered to have medium distribution. A polymer with a polydispersity value of 2.0-2.1 is c nsidered co have a broad discribution.

The ..omopolymerization of NCA of Asp.Bz and Che copolymerizations of NCAs of Asp.Bz, Glu.Bz, Phe, and Tyr were also carried out using MeBzNH₂ as iniciator (No. 2-11, 2-15, and 2-16) . Similar results were obtained for reactions initiated by

BzNH₂.

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TABLE 1

COPOLYMERIZATION OF NCAs INITIATED WITH AMINES _STORED AT ROOM TEMPERATURE FOR 4 DAYS

<td> POLYM. NO.</td><td> COMONOMER COMPOSITION</td><td> INITIATOR (IMl/IIJ)</td><td> SOLVENT</td><td> YIELD (%)</td><td> M_w</td>

<td> 2-1</td><td> Asp-Glu-Phe-Tyr (1:1:1:1)</td><td> BzNH₂ (5:1)</td><td> THF</td><td> 84.1</td><td> 830</td>

<td> 2-2</td><td> Asp-Phe (1:1)</td><td> BzNH₂ (5:1)</td><td> THF</td><td> 70.9</td><td> 730</td>

<td> 2-3</td><td> Asp-Tyr (1:1)</td><td> BzNH₂ (5:1)</td><td> THF</td><td> 88.6</td><td> 1000</td>

<td> 2-4</td><td> Asp-Tyr (2:1)</td><td> BzNH₂ (5:1)</td><td> THF</td><td> 89.3</td><td> 1050</td>

<td> 2-5</td><td> Glu-Tyr (1:1)</td><td> BzNH₂ (5:1)</td><td> THF</td><td> 84.9</td><td> 870</td>

<td> 2-6</td><td> Glu-Phe-Tyr (2:1:1)</td><td> BzNH₂ (5:1)</td><td> CH₂CI₂</td><td> 68.8</td><td> 790</td>

<td> 2-7</td><td> Glu-Phe-Tyr (1:1:1)</td><td> BzNH₂ (5:1)</td><td> CH₂CI₂ '</td><td> 53.7</td><td> 1000</td>

<td> 2-11</td><td> Asp</td><td> MeBzNH₂ (5:1)</td><td> THF</td><td> 88.3</td><td> 870</td>

<td> 2-15</td><td> Asp-Glu-Phe-Tyr (1:1:1:1)</td><td> MeBzNH₂ (5:1)</td><td> THF</td><td> 76.4</td><td> -</td>

<td> 2-16</td><td> Asp-Glu-Phe-Tyr (1:1:1:1)</td><td> MeBzNH₂ (5:1)</td><td> THF</td><td> 76.4</td><td> 630</td>

Example 4: Preparation of Proteinoids by the NCA Method

Using a-Methyl Tyrosine Ester as Initiator

This example illustrates the method of conducting NCA polymerizations, using a-methyl tyrosine ester (Tyr.Me) as the initiator. The reaction conditions are essentially the same as described in Example 4 except tecrahydrofuan (THF) solvent was used. The results are listed in Table 2.

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TABLE 2

PROTEINOID SYNTHESIS BY NCA INITIATED WITH AMINO

ACIDS STORED AT ROOM TEMPERATURE FOR 4 DAYS

<td> POLYM. NO.</td><td> COMONOMER COMPOSITION</td><td> INITIATOR (IMJ/dl)</td><td> SOLVENT</td><td> YIELD (%)</td><td> M_w</td>

<td> 2-8</td><td> Asp-Glu-Phe (1:1:1)</td><td> Tyr.Me (1:1)</td><td> CH₂CI₂</td><td> 100</td><td> 450</td>

<td> 2-9</td><td> Asp-Glu-Phe (1:1:1)</td><td> Tyr.Me (3:1)</td><td> ch₂ci₂</td><td> 71.4</td><td> 450</td>

<td> 2-10</td><td> Asp-Glu-Phe (1:1:1)</td><td> Tyr.Me (5:1)</td><td> ch₂ci₂</td><td> 68.0</td><td> 730</td>

<td> 2-12</td><td> Asp</td><td> Tyr.Me (1:1)</td><td> THF</td><td> 100</td><td> 460</td>

<td> 2-13</td><td> Glu-Tyr (1:1)</td><td> yff-Ala (2:1) Suc.An (2:1)</td><td> THF (reflux)</td><td> 67.4</td><td> 480</td>

<td> 2-14</td><td> Asp</td><td> Tyr.Me (6:1)</td><td> THF</td><td> 91.8</td><td> 890</td>

<td> 2-17</td><td> Phe</td><td> Tyr.Me (1:1)</td><td> THF</td><td> 73.0</td><td> ND</td>

<td> 2-18</td><td> Tyr</td><td> Tyr.Me (1:1)</td><td> THF</td><td> 65.7</td><td> ND</td>

<td> 2-19</td><td> Phe</td><td> Tyr.Me (5:1)</td><td> THF</td><td> 78.3</td><td> ND</td>

<td> 2-20</td><td> Tyr</td><td> Tyr.Me (5:1)</td><td> THF</td><td> 63.3</td><td> ND</td>

It was found that the initiation by Tyr.Me is very fast (No. 2-17 to 2-20) and all the NCA has been converted after 2 hours. From GPC data, it was observed that the molecular weight of the polymer increased with increasing ratio of [M] / [Tyr.Me] and the polydispersity is quite narrow. The existence of a Tyr.Me residue in the polymers was confirmed by *H NMR spectra. In conclusion, Tyr.Me is a novel and effective initiator for the polymerization of amino acid NCA's.

Sample No. 2-13 represents a polymerization initiated with β-alanine and terminated with succinic anhydride. As βalanine is insoluble in most organic solvents, the reaction was carried out in refluxing THF. As a result, the polydispersity of

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Example 5: Preparation of Proteinoids 5 bv the DPPA Method (#1)

This is an example of a direct polycondensation of

Asp.Bz in the presence of DPPA and triethylamine (TEA) as a base under various polymerization conditions ( (a) , (b) , (c) , and (d) ) . The molecular weight of the polymers, as well as polydispersity, was evaluated in each case by GPC. The polymers were characterized by IR and NMR spectroscopy.

Asp.Bz was prepared by che esterification of L-aspartic acid as follows: L-aspartic acid (26.6 g, 0.2 mole) was suspended in 300 ml of freshly distilled benzyl alcohol in a 500 ml round bottom flask, followed by addition of 45 ml of concentrated hydrochloric acid (12N) . The mixture was heated up to 60'C under vigorous stirring for 3 0 minutes. Thereafter, the reaction solution cooled to room temperature. Triethyl amine (about 56 ml) was added co neutralize (to a pH of about 7) the solution. The crude product was collected by filtration, washed with ethanol and acetone, dried in vacuo, and crystallized twice from hot water. 18 g of product was obtained (% yield = 44%). M.pc = 217-C.

Commercial DPPA was used without further purification.

TEA was discillated before use. Solvents for polymerization were purified by conventional methods. The direct polycondensation of Asp.Bz was carried out by stirring a dimethyl formamide (DMF) solution of the monomer in the presence of DPPA and TEA. The mixture was stirred for l h at 0-10°C followed by stirring at room temperature for two days. The resulting polymer was precipitated in a large amount of water, collected by filtration, and then dried in vacuo.

a. Effect of Monomer Concentration

Listed in Table 3 are the results for the polymerization of Asp.Bz in DMF at room temperature for two days.

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Poly(Asp.Bz)s were obtained from these direct polycondensations in high yield.

The molecular weight of the polymers was found to be dependent on the concentration of the monomer [M] . Low molecular weight polymers with broad distribution were obtained from a low [M] (Figure 2, curve ,A) . On the other hand, when [M] was greater than 0.2 g/mL, a polymer with a bimodal molecular weight distribution was obtained (Figure 2, curve B) . The lower molecular weight oligomers (-1000) ma/ be due to an intramolecu1ΰ lar termination between the terminal amino and the β-carboxylic groups. After several reprecipitations from THF/methanol, a polymer with a higher molecular wei- (M„ = 22,000) and narrow polydispersity (Μ^,/Μη = 1.68) was su. ^ssfully isolated from the polymer mixture prepared at [M] = lg/mL. The separation was also performed using GPC column with Bio-Beads.

TABLE 3

EFFECT OF THE MONOMER CONCENTRATION ON POLYMERIZATION

OF Asp.Bz BY DPPA IN DMF AT ROOM TEMPERATURE:

[DPPA] / [M] = 1.3; [TEA] [M] = 2.3

<td> [M] (g/ml)</td><td> YIELD (%)</td><td> M„X10'^3|c|</td><td> M_w/M_n</td>

<td> 0.025</td><td> 71.5^W</td><td> 1.4</td><td> 4.15</td>

<td> 0.033</td><td> 74.7¹’¹</td><td> 1,0</td><td> 3.50</td>

<td> 0.05</td><td> 67.2^,al</td><td> 1.1</td><td> 5.11</td>

<td> 0.10</td><td> 63.2^W</td><td> 0.91</td><td> 3.70</td>

<td> 0.20</td><td> 85.4“</td><td> 16.3 (60.7), 1.0 (39.3)</td><td> 1.84, 1.13</td>

<td> 0.50</td><td> 86.5^|b|</td><td> 11.0 (59.4), 0.92 (40.6)</td><td> 2.22, 1.08</td>

<td> 1.0</td><td> 97.6^|b|</td><td> 15.1 (71.4, 0.88 (28.6)</td><td> 1.81, 1.05</td>

a) The polymer was collected by centrifugation after polymerization for 2 days;

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b) The polymer was collected by filtration after polymerization for 2.5 days.

c) The values in parentheses are molar percentages.

b. Effect of Reaction Time and Temperature

The yield of the resulting polymer increased with the reaction time: 75% conversion in 5 h and 95% in 4 days (Figure 3, curve A). The molecular weight of the resulting polymer also increased with time in the initial phase (up to 4 h) and then became almost constant (Figure 4). The polymerization decreased with increasing temperature (Figure 3, curve B) . Polymers obtained at 60 and 80°C were of yellow color and insoluble in THF but soluble in DMF and DMSO. This may be due to the formation of an imide ring which has been reported to occur during thermal polycondensations of aspartic acid.

c. Effect of Molar Ratios [DPPA]/[M] and [TEA]/[M]

The dependence of the yield and the molecular weight of 20 the polymer on the molar ratios of [DPPA] / [M] , as well as [TEA]/ [M], was investigated (Table 4). The highest yield was obtained at a [DPPA] / [M] of 1.3 and a [TEA] / [M] of 2.3 (Figure 5) . These observations are in agreement with the results reported by Nishi et al. Higher molecular weight products were obtained in the range of [DPPA] / [M] = 1.3-2.0 and [TEA] / [M] =

2.0-3.0, respectively.

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TABLE 4

EFFECT OF THE MOLAR RATIOS OF DPPA AND TEA ON POLYMERIZATION OF Asp.Bz IN DMF AT ROOM TEMPERATURE: [M] = 0.50 g/ml

<td> [MJ/DPPA</td><td> IMJ/ITEAJ</td><td> YIELD (%)</td><td> M_nX10'^3WI</td><td> M_w/M_n</td>

<td> 0.5</td><td> 2.3</td><td> 16.3</td><td> 0.31</td><td> 4.09</td>

<td> 1.0</td><td> 2.3</td><td> 69.6</td><td> 3.1 (45.4), 0.39 (54.6)</td><td> 2.58, 1.48</td>

<td> 1.3</td><td> 2.3</td><td> 86.5</td><td> 11.0 (59.4), 0.92 (40.6)</td><td> 2.22, 1.08</td>

<td> 1.5</td><td> 2.3</td><td> 69.4</td><td> 15.9 (34.2), 0.83 (65.8)</td><td> 1.77, 1.21</td>

<td> 2.0</td><td> 2.3</td><td> 64.3</td><td> 13.1 (58.3), 0.89 (41.7)</td><td> 1.87, 1.09</td>

<td> 1.3</td><td> 1.5</td><td> 58.4</td><td> 6.0 (39.3), 0.63 (60.7)</td><td> 2.43, 1.37</td>

<td> 1.3</td><td> 2.0</td><td> 78.3</td><td> 13.3 (64.3), 0.92 (35.7)</td><td> 1.87, 1.19</td>

<td> 1.3</td><td> 3.0</td><td> 74.6</td><td> 13.6 (64.8), 0.83 (35.2)</td><td> 1.98, 1.18</td>

<td> 1.3</td><td> 3.5</td><td> 65.0</td><td> 8.3 (60.0), 0.80 (40.0)</td><td> 2.70, 1.10</td>

a) The value in parentheses are molar percentage.

d. Effect of Solvent

A comparison of the polymerizations in different solvents is shown in Table 5. It can be seen from this table that the yield and the molecular weight of the polymer are influenced by the solvents used. Higher yields were obtained in DMF while higher molecular weights were obtained in THF and in bulk. On the other hand, the polymerization in dioxane gave a lower molecular weight product, and therefore is preferred.

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TABLE 5

EFFECT OF THE SOLVENTS ON POLYMERIZATION OF Asp.Bz AT ROOM TEMPERATURE FOR 2 DAYS [M]/(DPPA] = 1.3, [M]/[TEA] = 2.3, [M] = 0.50 gr/ml

<td> SOLVENT</td><td> YIELD (%)</td><td> M_nX10'^3lb|</td><td> M_w/M„</td>

<td> DMF</td><td> 86.5</td><td> 11.0 (59.4), 0.92 (40.6)</td><td> 2.22, 1.08</td>

<td> DMSO</td><td> 70.6</td><td> 11.5 (78.9), 1.05 (21.1)</td><td> 1.87, 1.13</td>

<td> THF</td><td> 49.9</td><td> 29.6 (74.6, 1.14 (25.4)</td><td> 1.31, 1.13</td>

<td> ACETONITRILE</td><td> 71.1</td><td> 20.3 (79.3), 1.05 (20.7)</td><td> 1.65, 1.14</td>

<td> DIOXANE</td><td> 70.5</td><td> 4.7 (68.5), 0.82 (31.5)</td><td> 3.80, 1.13</td>

<td> NONE’¹</td><td> 61.2</td><td> 29.8 (82.8), 0.86 (17.2)</td><td> 1.32, 1.16</td>

a) Bulk polymerization.

b) The value in parentheses are molar percentage.

Example 6: Preparation of Proteinoids bv the DPPA Method (#2)

Copolymerizations of Asp.Bz with other amino acid monomers such as γ-benzyl glutamate (Glu.Bz), β-alanine (Ala), Phenylalanine (Phe)', and O-benzyl tyrosine (Tyr.OBz) in the presence of DPPA were carried out using the same procedure as that for the homopolymerization of Asp.Bz (Example 5) . Random copoly (amino acids) were obtained in high yield (&gt; 77%) as shown in Table 6. This indicates that the copolymerization of amino acids using DPPA is a useful approach to copolypeptide synthesis.

Bimodal molecular weight distributions were also observed in these cases similarly to the homopolymerization of Asp.Bz.

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TABLE 6

COPOLYMERIZATION OF α-AMINO ACIDS IN THE PRESENCE OF DPPA AS CONDENSING AGENT IN DMF AT ROOM TEMPERATURE FOR 2 DAYS

<td> POLYM. NO.</td><td> COMONOMER COMPOSITION</td><td> YIELD (%)</td><td> M_w</td><td> M_w/M_n</td>

<td> Co. 1 DPPA</td><td> Asp.Bz-Glu.Bz (1:1)</td><td> 97.4</td><td> 15900, 1080</td><td> 1.76, 1.13</td>

<td> Co.2DPPA</td><td> Asp.Bz-/?-Ala (1:1)</td><td> 91.2</td><td> 1590</td><td> 1.18</td>

<td> Co_:3DPPA</td><td> Asp.Bz-Phe (1:1)</td><td> 89.7</td><td> 13700, 800</td><td> 1.89 1.25</td>

<td> Co.4DPPA</td><td> Asp.Bz-Tyr.OBz (1:1)</td><td> 87.3</td><td> 9000, 1000</td><td> 1.78, 1.17</td>

<td> Co.5DPPA</td><td> Asp.Bz-Glu.Bz-Phe-Tyr.OBz (1:1:1:1)</td><td> ” 92.5</td><td> 16800, 960</td><td> 1.66, 1.14</td>

Example 7: Reductive Debenzylation of Proteinoids

Produced by the DPPA Method

The example illustrates a preferred method for the removal of benzyl protective groups in poly (Asp.Bz) and poly(Glu.Bz) by catalytic hydrogenation.

The hydrogenation of the polymers was carried out according to the following procedure: To a solution of the polymer in THF/methanol (1:1, v/v) , Pd on active carbon (10%) was added in the amount of 1/10 of the polymer weight. After the replacement of air by nitrogen, hydrogen gas was introduced into the system and maintained with a balloon. The reaction mixture was stirred at room temperature overnight. After removing the catalyst by filtration and concentrating the solution, the mixture was poured into a large amount of petroleum ether to precipitate the polymer. The polymer obtained was then dried in vacuo.

The completion of the hydrogenation was confirmed by *H NMR of the polymer. In most cases, useful water-soluble polymers

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Example 8: Preparation of Empty Proteinoid..carriers with Glu, Asp, Tyr, Phe Proteinoid_

This Example illustrates a method for the preparation and cleaning of empty proteinoid carriers.

PROCEDURE

1

Reagents:

a. Proteinoid powder prepared as described in Example 1

b. Anhydrous citric acid (USP)

c. Gum acacia NF

d. Deionized water

e. Glatial acetic acid

2. Equipment:

a. Ph meter

b. Water bath, 40°C

3. Preparation of Solutions:

a. Proteinoid solution - Dissolve lOOmg proteinoid in 1ml deionized water (or multiples thereof). Filter through a Whatman #1 filter paper (if necessary) and keep at

40°C in a water bath. This is solution A.

b. 1.7 N citric acid with 0.5% acacia - Dissolve 5g of acacia and 109g of citric acid in 1 liter ’ deionized water. Incubate at 40°C. This is solution B.

³⁰ ..

4. Preparation of Proteinoid carriers :

a. Add all of solution A to solution B rapidly in one step while swirling solution B by hand, in a 40°C water bath.

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Example 9: Preparation of Murine IgG Monoclonal Antibodycontaining Proteinoid Carrier

This experiment describes encapsulation of antireovirus monoclonal antibody (mAb) 9BG5, an mAh directed against the sigma-1 gene product (Hemaglutinin, HA3) of the Reovirus Type

3. HA3 binds to the cell surface receptor for Reovirus type 3, and mAb 9GB5 interferes with viral binding to the receptor.

Mouse IgG monoclonal antibody 9BG5 was prepared and purified as described W.V. Williams et al. (1991) J. Biol. Chem. .

Vol. 266(8), pages 5182-5190, as well as references cited therein, using a purified Reovirus type 3 preparation (W.V. Williams et al. (1988) Proc. Natl. Acad. Sci. U.S.A, Vol. 85, pages 6488-6492) . The purified 9BG5 used in this Example had a protein concentration of 1.5 mg/ml in phosphate buffered saline (pH 7.2).

Proteinoid carriers encapsulating mAb 9BG5 were prepared having final concentrations of Glu/Asp/Tyr/Phe proteinoid (1:1:1:1 mole ratio of Glu, Asp,Tyr, and Phe in the reaction mixture) 50 mg/ml, mAb 0.7 mg/ml and gum arabic 0.5% in 0.85 N citric acid. Empty proteinoid carriers were, prepared to contain the same final concentrations, except mAb was omitted. Aliquots (0.5 ml) , in duplicate, of both mAb and empty proteinoid carriers preparations were centrifuged at 5000 RPM. Pellets and supernatants were frozen prior to analysis by Western blotting to determine antibody encapsulation efficiency.

Figure 6 is an x-ray film of a western blot analysis of purified mAb ^c' emp/.y proteinoid carriers (no mAb added) , and proteinoid ca_-^ers containing 9BG5. The analysis was done by immunoblot ting with anti-ir. ase IgG which specifically reacted with mAb 9BG5. The lanes correspond to the following:

Lane Sample

2 /xg 9BG5 mAb

1 μ&lt;3 9BG5

0.25 /xg 9BG5

MW molecular weight markers

Empty proteinoid carrier supernatant after encapsulation

Empty proteinoid carrier pellet

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The data indicates that the 9BG5 proteinoid carriers contained about 40% of the mAb in the pellet and the remaining

60% did not incorporate in the proteinoid carriers and was left in the supernatant. The empty proteinoid carriers did not contain antibody in the supernatant or the pellet as was expected.

The relative mobility (molecular weight) of the pure 10 mAb is slightly different than the mAb in the proteinoid carriers. This is most likely due to different salt concentrations in the samples, because the encapsulation process employed

0.8 M salt solution.

Example 10: Effect of Additives on Stability of Proteinoid

Carriers with Encapsulated Murine mAb 9BG5

Various proteinoid carrier formulations were screened, 20 with or without additives, to determine optimal carrier-forming conditions and concentrations of mAb required for carrer • formation.

The mAb 9BG5 preparations used to prepare the encapsulated proteinoid carriers had a protein concentration of approximately 2 mg/ml in phosphate buffered saline.

Final proteinoid concentration was 50 mg/ml and 5% (w/w) gum acacia (gum) or gelatin (gel). All proteinoid carriers were prepared in 0.85 N citric acid. Empty carriers were included for use as controls, and they were prepared in the same manner with the omission of mAb. Duplicate (0.5 ml) aliquots of proteinoid carrier suspension were centrifuged at 5000 RPM. Pellets and supernatants were frozen in dry ice prior to analysis.

Table 7 lists samples that were prepared. Numbers in parenthesis indicate amount of mAb added.

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TABLE 7

<td> SAMPLE</td><td> PROTEINOID</td><td> ADDITIVE</td><td> ' ' · ........ 1 FINAL PROTEIN (MG/ME)</td>

<td> 1</td><td> 326</td><td> Gum </td><td> 0</td>

<td> 3</td><td> 326</td><td> Gel</td><td> 0</td>

<td> 5</td><td> 334</td><td> Gum</td><td> 0</td>

<td> 7</td><td> 334</td><td> Gel</td><td> 0</td>

<td> 9 , 10</td><td> 326</td><td> Gum</td><td> 0.5</td>

<td> 11,12</td><td> 326</td><td> Gum</td><td> 0.25</td>

<td> 13</td><td> 326</td><td> Gel</td><td> 0.25</td>

<td> 15, 16·</td><td> 334</td><td> Gum</td><td> 0.25</td>

<td> 17, 18</td><td> 334</td><td> Gel</td><td> 0.25</td>

In order to test resistance to freeze and thawing on the integrity of the proteinoid carriers containing mAb, one of each pair of duplicate pellets were washed by gentle resuspension in 0.25 ml of 0.85 N citric acid. The pellets were then analyzed next to the unwashed pellets to test whether any mAb was lost in the washing.

The samples were analyzed by conventional Western blotting as described in Exc .e 9. Pellets were dissolved in sodium dodecyl sulfate with 0.05 N NaOH and analyzed under reducing conditions (breaks up the mAb into 50 kDa and 25 kDa bands). Aliquots (50μ1) of supernatants were analyzed under non25 reducing conditions (expected intact 150 kDa mAb) . This was done to determine differentially whether the mAb left ’behind is denatured or intact. .

As can be seen from the X-ray film from the Western Blots (Figure 7), pellets of samples 9 and 10, and 11 and 12

0 contain between 5 and 10 ^g of mAb. The washed samples did not lose any significant amount of mAb, suggesting that the proteinoid carriers remained intact after freeze-thawing.

The supernatants of samples 9 and 11 had no significant amount of mAb, indicating that unincorporated material was lost

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Sample 17 had some mAh encapsulated which was lost after washing (see number 18). This sphere preparation was not resistant to freeze-thawing. Additionally, a band at a MW of 150 kDa for sample 17 supernatants indicates that a significant amount of mAh is left behind after proceinoid carrier formation.

Based on these results, it appears that the mAb remains intact and therefore the encapsulating procedure does not degrade it. The empty proteinoid carrier controls did not produce any bands, as expected because they have no mAb.

Example 11: Efficacy of Encapsulated Murine

IgG Monoclonal Antibody

In this Experiment, a mAb 9BG5 proteinoid carrier 15 preparation and unencapsulated mAb 9BG5 were evaluated in rats. The mAb 9BG5 (1 mg/ml) , prepared as described in Example 9, was encapsulated in Glu/Asp/Tyr/Phe proteinoid (1:1:1:1 mole ratio of

Glu, Asp,Tyr, and Phe in the reaction mixture) protein carrier formulation with gum arabic. The mAb proteinoid carriers suspension contained 0.25 mg/ml mAb and 50 mg/ml proteinoid in 0 85 N citric acid-0.5% gum. Empty proteinoid carriers were prepared similarly, but did not contain mAb. Since 30% of the mAb was found to be encapsulated, the mAb proteinoid carriers were estimated to contain 0.075 mg/ml mAb and this value was used to determine dosages. The mAb proteinoid carriers were examined microscopically and appear to be a fairly homogeneous preparation.

For animal dosing, appropriate aliquots of'proteinoid carriers were centrifuged at 5000 RPM for 15 minutes, and pellets were resuspended in 1.0 ml of 0.85 N citric acid-0.5% gum.

A purified mAb solution (0.95 mg/ml mAb in 0.85 N citric acid-0.5% gum) was used for oral gavage. This solution was prewarmed to 40¹C prior to administration. For IV administration, a purified mAb solution (1 mg/ml mAb in phosphate buffer saline) was used.

The amounts and administration routes employed in the

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1. Empty proteinoid carriers (no mAh): 1 ml aliquot containing 50 mg empty proteinoid carriers by oral gavage (rats # 2312 and 2313) . 5 2 . mAb 9BG5 proteinoid carriers : 3.7 mg mAb/ kg body weight of rat by oral gavage (rat # 2287, 2288, 2290, and 2291).

3. unencapsulated mAb 9GB5: 0.73 mg/ kg body weight of rat by intravenous administration (rats #2292, 2293, and 2311).

4. unencapsulated mAb 9BG5: 3.7 mg/ kg body weight of rat by oral gavage (rats #2314 and 2315).

Baseline blood samples (1 ml aliquots) were withdrawn from each rat just prior to dosing (0 time). After dosing, blood samples were drawn at 1 h, 6 h and 24 h. The blood samples were processed immediately and sera were stored frozen at -20°C.

Thawed serum taken from the experimental animals were analyzed by conventional ELISA techniques, in triplicate, using purified reovirus type 3 and V_tSH dimeric peptides immobilized in multi-well plates (W.V. Williams et al (1991) J. Biol. Chem.. Vol. 266(8), pages 5182-5190). Control plates included wells having no immobilized reovirus and V_LSH peptides to which mAb (lmg/ml) was added. VLSH peptide (W.V. Williams et al. ibid, Table 1) is a synthetic variant of VL peptide, the latter which corresponds to a portion of the light chain variable CDR II region of 87.92.6 antibody. The 87.92.6 antibody displays idiotypic and anti-idiotypic behavior towards reovirus type 3 receptor and niAb 9BG5, respectively (W.V. Williams et al. ibid). The bound protein content of each well were measured by standard protein methods, e.g., Lowry method, and the results for each multi-well plate are shown'in Figures 8(a-c), respectively.

Figures 8 (a-c) illustrate the levels of serum proteins which bound to immobilized reovirus ty” a 3 and V_LSH as detected by measurement of protein concentration. These Figures show that the serum levels of bound proteins, after 24 hours post-dosing, were highest for animals orally administered mAb proteinoid carriers and animals administered unencapsulated mAb by the IV

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PCT/US93/05723 route. Lower levels of bound serum proteins were found in animals orally adminstered uncapsulated mAb. Serum taken from the animals receiving empty proteinoid carriers (no mAb) showed non-specific serum IgG protein binding, as expected, under the assay conditions.

Figure 9 show mAb binding under conventional ELISA procedures using immobilized reovirus type 3 and V_LSH proteins. Serial dilutions of mAh treated with 0.85 N citrate-0.5% gum (Figure 9(a) or phosphate buffered saline (Figure 9 (b) were employed. The Figures show that the bound protein levels were higher for mAb in citrate buffer than for mAb in phosphate. Without being bound by any theory of operation for this invention, it is believed that the binding enhancement may be due to changes in the three dimensional conformation resulting from citrate-protein binding.

In summary, serum levels of mAb, as reflected by the absorbance of bound proteins, were greater in animals receiving encapsulated mAb by the oral route or unencapsulated mAb by the IV route, than an animal receiving orally administered unencap20 sulated mAb.

Example 12: Preparation of Proteinoid carrier containing

Heparin

This Example describes a method for the preparation and cleaning of heparin proteinoid carriers.

PROCEDURE

Reagents: '

a. Proteinoid powder prepared as described in Example 1

b. Heparin

c. Anhydrous citric acid (USP)

d. Gum acacia NF e . Deionized water

f. Desiccant

g. Liquid nitrogen

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Equipment:

a. Magnetic stirrer

b. Buret

c. Mic’-cscope d. Cli....cal centrifuge

e. Dialysis membrane tubing (Spectrum 6, 10 mm, 50,000

M.W. Cutoff)

f. pH meter

g. Lyophilizer (Labconco #75035)

h. Lyophilizing flasks (150-300 mL)

i. Rotating shell freezer .

j. Isopropanol/dry ice bath or liquid N₂

k. Mortar and pestle

l. Storage containers (500 mL)

m. Eppendorf pipet (0-100 uL)

n. Plastic closures for dialysis tubing (Spectrum)

o. 2 mL syringe with 0.45 um Acrodisk

3. Preparation of Solutions:

a. Proteinoid Solution A* (80 mg/ml):

Dissolve 160 mg proteinoid in 1 ml of deionized water. Using a 2 ml syringe fitted with a 0.45 um Acrodisk, the proteinoid solution was filtered into a 10 ml test tube and kept at 40° C.

b. Solution Β (1.7 N citric acid with 1% gum):

Dissolve 10 g of gum acacia and 109 g of citric acid in 1 liter of deionized water.

c. Solution C (Heparin solution):

Dissolve heparin in Solution B at 150 mg/mL and keep at 40° C.

or multiples thereof.

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a. Add all of solution A to solution C quickly while swirling solution C slowly, by hand, in a 40°C water bath. 5

5· Dialysis of Heparin Proteinoid carriers:

It has been found the presence of citric acid in the encapsulated proteinoid carriers interferes with a subsequent lyophilization process. Hence, proteinoid carrier encapsulates prepared with citric acid solutions are preferably dialyzed against 5% acetic acid solution for at least two hours with at least four changes of the dialysis solution to remove citric acid by an exchange process. Thus,

a. Transfer the suspension with a syringe (no needle) to dialysis tubing and seal with plastic closures. Tubing should be no more than 70¾ full.

b. Discard any amorphous material sedimented and/or aggregated on the surface.

c. Dialyze the proteinoid carrier suspension against acetic acid solution (using 20 mL of acetic acid solution per ml of proteinoid carrier suspension) while stirring the acetic acid solution with a magnetic stirrer.

d. Replace the acetic acid solution every hour. Continue dialyzing for a total of 3 hours.

6. Lyophilization:

a. Add one part of 50¾ trehalose (Sigma Chemical Co., St. Louis, MO, USA) into nine parts of dialyzed proteinoid carrier solution. Flash freeze proteinoid carriers in a freeze-drying flask using the shell freezer adjusted to rotate at ca. 190 rpm and immersed in a liquid

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b. Freeze dry for 24 hours or until dry as evidenced by lack of self-cooling. ...

c. Record weight of dry proteinoid carriers.

d. Grind to a fine powder with mortar and pestle.

e. Transfer proteinoid into an amber container, seal with desiccant, and store at room temperature. .

7. Resuspension:

a. Weigh the lyophilized powder and calculate the amount of proteinoid in the powder.

b. Add aqueous 0.85 N citric acid into the lyophilized powder at 40°C. The final concentration of proteinoid in solution is 80 mg/ml.

Example 13: Preparation of Insulin-containing Proteinoid

Carrier

This Example illustrates a method for the preparation 25 of insulin proteinoid carriers.

PROCEDURE

Reagents :

a. Proteinoid powder

b. Anhydrous citric acid (USP)

c. Gelatin (USP)

d. Porcine insulin (Novo Nordisk)

e. Deionized water (USP)

2. Equipment:

a.

Water bath

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b. 0.2 micron Acrodisk filter

c. Sterile syringe (lOcc)

d. Glass or plastic vessel of appropriate volume for desired amount of proteinoid carrier-solution.

3. Preparation of Solutions:

a. 1.7 N citric acid with 5.0% gelatin:

Dissolve 109 mg anhydrous citric acid and 50 mg gelatin per 1 ml of deionized water at desired volume” and incubate in water bath at 40 °C until gelatin is completely dissolved. This may be prepared and stored at 40°C for later use.

h&gt;. Insulin solution:

Dissolve 12 mg insulin per 1 ml of 1.7 N citric acid with 5% gelatin at 40°C at desired volume.

c. Proteinoid solution:

Dissolve 100 mg proteinoid per 1 ml deionised water at room temperature and desired volume. Using syringe and 0.2 micron Acrodisk, filter the solution to ensure a clear liquid and incubate in a water bath at 40°C. See Section 5b.

4. Preparation of Proteinoid carriers :

a. Proteinoid solution and insulin solution are combined at equal volumes sufficient to produce the final desired volume of proteinoid carriers.

b. Rapidly add the filtered proteinoid solution to the insulin solution at 40 °C while simultaneously and constantly swirling the insulin solution to ensure a _” Proteinoid and Insulin solutions should each be prepared at one-half the total volume of the final microsphere solution desired.

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Example 14: Procedure for Preparation of Erythropoietin

Containing Proteinoid carriers

Encapsulation of human erythropoietin (EPO) in proteinoid carriers was performed in the same manner described in Example 13. EPO was obtained from Genetic Institute (Cambridge, MA, USA, now available from Amgen Corp., Thousand Oaks, CA, USA) . A solution of Gln/Asp/Tyr/Phe (1:1:1:1 mole ratio of Gin, Asp,

Tyr, and Phe in the proteinoid reaction mixture) proteinoid and a 150 ug/mL EPO solution in 1.7 N citric acid with 1% gum was used in preparing the EPO-containing proteinoid carrier.

Example 15: Evaluation of Erythropoietin-containing 15 Proteinoid Carrier_

In this Example, an EPO-containing protein carrier, prepared as described in Example 14, was evaluated in rats. An EPO experimental synopsis is given below.

Rats weighing 150-200 grams are anesthetized with ketamine (£.5mg/kg) and thorazine 3.75mg/kg) with intramuscular injection. The rat is then administered either unencapsulated erythropoietin or encapsulated erythropoietin by oral gavage. In orief, an '8 french nelaton catheter is inserted down the esophagus of the rat until the 10cm mark on the catheter is even with the incisors. The test or control solution is drawn up into a syringe and attached to the catheter. Holding the animal upright, the solution is expressed into the stomach of the rat. The experimental results are summarized in Figures 10-12.

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ERYTHROPOIETIN EXPERIMENTAL SYNOPSIS

<td></td><td> Batch</td><td> Dose</td><td> Rats Resnondinq</td><td> Comments</td>

<td> 5</td><td> Control</td><td> l5Mg/kg</td><td> 0/4</td><td> Fasted 15 hours.</td>

<td></td><td> 251&lt;3K</td><td> 15μg/kg</td><td> 0/4</td><td> Access to bedding.</td>

<td></td><td> 254&lt;3K</td><td> 15μg/kg</td><td> 2/4</td><td> Gavaged</td>

<td></td><td> Control</td><td> 15μg/kg</td><td> 0/2</td><td></td>

<td> 10</td><td> 251&lt;3K</td><td> 15μg/kg</td><td> 0/2</td><td> Fasted 36 hours.</td>

<td></td><td> 254&lt;3K</td><td> 15μg/kg</td><td> 1/4</td><td> 5% sucrose.</td>

<td></td><td> 270K</td><td> 15μg/kg</td><td> 1/3</td><td> No bedding.</td>

<td></td><td> 270G</td><td> ^g/kg</td><td> 3/3</td><td> Gavaged.</td>

<td> 15</td><td> Control</td><td> ^g/kg</td><td> 1/5</td><td> Fasted 24 hours.</td>

<td></td><td> 264CP</td><td> 15μg/kg</td><td> 1/4</td><td> Access to bedding.</td>

<td></td><td> 270G</td><td> ^g/kg</td><td> 1/6</td><td> Gavaged.</td>

<td></td><td> Control</td><td> lC^g/kg</td><td> 0/5</td><td> Fasted 24 hours.</td>

<td> 20</td><td> 270G</td><td> lC^g/kg</td><td> 3*/6</td><td> No bedding.</td>

<td></td><td> Control</td><td> 3(^g/kg</td><td> 0/3</td><td> Fasted 24 hours.</td>

<td></td><td> Control</td><td> 6C^g/kg</td><td> 1/4</td><td> No bedding .</td>

<td></td><td> 270G</td><td> 3C^g/kg</td><td> 1/3</td><td> Direct injection</td>

<td> 25</td><td> 270G</td><td> 6(^g/kg</td><td> 1/4</td><td> into the stomach.</td>

<td></td><td> Control</td><td> 5C^g/kg</td><td> 0/3</td><td></td>

<td></td><td> Control+</td><td></td><td></td><td></td>

<td></td><td> Pepsin</td><td> 5C^g/kg</td><td> 0/4</td><td> Direct injection</td>

<td> 30</td><td> 270G</td><td> 5C^g/kg</td><td> 2/4</td><td> into the intestine.</td>

<td></td><td> 270G +</td><td></td><td></td><td></td>

<td></td><td> Pepsin</td><td> 5C^g/kg</td><td> 0/4</td><td></td>

<td></td><td> Control</td><td> 10C^g/kg</td><td> 1/5</td><td> Multiple Dosing</td>

<td> 35</td><td> 270G</td><td> 10C^g/kg</td><td> 1/5'</td><td> (5 dosing intervals</td>

<td></td><td> I.V.</td><td> 5C^g/kg</td><td> 2/2</td><td> at t 1/2)</td>

<td></td><td> S.C.</td><td> 50gg/kg</td><td> 2/2</td><td> Gavage by stomach</td>

<td></td><td></td><td></td><td></td><td> tube.</td>

<td> 40</td><td></td><td> *Rats were</td><td> foaming at nostrils.</td><td></td>

<td></td><td></td><td> Figure 10</td><td> illustrates levels of</td><td> erythropoietin (EPO)</td>

<td></td><td> detected</td><td colspan="3"> in rat serum taken from rats administered Gln/Asp/Tyr/-</td>

Phe proteinoid carrier encapsulated EPO (15^g EPO/kg body weight) 45 and encapsulated EPO (15/xg EPO/kg body weight) at t = 0.5, 1, and hours. Serum erythropoietin levels were determined over time with an erythropoietin enzyme immunoassay kit (Amgen, Thousand

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Oaks, CA, USA). The results show that EPO serum levels in rats administered erythropoietin proteinoid carriers were relatively higher at all time points compared to rats (control) which received unencapsulated material. At t = 2 hours, the EPO levels remained at approximately 300 pg/mL serum in rats administered erythropoietin proteinoid carriers while the control rats had undetectable EPO levels.

Figure 11 illustrates EPO serum levels in rats that were administered either erythropoietin (50^g/kg) or Gln/Asp/Tyr10 /Phe proteinoid (1:1:1:1 mole ratio of Gin, Asp,Tyr, and Phe in the reaction mixture) proteinoid carrier encapsulated erythropoietin (50/zg/kg) directly into the proximal duodenum. Serum erythropoietin levels were determined over time with the aforementioned erythropoietin enzyme immunoassay kit. The results show that EPO serum levels in rats administered erythropoietin proteinoid carriers steadily increased at a rate of approximately 50 pg/mL per hour over a range of two hours. In contrast, rats (control) which received unencapsulated EPO had EPO levels peaked at 100 pg/mL at 1 hour following administration and steadily decreased to about 50 pg/mL at the end of 2 hours.

Figure 12 illustrates EPO serum levels in rats who were orally gavaged with either Gln/Asp/Tyr/Phe proteinoid (1:1:1:1 mole ratio of Gin, Asp,Tyr, and Phe in the reaction mixture) proteinoid carrier encapsulated or unencapsulated erythropoietin (100/zg/kg) ; or received a subcutaneous injection of either 2^g/kg or 10^g/kg. Serum erythropoietin levels were determined over time with the aforementioned erythropoietin enzyme immunoassay kit. The results show that EPO serum levels in rats (#640-645) orally administered erythropoietin proteinoid carriers were relatively higher up to t = 2 hours, compared to rats (EPO) which received unencapsulated material.

The results obtained in this Example provide evidence that proteinoid encapsulation markedly improved the oral bioavailability of EPO.

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Example 16: Preparation of Calcitonin-containing

Proteinoid carrier

Encapsulation of salmon calcitonin in proteinoid proteinoid carriers was performed in the same manner described in

Example 13. Calcitonin, a peptide hormone which acts predominantly on bone to lower serum calcium concentration, was obtained from Sandoz (Basil, Switzerland). Calcitonin proteinoid carriers were prepared by mixing a 1:1 volume ratio of a lOOmg/ml aqueous solution of Gln/Asp/Tyr/Phe proteinoid (1:1:1:1 mole ratio of

Gin, Asp, Tyr, and Phe used in the proteinoid reaction mixture) and a 150 ug/mL calcitonin solution in 1.7 N citric acid solution with 1% gum acacia, as described in Example 13. The efficiency of calcitonin encapsulation was approximately 40%. Calcitonin concentration was determined directly by HPLC after dissolving the calcitonin proteinoid carriers in 60% aqueous acetonitrile.

Example 17: Evaluation of Calcitonin-containing

Proteinoid carriers in Monkeys

In this Example, the calcitonin proteinoid carriers, prepared as described in Example 16, were evaluated in cynomolgus monkeys. Male cynomolgus monkeys weighing 4-5 kc were fasted overnight, anesthetized (approximately lOmg/kg ketamine HCl) and placed into a primate restraint chair for dosing and blood sampling. A single oral dose of calcitonin proteinoid carriers (0.25 mg/kg body weight) was administered to each of four monkeys by nasogastric gavage. The dosage was based on the body weight taken on the morning of dosing. Blood samples were collected from saphenous vein catheters at hourly intervals, starting at t = 0 prior to administration of the proteinoid carriers, and hourly, from 1 to 7 hours post-dose for serum calcium determination. The hypocalcemic response following oral calcitonin administration was used as an index of pharmacological response. Serum calcium concentrations were quantitated by a conventional O-cresolphthalein complexone method.

Figure 13 demonstrates the response obtained in cynomolgus monkeys following naso-gastric gavage of microencapWO 93/25583

PCT/US93/05723 sulated calcitonin. Significant changes from baseline serum calcium concentration were observed. Six hours following dosing, serum calcium concentrations decreased by 13 /zg/ml. A significant pharmacological response was still apparent seven hours after the administration of calcitonin proteinoid carriers.

Example 18: Evaluation of Calcitonin-containing Proteinoid

Carriers in Rats

In this Example, the calcitonin proteinoid carriers prepared in accordance with Example 16 are evaluated in fasted male Spraque Dawley rats weighing 100-150g. Calcitonin proteinoid carriers and calcitonin were administered by either oral gavage or intraduodenal injection. The rats are divided into the following groups:

’

1. calcitonin proteinoid carriers: 60 ug calcitonin/kg body weight by oral gavage (3 rats);

2. calcitonin proteinoid carriers: 3 ug calcitonin/kg body weight by intraduodenal gavage (3 rats);

3. calcitonin: 60 ug calcitonin/kg body weight by oral gavage (3 rats) (Control) ,.

4. calcitonin: 3 ug calciconin/kg body weight by intraduodenal gavage (3 rats) (Control).

Oral gavage dosing of rats is performed. Calcitonin proteinoid carriers are prepared immediately prior to dosing and Groups 1 and 2 each receive an appropriate dosage of the proteinoid carrier suspension. Groups 3 and 4 receive the unencapsulated calcitonin (no proteinoid carriers) . Approximately 0.5 ml of blood is serially withdrawn from the tail artery of each rat just prior to dosing (0 time) and 1 h, 2 h and 3 h pose-dosing. £ ~um from the blood samples are stored at -20°C for serum calcium, concentration determination.

Figure 14 is the serum concentration-time curve for orally administered microencapsulated calcitonin and un ncapsu35 lated calcitonin in rats. Experimental results in rats demonstrate a significant increase in pharmacological response (i.e.,

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PCT/US93/05723 decreasing serum calcium levels) when proteinoid encapsulated calcitonin is compared to the unencapsulated vehicle control group. One hour afer dosing, serum calcium concentrations decreased 23 Mg/ml in the rats receiving encapsulated calcitonin compared to a decrease of only 6.5 Mg/ml in the control group. Furthermore, the responses were dose-dependent (data not shown) .

The results of intraduodenal injection of encapsulated or unencapsulated calcitonin in rats is shown in Figure 15. The results demonstrate a time-dependent decrease in serum calcium levels for the encapsulated preparation. The control group showed no response. One hour after intraduodenal administration, serum calcium levels in the calcitonin proteinoid carrier group decreased by Ιθμυ/ηιΐ, whereas unencapsulated calcitonin was unchanged. These results indicate that transmembrane transport of calcitonin is enhanced by proteinoid encapsulation.

The results obtained in this Example and in Example 17 provide evidence that proteinoid encapsulation markedly improves the oral bioavailability of calcitonin. The data also indicate that the oral drug delivery system is not species -dependent.

Example 19: Preparation and Evaluation of Factor IX-containing Proteinoid Carrier

Factor IX is a vitamin K-dependent blood coagulation proenzyme, MW 56 kD. Factor IX deficiency, known as hemophilia

B, occurs in approximately 1 out of every 25,000 males. To date, treatment of this disorder is accomplished by intravenous administration of Factor IX, although a recent report details efforts to supplement by subcutaneous injection (Thompson (1986) Blood, Vol. 67(3), pages 565-572).

Encapsulation of Factor IX (FIX) in proteinoid carriers was performed, following the procedure described in Example 13, by mixing (1:1 v/v) 100 mg/mL of Glu/Asp/Tyr/Phe proteinoid (1:1:1:1 mole ratio of Glu, Asp, Tyr, and Phe used in the proteinoid reaction mixture) in deionized water and an aqueous solution of FIX. Two proteinoid carrier suspensions were prepared and evaluated in vivo separately as described in

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Examples 20 and 21.

FIX proteinoid carrier suspension A contained 50 mg/ml of proteinoid and 500 U/'ml FIX (FIX is available from the American Red Cross, Rockville, Maryland, USA) solution containing

4% acetic acid, 2% gum acacia, 0.2% PEG 14 (available from Union

Carbide, Danbury, CN, USA/, 14 mM CaCl₂, final pH 3.81.

The second suspension, FIX proteinoid carrier suspension B, contained 50 mg/ml proteinoid and 116 U/ml FIX solution containing 3.8% acetic acid, 1.5% gum acacia, 0.15% PEG 14, 11 mM

CaCl₂, final pH 4.58.

The stability of FIX proteinoid carrier preparations was assessed over a short time course in vitro. The protein carriers encapsulating FIX were examined by optical microscopy and laser light scattering. Aliquots of proteinoid carrier suspension were withdrawn every 30 minutes for 1.5 hours, FIX proteinoid carriers were isolated by centrifugation at 4500Xg and dissolved in activated partial thromboplastin time (APTT) assay buffer (0.05M histidine-0.01M NaCl-0.1% bovine serum albumin0.01% TWEEN-40, pH 7.47) to release soluble FIX and proteinoid.

Quantitation of FIX activity by APTT employed both FIX standards (0.025, 0.05, and 0.1 U/ml) and empty proteinoid carrier suspension as control. APTT assay kits are commercially available, e.g. Sigma Diagnostics (St. Louis, MO, USA).

Based on the above analysis, it was determined that FIX proteinoid carriers of greater stability are obtained by encapsulating FIX at a higher pH, e.g., pH 4.9. Furthermore, the efficiency of encapsulation is approximately 20% of available FIX units and activity levels remain constant for at least 1.5 hours when FIX proteinoid carrier pellets are stored at about 4’C.

Example 20: Evaluation of FIX-containing Proteinoid carriers (A) in Rats

In this Example, FIX proteinoid carrier suspension A, prepared as described in Example 19, were evaluated in male

Sprague Dawley racs (ave. weight 300g). Appropriate aliquots of suspension were centrifuged at 4500Xg to pellet the FIX protein

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PCT/US93/05723 carriers, which were subsequently resuspended in the same buffer for animal dosing. The rats are divided into two groups as follows :

1. Oral FIX proteinoid carriers (FIX sph PO) : 2709 U FIX/kg body weight by intragastric gavage (4 rats);

2. Intravenous FIX (no proteinoid carriers) (FIX IV): 200 U/kg body weight by intravenous injection. 32 rats received 0.7 ml FIX in 0.11 NaCl-0.02M sodium citrate, pH 6.85 by tail vein injection.

The FIX proteinoid carrier suspension and solution are prepared immediately prior to dosing. One ml of blood was withdrawn from each rat just prior to dosing (0 time) and 1 h, h and 4 h (post-dosing), a citrate anticoagulant was added to the blood, and plasma from the blood samples were stored at 15 70°C.

Plasma samples were assayed by a modified APTT assay using FIX coagulated deficient plasma (assay kit is available from Ortho Diagnosis (Raritan, New Jersey, USA) . Changes in clotting times were calculated by subtracting individual baseline

0 (0 hr) values from subsequent clotting time values. The data shown in Figure 16 are the mean values for a given group. Values below baseline indicate the presence of exogenous FIX.

As shown in Figure 16, significant amounts of FIX was delivered co blood via oral administration of FIX proteinoid carriers. The relative plasma level is lower in the FIX proteinoid carriers group, however the dimunition in clotting time at 0.5, 1.0 and 2.0 hours is notable. This is achieved by oral dosing with approximately 14 times the IV dose. Moreover, these results are particularly interesting since Factor IX is an acid labile protein whose half-life is approximately less than one hour at 37· C at pH 5.0. The FIX proteinoid carriers in this experiment were at pH 3.81 and encapsulated 14.8% of the available FIX units during preparation. The results support that FIX proteinoid carriers remain viable in the GI tract to facilitate delivery.

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Example 21: Evaluation of FIX-containing Proteinoid Carriers (B) in Rats

In this Example, FIX proteinoid carrier suspension B, prepared as described in Example 19, were evaluated in male

Sprague Dawley rats (ave. weight 300g). Resuspended FIX proteinoid carriers were prepared as described in Example 20. The rats are divided into two groups as follows:

1. Oral FIX proteinoid carriers (FIX sph PO) : 1006U FIX/kg body weight by intragastric gavage (5 rats) .

2. Intravenous FIX (no proteinoid carriers) (FIX IV) : 185 U/kg body weight by intravenous injection. 3 rats received 0.3 ml FIX in 0.11 NaCl-0.02M sodium citrate, pH 6.85 by tail vein injection.

3. Oral FIX ino proteinoid carriers) (FIX unencap PO): 2760U

FIX/kg body weight by intragastric gavage. 4 rats received

1.0 ml of FIX in saline solution containing 3.8% acetic acid, pH 6.85.

The FIX proteinoid carrier suspension and solutions were prepared immediately prior to dosing. Plasma samples were obtained and assayed as described in Example 20. Changes in clotting times were calculated by subtracting individual baseline (0 hr) values from subsequent clotting time values. The data shown in Figure 17 are the mean values for a given group. Values below baseline indicate the presence of exogenous FIX. The FIX proteinoid carriers, prepared at pH 4.58, encapsulated 23.1% of the FIX units.

As shown in Figure 17, at oral dose levels of only 5 times that of the IV dose, significant oral delivery was observed. In addition, native FIX (pH 6.85) dosed at 15 times the IV dose level resulted in no detectable levels of exogenous FIX in the plasma.

Thus, the results shown in this Example and in Example 20 support that oral delivery of FIX can be accomplished via the use of FIX proteinoid carriers . These proteinoid carriers appear to adequately protect FIX during transit through the Gl tract and deliver FIX to the blood stream.

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Example 22: Preparation of alpha-Interferon (IFN)-containing

Proteinoid carrier

In this Example, a study was undertaken to evaluate the protective capability of proteinoid carriers on enzymatic degradation under simulated gastrointestinal conditions. The in vitro stability of IFN in proteinoid carriers was examined in simulated gastric fluid (SGF) containing pepsin in 0.08 N HC1 and simulated intestinal fluid (SIF) containing pancreatin in phosphate buffer. The reagents and stability assay procedure are described in the United States Pharmacopoeia (Vol. XXII, 1990, pages 1788 and 1789).

Preparation of IFN-containing proteinoid carriers

Encapsulation of IFN in proteinoid carriers was performed in the same manner described in Example 13. Alpha-IFN is available from a number of commercial sources . One commercial IFN product includes Roferon-A (Hoffman LaRoche) . IFN proteinoid carriers were prepared with an aqueous solution of Glu/Asp/Tyr/Phe proteinoid (1:1:1:1 mole ratio of Glu, Asp, Tyr and Phe used

0 in the proteinoid reaction mixture) , and an IFN solution containing 1.7 N citric acid solution with 5% gelatin. The IFN proteinoid carrier suspension contained 80 mg/ml proteinoid, 600 ug/ml IFN, 0.6N citric acid, and 2.5% gelatin, pH 3.0.

Stability of IFN proteinoid carriers in SGF

SGF (2 ml) was added into 1 ml of IFN proteinoid proteinoid carrier suspension. The solution was incubated at 40 - C with shaking, and aliquots were taken serially after SGF addition as described in the U.S. Pharmacocopia (ibid). An equal volume of stopper solution (pepstatin A in phosphate buffer, was added to each aliquot immediately after sampling to stop the enzymatic degradation and to open the proteinoid carriers. The IFN concentration in all samples was then determined by HPLC. As a comparison, the stability of IFN alone in SGF was evaluated. The experiment were performed as described above, without the proteinoid carriers. As another control, the

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PCT/US93/05723 stability of IFN proteinoid carriers was evaluated in 0.08 N HC1.

Stability of IFN-containing proteinoid carriers in SIF

SIF (2 ml) was added into 1 ml IFN proteinoid carriers.

The solution was incubated at 40 C with shaking and samples were taken serially as described in the United States Pharmacocopia (ibid). An equal volume of stopper solution (aprotinin and trypsin/chymotrypsin inhibitor in phosphate buffer) was added to each aliquot immediately after sampling to stop the enzymatic degradation. The IFN concentration was analyzed by HPLC.

To study the study the stability of IFN alone in SIF,

600 ug of IFN was dissolved in 0.85 N citric acid or 0.01 M phosphate buffer. SIF (2 ml) was added to 1 ml IFN solution. The solution was sampled and analyzed as described above.

Results and Discussion (a) Protective Effects of Proteinoid carriers in SGF

As shown in Figure 18, after 1 hour of SGF incubation, approximately 50% of IFN remained intact. After incubation in 20 SGF for 6 hours, approximately 20% of IFN was not degraded. As expected, IFN alone (in the absence of proteinoid carriers), was found to be completely destroyed by pepsin in SGF within 20 minutes .

Another control was performed using IFN alone in 0.08

N HC1. IFN alone was stable in SGF without pepsin (0.08 HC1). There was only a slight decrease after 2 hour incubation. This suggests that IFN was rather stable in HC1 at pH 1.2 up to six hours (Figure 19).

The results suggest that proteinoid carriers can retard

IFN from pepsin digestion, while IFN alone cannot survive in tne stomach for more than 20 minutes. These observations demonstrate the protective ability of proteinoid carriers on enzymatic digestion of protein drugs in the stomach.

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PCT/US93/05723 (b) Protective Effects of Proteinoid carriers in SIF

As shown in Figure 20, IFN proteinoid carriers were much more stable than IFN alone (rn the absence of proteinoid) in SIF. IFN alone at pH 7.4 was completely degraded within 10 minutes when incubated with SIF. However, approximately 70% of the IFN/proteinoid carriers survived after 6 hours in SIF, indicating that considerable stability is provided by the proteinoid proteinoid carrier.

IFN alone was slightly more stable in SIF at pH 3 10 than at pH 7.4. After 6 hr incubation in SIF at pH 3, there was approximately 10% of the IFN remaining. The stability of IFN in SIF at pH 3 is attributed to the low pH, which appears to suppress enzymatic activity of the intestinal proteases.

Example 23: Evaluation of Heparin-containing Proteinoid carriers in Rats

In this Example, a study was undertaken to ascertain whether proteinoid carriers are required for protective capability or whether (1) proteinoids (soluble proteinoids--not in carrier form) may be used and whether (2) alternative methods of carrier loading, such as incubating the therapeutic compound with preformed proteinoid carriers, are useful.

Preparation of Heparin-containing proteinoid carriers

Encapsulation of heparin in proteinoid carriers was performed in the same manner described in Example 12. Heparin (USP grade) was used and this material is available from a variety of commercial sources including Eli Lilly (Indianapolis, USA). Heparin proteinoid carriers were prepared, following the procedure of Example 12, using a 1:1 volume ratio of 150 mg/ml of Glu/Asp/Tyr/Phe/Orn₀j (1:1:1:1:0.5 mole ratio of Glu, Asp, Tyr, Phe, and Orn used in the proteinoid reaction mixture) proteinoid in deionized water, and an 20mg/mL aqueous heparin solution containing 1.7 N citric acid solution and 0.5% gum acacia. The heparin proteinoid carrier suspension was dialyzed in acetic acid solution as described in Example 12 . Heparin proteinoid carriers

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PCT/US93/05723 were then centrifuged at 4800Xg (15 minutes) and total heparin was measured by assaying the pellet and the supernatant with a modification of the Azure A method (Gundry et al. Amer. J. of Surgery (1984) Vol. 148, pages 191-194). Proteinoid was assayed by dissolving the proteinoid carriers with 0.1 N NaOH and measuring absorbance at 294 nm.

Preparation of heparin-spiked empty proteinoid carriers

Empty proteinoid carriers were prepared following the same procedure described above for the heparin proteinoid carriers, with the modification being that no heparin was present. The lyophilized empty proteinoid carriers were resuspended in 0.85N citric acid and 0.5% gum containing heparin at a concentration of 20 mg/ml. The amount of heparin co15 isolated with the proteinoid carriers was measured as described above.

Experimental Procedure

Male Spaque Dawley rats weighing approximately 350g were dosed by oral gavage or intraduodenal (ID) injection (just anterior to the pyloric sphincter and into the duodenum) . Rats were dosed orally or ID with one of the following: lyophilized heparin proteinoid carriers, heparin-spiked empty proteinoid carriers, proteinoid/heparin in water, heparin in 0.85N citric acid and 0.5% gum and heparin alone in water. In both oral and ID injection experiments, weight ratios of neparin:proteinoid were constant. The total heparin dose in the oral studies was 100 mg/kg body weight; in ID injections studies, it was 50 mg/kg. The proteinoid dose was 40' mg/kg for oral gavages and 20 mg/kg for ID injections. The dosing volume was approximately 0.3 to 0.5 ml. Approximately 0.5 ml of blood is serially withdrawn from the tail artery of each rat just prior to dosing (0 time) and 1 h, 2 h and 4 h post-dosing. Serum from the blood samples are stored at -20°C for heparin activity determination.

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Results and Discussion

The results obtained suggest that heparin alone as well as soluble proteinoid and heparin (both in water, dosed orally or by ID injection) did not appear to be absorbed -from the GI tract in amounts sufficient to increase APTT values (Figure 21) . Heparin in citric acid elicited some increase in APTT values, but only when dosed directly into the duodenum.

Heparin proteinoid carriers gave the highest APTT values, indicated increased absorption of heparin when dosed orally, as well as when directly injected into the duodenum (Figures 22 and 23) . While the observed activity was lower than observed with heparin proteinoid carriers (Figure 23), heparinspiked empty proteinoid carriers showed increased APTTs over baselines. Both types of proteinoid carriers showed a much greater increase in APTT values than that observed with citric acid/heparin.

The 'results obtained in this Example suggest that, in the proteinoid system, proteinoid carriers are necessary for the observed increase in heparin absorption, as soluble proteinoid did not show detectable activity within the experimental limits.

Example 24: Preparation and Evaluation of Ml-containing

Proteinoid carrier

In this Example, influenza virus antigen-containing proteinoid carriers were prepared and evaluated in rats.

Preparation of Ml Proteinoid carriers ·

Encapsulation of Ml in proteinoid carriers was 30 performed in the same manner described in Example 13. Ml protein, a major internal component of influenza virus, was obtained by purification of a swine influenza vaccine donated by Drug Directorate, Health Protection Branch, Bureau of Biologies, Ottawa, Ontrario Cannada. The vaccine was prepared with the high-yielding recombinant strain X-53Aa, which derives its HA and NA from the parent strain A/NJ/11/76(H1N1) and its internal

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PCT/US 93/05723 proteins, including Ml, from the parent strain A/PR/8/34 IR.B. Couc et al. (1983) Ann. Rev. Microbiol., Vol. 37, pages 529-549 and B.R. Murphy (1982) Infec. Immun., Vol. 36, pages 1102-1108). Ml was purified as described by Khan et al ( (19-82) J. Clin.Micro5 biol.. Vol. 16, pages 813-823). Ml proteinoid carriers were prepared, by mixing (at 40’C), equivolumes of an aqueous solution of lOOmg/ml of Glu/Asp/Tyr/Phe proteinoid in deionized water and a lOmg/mL solution of Ml protein in 1.7N citric acid and 5% gum arabic (pH 2.0). The final Ml concentration in the suspension was l.Omg/ml.

Preparation of H -NA-containing Proteinoid carriers and unencapsulated antigens

HA-NA antigen was isolated according to the procedure 15 of Gallagher et al. ((1984) J.Clin.Microbiol. . Vol. 20, pages 8093). Influenza virus (A/PR8/34) was centrifuged at 90,000 G for 60 min. The viral pellet was solubilized with 0.05M acetate buffer (pH 7.0) containing 7.5% octylglucoside and re-centrifuged under the same conditions. The resulting supernatant contained approximately 90% HA and 10% NA as determined by SDS-PAGE.

HA-NA proteinoid carriers were prepared following the same protocol as for the Ml proteinoid carriers but substituted Ml for HA-NA. The final concentration of HA-NA in the suspension was also 1.0 mg/ml.

Empty proteinoid carriers were prepared following the sampe procedure described for the Ml proteinoid carriers, with the only modification being that a 1.7 N citric acid/gum solution was used in place of the Ml/citric acid/gum solution.

Unencapsulated antigens, Ml and HA-NA, was diluted in

1.7 N citric acid, 10 mg/ml gum arabic to the same final lmg/ml concentration.

Experimental procedure .

Male Spraque Dawley rats (about 350g weight) were used in this experiment. Oral dosage was by gavage. Four groups of

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PCT/US93/05723 five rats each (the subcutaneous control group had 4) were dosed as follows: Group 1 was dosed orally with lmg of Ml proteinoid carriers per rat (1 ml), Group 2 was dosed orally with 1 mg per rat of empty proteinoid carrier, Group 3 was -dosed with 1 mg of unencapsulated Ml per rat of empty carrier, Group 3 was dosed with 1 mg of unencapsulated Ml per rat in 1 ml and Group 4 was dosed subcutaneously (SC) with 25 ug per rat of Ml in 0.3 ml. Blood samples (300 ul) were taken from each rat by tail bleeeding before dosing and at 1, 2, 3 and 4 hours post-dose (to assay for antigen) and at 14, 28, and 42 days post-dose (for antibody assay). Solutions for subcutaneous control-Ml in TRIS (no SDS) was diluted to a concentration of 167 ug/mL. An equal amount of Freunds Complete Adjuvant (FCA, Sigma) was added and the mixture was thoroughly homogenized. The final concentration of Ml in the mixtue was 83.3 ug/ml. HA-NA solutions for subcutaneous administration were prepared in the same manner except that phosphate buffered saline replaced TRIS-SDS buffer.

• The same immunization and bleeding schedule was follwed when dosing with HA-NA proteinoid carrier, with the following modifications: all rats received an oral booster with HA-NA proteinoid carrier (250 ug/rat) 42 days after the first oral dose and blood samples were again taken 14 days after the booster dose. Serum derived from the samples were stored at -20’C until assayed.

Serum anti-Ml and anti-HA-NA specific IgGs were assayed by an ELISA method as described Khan et al. ((1982) J.

Clin. Microbiol., vol. 16, pages 813-820).

Results and Discussion

Attempts to measure antigen in plasma samples were unsuccessful. Ml antigen could not be detected in rat plasma samples taken 1-4 hours post-dosing in all groups, including the subcutaneous control.

Plasma samples from rats dosed orally with empty proteinoid' carriers showed no significant antibody titer against

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TABLE 8

ANTI M PROTEIN ANTIBODY TITERS IN SERUM FROM RATS DOSED WITH M PROTEINOID CARRIERS VS CONTROLS

<td></td><td></td><td> 14 day-</td><td> 28 day</td><td> 42 day</td>

<td> Dosing</td><td> rat #</td><td> titer</td><td> titer</td><td> titer</td>

<td> oral M protein</td><td> 197</td><td></td><td> &lt;30</td><td> &lt;30</td><td> &lt;30</td>

<td> unencapsulated</td><td> 198</td><td></td><td> &lt;30</td><td> &lt;30</td><td> &lt;30</td>

<td> 1 mg/rat</td><td> 199</td><td></td><td> &lt;30</td><td> &lt;30</td><td> &lt;30</td>

<td></td><td> 200</td><td></td><td> &lt;30</td><td> &lt;30</td><td> 35</td>

<td></td><td> 201</td><td></td><td> &lt;30</td><td> &lt;30</td><td> 56</td>

<td rowspan="2"> empty carrier</td><td> 203</td><td></td><td> &lt;30</td><td> &lt;30</td><td> 82</td>

<td> 204</td><td></td><td> &lt;30</td><td> &lt;30</td><td> 70</td>

<td></td><td> 205</td><td></td><td> &lt;30</td><td> &lt;30</td><td> 60</td>

<td></td><td> 206</td><td></td><td> &lt;30</td><td> &lt;30</td><td> 86</td>

<td></td><td> 207</td><td></td><td> &lt;30</td><td> &lt;30</td><td> 45</td>

<td> M proteinoid</td><td> 209</td><td></td><td> &lt;30</td><td> &lt;30</td><td> 64</td>

<td> carriers</td><td> 210</td><td> 2</td><td> ,150</td><td> 820</td><td> 5,200</td>

<td> 1 mg/rat total</td><td> 211</td><td></td><td> 860</td><td> 430</td><td> 1,150</td>

<td></td><td> 212</td><td></td><td> 760</td><td> 1,850</td><td> 3,000</td>

<td></td><td> 213</td><td></td><td> &lt;30</td><td> &lt;30</td><td> 62</td>

<td> subcut. control</td><td> 215</td><td> 40</td><td> ,000</td><td> 62,000</td><td> 330,000</td>

<td> 0.025 mg/rat</td><td> 217</td><td></td><td> 34</td><td> 8,000</td><td> 54,000</td>

<td> in FCA</td><td> 218</td><td></td><td> 430</td><td> 8,000</td><td> 125,000</td>

<td></td><td> 219</td><td></td><td> 270</td><td> 6,600</td><td> 78,000</td>

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PROTEINOID BATCHES

Appendix a ρ I of 9

<td rowspan="2"> Bt. Ko.</td><td rowspan="2"> /AA</td><td rowspan="2"> COMPOSITION”</td><td colspan="3"> ADDITIVE TEKP TIHE</td><td rowspan="2"> SPHERE RATING ...</td><td rowspan="2"> BATCH SIZE Operator Date colar</td>

<td colspan="2"> C</td><td> (hr)</td>

<td> 085</td><td> 3</td><td> GUJ2 ASP2 ILEO</td><td> —</td><td> 170</td><td> 3.0</td><td> IHS5 HT1</td><td> 0.0 .</td>

<td> 086</td><td> 3</td><td> GL02 ASP2 VAL .</td><td> — </td><td> 170</td><td> 3.0</td><td> IKS 4 HTO HEPO</td><td> 0.0</td>

<td> 087 ·</td><td> 3</td><td> GLO ASP LEO</td><td> —</td><td> 170</td><td> 3.0</td><td> IKS5 HT3 HEP5</td><td> 0.0</td>

<td> 088</td><td> 2</td><td> GL02 ASP2 EQO</td><td> SEE</td><td> WHO</td><td> 0.0</td><td></td><td> 0.0</td>

<td> 089</td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 170</td><td> 3.0</td><td> IHS5 HTO</td><td> 0.0</td>

<td> 090</td><td> 3</td><td> GL02 ASP2 VAL</td><td> —</td><td> 170</td><td> 3.0</td><td> IHS3 HTO HEP1</td><td> 0.0</td>

<td> 091</td><td> .3</td><td> GLO ASP LEO</td><td> —</td><td> 170</td><td> 3.0</td><td> IHS2 HT1</td><td> 0.0</td>

<td> 092</td><td> 3</td><td> GLO ASP TER</td><td> —</td><td> 170</td><td> 3.0</td><td> IHS2 HTO</td><td> 0.0</td>

<td> 093</td><td> 4</td><td> GL02 ASP2 VAL PRO</td><td> —</td><td> 170</td><td> 3.0</td><td> DJS2 HT2</td><td> 0.0</td>

<td> 094</td><td> 3</td><td> GLO ASP CYS-H</td><td> —</td><td> 170</td><td> 3.0</td><td> msi HT1</td><td> 0.0</td>

<td> 095</td><td> 4</td><td> PRO SER THR CYS</td><td> —</td><td> 170</td><td> 3.0</td><td></td><td> 0.0</td>

<td> 096</td><td> 3</td><td> GLO ASP VAL2</td><td> —</td><td> 170</td><td> 3.0</td><td> IKS3 HTO HEP4</td><td> 0.0</td>

<td> 097</td><td> 3</td><td> GLO ASP VAL</td><td> —</td><td> 170</td><td> 3.0</td><td> IHS2 HT1</td><td> 0.0</td>

<td> 098</td><td> 3</td><td> GLO ASP CYS-B</td><td> —</td><td> 1/0</td><td> 3.0</td><td> IHS4 HT1</td><td> 0.0</td>

<td> 099</td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 170 -</td><td> 3.0</td><td> IHS4</td><td> 0.0</td>

<td> 186 -cp</td><td> 4</td><td> PYGLO ASP TYR FEE</td><td> PA</td><td> 176</td><td> 4.0</td><td> IHSO HT4 HEP5</td><td> 0.3</td>

<td> 199 -cp</td><td> 4</td><td> GLO ASP TYR PHE</td><td> H20</td><td> 100</td><td> 99.0</td><td> HTO mso HEPO</td><td> 0.0</td>

<td> 202A-cp</td><td> 4</td><td> GL02.4 ASP2 VAL2 GLY</td><td> —</td><td> 170</td><td> 4.0</td><td> IHS3 HTO</td><td> 0.6</td>

<td> 202B-cp</td><td> 4</td><td> GLO2.4 ASP2 VAL2 GLY</td><td> —</td><td> 170</td><td> 4.0</td><td> HTO IHS3</td><td> 0.6</td>

<td> 206A-cp</td><td> 4</td><td> GLU ASP-TYR PHE</td><td> SOLTA</td><td> 175</td><td> 4.5</td><td> IHS4 HT4 HEP3</td><td> 0.6</td>

<td> 20' cp</td><td> 4</td><td> GLO ASP_TYR PHE</td><td> SOLA</td><td> 175</td><td> 4.5</td><td></td><td> 0.6</td>

<td> 2C- K</td><td> 4</td><td> GLO ASP-TYR PHE</td><td> SF A</td><td> 175</td><td> 4.5</td><td></td><td> 0.6</td>

<td> 207· · ,p</td><td> 4</td><td> GLO ASP-TYR PHE</td><td> SOLTA</td><td> 175</td><td> 10.0</td><td> J.HS5 HT4 HEP4</td><td> 2.0</td>

<td> 207B-cp</td><td> 4</td><td> GLO ASP-TYR PHE</td><td> SOLTA</td><td> 175</td><td> 10.0</td><td> HT5 IHS4 HEP4</td><td> 2.0</td>

<td> 21H-cp</td><td> 4</td><td> GLO ASP-VAL LYSFB</td><td> SOLTA</td><td> 190</td><td> 4.3</td><td> IHS5 KTS HEP5 v</td><td> 0.3</td>

<td> 231£-cp</td><td> 4</td><td> GLO ASP-VAL LYSFB</td><td> SOLTA</td><td> 190</td><td> 4.5</td><td></td><td> 0.3</td>

<td> 2-jA-cp</td><td> 3</td><td> GL02-TYR.FHE</td><td> SOLTA</td><td> 185</td><td> 5.0</td><td> IHS4 HT3 EEP4</td><td> 0.3</td>

<td> 212B-cp</td><td> 3'</td><td> GL02-TYRPHE</td><td> SOLTA</td><td> 185</td><td> 5.0</td><td></td><td> 0.3</td>

<td> 214 -cp</td><td> 3</td><td> GLO LYSFB-ARG</td><td> SOLTA</td><td> 180</td><td> 7.0</td><td> IKSO HTO HEPO</td><td> 0.0</td>

<td> 223 -cp</td><td> 4</td><td> LYSFB2 ARG2 LEO PGLO</td><td> SOLTA</td><td> 180</td><td> 8.0</td><td> IHSO HTO HEP2</td><td> 0.3</td>

<td> 227A-cp</td><td> 2</td><td> VAL2 GLY2</td><td> SOLT</td><td> 180</td><td> .1.5</td><td> MSO HTO HEPO</td><td> 0.1</td>

<td> 227B-cp</td><td> 2</td><td> VAL2GLY2</td><td> SOLTA</td><td> 180</td><td> 1.5</td><td> HTO IHSO HEPO</td><td> 0.1</td>

<td> 228A-cp</td><td> 3</td><td> VAL2GLY2PGL0</td><td> SOLT</td><td> 180</td><td> 2.5</td><td> IHSO HTO HEPO</td><td> 0.1</td>

<td> 228B-cp</td><td> 3</td><td> VAL2GLY2PGL0</td><td> SOLTA</td><td> 180</td><td> 2.5</td><td> HTO IHSO HEPO</td><td> 0.1</td>

<td> 248 -cp</td><td> 3</td><td> GLO ASP LEO</td><td> —</td><td> 190</td><td> 4.0</td><td> IHS3· HTO HEPa</td><td> O'.O</td>

<td> 265A-cp</td><td> 4</td><td> GLO ASP-TYR PHE</td><td> SOL</td><td> 155</td><td> 4.0</td><td> IHS4 HT4 HEP5 '</td><td> 1.0</td>

<td> 265B ·</td><td> 4</td><td> GLO ASP -TYR PHE</td><td> SOLFOLAKE</td><td> 155</td><td> 4.0</td><td></td><td> 1.0</td>

<td> 265C</td><td></td><td></td><td></td><td></td><td> .0</td><td></td><td> .0</td>

<td> 296A-cp</td><td> 4</td><td> GLO LYSH PHE ASP</td><td> SOL-K</td><td> 180</td><td> 3.0</td><td> IHS4 HT2 HEPO</td><td> 0.6</td>

<td> 296B-cp</td><td> 4</td><td> GLO LYSH PHE ASP</td><td> SOL-H</td><td> .180</td><td> 3.0</td><td></td><td> 0.6</td>

<td> 298 -cp</td><td> 4</td><td> GLO ASP-TYR PHE</td><td> SOL-H</td><td> 190</td><td> 1.5</td><td> IHSla KT3 HEP4</td><td> 0,5 ·</td>

<td> 301 -cp</td><td> 4</td><td> GLO ASP-TYR PHE</td><td> SOL</td><td> 175</td><td> 8.0</td><td> IHS4 KT2 HEP3</td><td> •2.0</td>

<td> 302 -cp</td><td> 4</td><td> GLO ASP-TYR PHE</td><td> HHePO</td><td> ISO</td><td> 1.5</td><td> IHS4 HT2 HEP3</td><td> 0.3</td>

<td> 308 -cp</td><td> 4</td><td> GLO ASP TYR PHE .</td><td> HKP</td><td> 170</td><td> 1.0</td><td> IHS4 HT4 HEP4</td><td> 0.3</td>

<td> 309 -CP</td><td> 4</td><td> -GL01.3 ASP1.3 TO PHEl.j</td><td> i -SOLFOLAKE</td><td> 190</td><td> 1.5</td><td> IHS4aHT3oaHEP4a</td><td> 0.3</td>

<td> 310 -cp</td><td> 4</td><td> -GLO ASP TYR PEE</td><td> SOLTALAKE</td><td> 190</td><td> 4.0</td><td> IKS4 HT2 HEPS</td><td> 1.0</td>

Glossary:. · a=aaorphous o=oil * varying teEperature

-+ =cook tise change

PCT/US93/05723

PROTEINOID BATCHES

Appendix a Paae 2 of 9 /AA COMPOSITION

ADDITIVE TEKP TIKE SPHERE

<td></td><td></td><td></td><td> C</td><td> (hr)</td><td> RATING</td><td> solar</td>

<td> 2</td><td> GLD2 ASP2 EQO</td><td> —</td><td> .- lot)</td><td> 1.5</td><td> «.·</td><td> 0.0</td>

<td> 3</td><td> ASP2 ARG ILEO</td><td> —</td><td> 170</td><td> 0.0</td><td> HTO</td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 175</td><td> 3.0</td><td></td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> PA</td><td> 170</td><td> 3.0</td><td></td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> GLYC</td><td> 170</td><td> 3.0</td><td> HTO</td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> GLYC</td><td> 170</td><td> 3.0</td><td> IHS4 KT4</td><td> 0.0</td>

<td> 2</td><td> GUJ2 ASP2 EQO</td><td> GLYC</td><td> 170</td><td> 3.0</td><td> KTO</td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> PA</td><td> 170</td><td> 3.0</td><td> KT1</td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> GLYC</td><td> 190</td><td> 6.0</td><td> ’«TO</td><td> 0.0</td>

<td> 2</td><td> GLD2 ASP2 EQO</td><td> .PA</td><td> 190</td><td> 6.0</td><td> KTO</td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 190</td><td> 6.0</td><td> KTO</td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 190</td><td> 3.0</td><td> KTO</td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO·</td><td> —</td><td> 170</td><td> 3.0</td><td> KTO</td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 170</td><td> 6.0</td><td></td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> ; -</td><td> 170</td><td> 6.0</td><td> KTO</td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 170</td><td> 4.0</td><td> IHSO KTO</td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO -</td><td> —</td><td> 200</td><td> 3.5</td><td> TKS4 HTO</td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 150</td><td> 3.5</td><td> HT-VERY SH</td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 110</td><td> 4.3</td><td> KTO</td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 150</td><td> 3.5</td><td> HTO</td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 180</td><td> 5.0</td><td></td><td> 0.0</td>

<td> 2</td><td> GLD2 ASP2 EQO</td><td> —</td><td> 150</td><td> 3.0</td><td> IHSO HTO</td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 160</td><td> 3.0</td><td> HTO</td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 165</td><td> 3.0</td><td> HT ί HO AK</td><td> 0,0</td>

<td> 2</td><td> GL02 LEO</td><td> —</td><td> 170</td><td> 3.0</td><td> KTO</td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 170</td><td> 3.0</td><td></td><td> 0.0</td>

<td> 2</td><td> GL02 LEO</td><td> —</td><td> 170</td><td> 3.0</td><td> IKS2 HTO</td><td> 0.0</td>

<td> 3</td><td> GL02 ASP2 LEO</td><td> —</td><td> 170</td><td> 3.0</td><td> IHS5 ΒΕΡΟ B</td><td> 0.0</td>

<td> 2</td><td> GL02 GLY' ·</td><td> —</td><td> 170</td><td> 3.0</td><td> KTO</td><td> 0.0</td>

<td> 2</td><td> ASP2 LEO ‘</td><td> —</td><td> 165</td><td> 3.0</td><td></td><td> 0.0</td>

<td> 2</td><td> ASP2 LEO</td><td> —</td><td></td><td> 0.0</td><td></td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 170</td><td> 3.0</td><td> HS5 S AHORPHOO</td><td> 0)0</td>

<td> 3</td><td> GL02 ASP2 LEO</td><td> —</td><td> 170</td><td> 6.0</td><td> EE</td><td> 0.0</td>

<td> 3</td><td> GLO ASP3 LEO</td><td> —</td><td> 170</td><td> 2.6</td><td></td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 170</td><td> 3.0</td><td> IHSO HTO</td><td> 0.0</td>

<td> 3</td><td> GLO ASP PRO</td><td> —</td><td> 170</td><td> 4.0</td><td> BiSO HTO ΒΕΡΟ</td><td> 0.3</td>

<td> 2</td><td> CUE ASP2 EQO</td><td> —</td><td> 170</td><td> 3.0</td><td> IHS5</td><td> '0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 170</td><td> 3.0</td><td> KTO HO AHO</td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 170</td><td> 4.5</td><td> IHS5</td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 170 ·</td><td> 4.0</td><td> IHS5</td><td> 0.0</td>

<td> 4</td><td> GLO ASP PRO LYS3</td><td> —</td><td> 170</td><td> 4.5</td><td> LOST BATCH</td><td> 0.0</td>

<td> 3</td><td> GL02 ASP2 ILEO</td><td> —</td><td> • 170</td><td> 4.0</td><td> IHS4 KTO ΒΕΡΟ</td><td> 0.0</td>

<td> 2</td><td> ARG LYS EQO</td><td> —</td><td> 170</td><td> 3.0</td><td></td><td> 0.0</td>

<td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 170</td><td> 4.0</td><td> IHS4 HTO</td><td> 0.0</td>

<td> 3</td><td> GL02 ASP2 ILEO</td><td> —</td><td> 170</td><td> 6.0</td><td> IHS4 HT1 BEP4</td><td> 0.0</td>

<td> 3 ·</td><td> GL02 ASP2 ILEO</td><td> —</td><td> 170</td><td> 3.0</td><td> IHS4 HTO</td><td> 0.0</td>

BATCH SIZE Operator Date amorphous o=oil * =varying tenperature t =cook tine change

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Appendix Page 3 o;

<td></td><td></td><td colspan="2"> PRO7EIH0ID BATCHES</td><td></td>

<td> Bt. HO.</td><td> /AA COMPOSITION</td><td> ADDITIVE TEHP</td><td> TIKE SPHERE</td><td> BATCH SI2E Operator</td>

<td></td><td></td><td> C</td><td> (hr) RATING -</td><td> solar</td>

<td> 31Ϊ -cp</td><td> 4 -GL02 LYSH2 PHE2 ASP</td><td> SULFALAHE 190</td><td> 1.7 IHD4O KT3o HEP3</td><td> 1.7</td>

<td> 312 -cp</td><td> 4 -GL02 LYSH2 PHE2 ASP</td><td> SOLTALAHE 190</td><td> 0.7 IHS4 HTO HEP4a</td><td> 17.9</td>

<td> 313 -cp</td><td> 4 · SL02-LYSH2 PHE2 ASP</td><td> SOLFALAHE 180</td><td> 3.0 IHS3 HT3 HEP3ao</td><td> 0.6</td>

<td> 314 -cp</td><td> 4 -ASP TYR PHE PGLO</td><td> SOLFOLAHE 190</td><td> 2.5 IHS2a HT4aHEP4a</td><td> 0.6</td>

<td> 315 -cp</td><td> 4 GLO ASP-VAL LYSFB</td><td> sulfolane 190</td><td> 4.0 IHS4 HT4 BEP3</td><td> 0.3</td>

<td> 316 -cp</td><td> 4 GLO ASP-TYR PHE</td><td> sulfolane 180</td><td> 21.0 IHS4 HT3a HEPa</td><td> 0.3</td>

<td> 317 -CP</td><td> 4 GLH-ASP TYR PHE</td><td> SOLFALAHE 175</td><td> 4.0 IKS5 HT5 HEP5</td><td> 0.3</td>

<td> 318 -cp</td><td> 5 GL02 ASP2 TYR2 PHE2 ORH</td><td> sulfalane 180*</td><td> .0 HT1 IHS4 HEP3a</td><td> ’ 1.0</td>

<td> 319 -cp</td><td> 4 -TYR PHE ASP PGLO</td><td> SOLFALAHE 190</td><td> 2.5 EHS4aHT4 HEP4a</td><td> 0.3</td>

<td> 320 -CP</td><td> 4 -TYR PHE PGLO ASP</td><td> Sulfolane 190</td><td> 1.5 IHS4aHT4 HEP4</td><td> 0.3</td>

<td> 321 -cp</td><td> 5 GL02 ASP2 TYR2 PHE2 ORH</td><td> SOLFOLAHE 180*</td><td> 3.0 IHS3aHT2aHEP4a</td><td> 1.0</td>

<td> 322 -cp</td><td> 4 GL02 LYSH2 PHE2 ASP-</td><td> SOLFOLAHE 192</td><td> 1.2 IHS2 HTO HEP2</td><td> 0.6 ·</td>

<td> 323 -cp</td><td> 4 GLO ASP TYR PHE-</td><td> SOLFOLAHE 190</td><td> .0 ABORT</td><td> 16.0</td>

<td> 324 -cp</td><td> 4 -GLO ASP TYR PHE</td><td> SULrOLAHI 190</td><td> 3.0 IHS4 HT4 HEP5a</td><td> 2.0</td>

<td> 325 -cp</td><td> 5 GL02 ASP2 TYR2 PHE2 ORH</td><td> SOLFOLAHE 180*</td><td> 3.0 IHSSa nT2a HEP</td><td> 1.0</td>

<td> 326 -CP</td><td> 4 -GLO ASP TYR PHE</td><td> SOLFOLAHE 190</td><td> 6.5 IHS3a HTOa EEP3</td><td> 16.0</td>

<td> 326.-CP</td><td> 4 -GLO ASP TYR PHE</td><td> SOLFOLAHE</td><td> .0 IHS4a HT4 HEP4a</td><td> .0</td>

<td> 327 -CP</td><td> 4 -GLO ASP TYR PHE</td><td> SOLFOLAHE 190</td><td> 4.0 IHS4a HT5a HEP3</td><td> 17.0</td>

<td> 328 -CP</td><td> 4 -GLO ASP TYR PHE</td><td> SOLFOLAHE 190</td><td> 3.0 IHS5a HT3aHEP3a</td><td> 17.0</td>

<td> 328 -7e</td><td> 4 -GLO ASP TYR PHE</td><td> SOLFOLAHE</td><td> .0 IHS3a HTOa HEP4</td><td> .0</td>

<td> 329 -cp</td><td> 4 -GLH ASP TYR PHE</td><td> SOLFOLAHE 175</td><td> 6.5 IHS5a HT3a HEP5</td><td> 1.0</td>

<td> 330 -cp</td><td> 2 ASP PHE</td><td> SOLFOUHE 180</td><td> 3.0 IHSOa HTla ΒΕΡΟ</td><td> 0.5</td>

<td> 331 -CP</td><td> 2 ASP2 PHE</td><td> SOLFOLAHE 180</td><td> 3.0 IHSOa HTOa HEPO</td><td> 0.5</td>

<td> 332 332</td><td> 2 ASP3 PHE</td><td> SOLFOLAHE 180</td><td> 3.0 IHS.,.&gt;HTlaHEPOc</td><td> 0.5</td>

<td> 333 -7a</td><td> 4 -GLO ASP TYR PHE</td><td> SOLFOLAHE 190</td><td> 5.0 IHS2aHT4a HEP5a</td><td> 17.0</td>

<td> 33&lt;-7ov</td><td> 4 -GLO ASP TYR PHE</td><td> SOLFOUHE 190</td><td> 5.0 IHS4aHT5a HEP4a</td><td> 17.0</td>

<td> 335 -CF</td><td> 2 -ASP PHE2</td><td> SGLFOLAKE ISO</td><td> 3.0 IHSlaHT2aHEPla</td><td> 0.5</td>

<td> 336 -11</td><td> 5 -GL02 ASP2.TYR2 PHE2 ORH</td><td> SOLFOLAHE 180</td><td> .0 IHS3a HT3a BEP4</td><td> 2.0</td>

<td> 337 337</td><td> 2 -ASP2 TYR</td><td> SOLFOLAHE 180</td><td> 6.5 IHS2aHT0cHEP0c</td><td> 0.5</td>

<td> 338 -CP</td><td> 2 -ASP TYR</td><td> SOLFOLAHE 180</td><td> 3.0 IHSO HTO HEPO</td><td> 1.0</td>

<td> 339 -CP</td><td> 2 -ASP3 TYR</td><td> SOLFOLAHE 180</td><td> 3.0 IHSOa HTO HEPO</td><td> 0.5</td>

<td> 340</td><td> 4 -GLO ASP TYR PHE</td><td> SOLFOLAHE</td><td> .0</td><td> 1.5</td>

<td> 341</td><td> 4 -GLO ASP TYR PHE</td><td> SOLFOLAHE</td><td> .0</td><td> 17.0</td>

<td> 342 342</td><td> 2 -ASPTYR2</td><td> SOLFOLAHE</td><td> .0 IHSOKTOHEPO</td><td> .0.5</td>

<td> 342 -CP</td><td> 2 -ASP TYR2 ·</td><td> SOLFOLAHE</td><td> .0 IHSOa HTO HEPO</td><td> 0.5</td>

<td> 343</td><td> 4 -GLO ASP TYR PHE</td><td> SOLFOLAHE</td><td> .0</td><td> 17.0</td>

<td> 344</td><td> 4 -GLO ASP TYR PHE</td><td> SOLFOLAHE</td><td> .0 ‘</td><td> 2.0</td>

<td> 345 -σ</td><td> 2 - ASP2 PHE</td><td> SOLFOLAHE</td><td> .0 IHSOa HTO HEPO .</td><td> .0</td>

<td> 346 -CP</td><td> 4 -GLH ASP TYR PHE</td><td> SOLFOLAHE</td><td> .0 INS0aHTlaHEP2A</td><td> .0</td>

<td> 347</td><td> 4 - GL02 ASP2 TYRS PHE5</td><td> SOLFOLAHE</td><td> .0 ’ </td><td> .0</td>

<td> 348</td><td> 2 -ASP2 :e</td><td> SOLFOLAHE .</td><td> .0</td><td> .0</td>

<td> 349</td><td> 2 -PHE AoP2</td><td> SOLFOLAHE</td><td> .0</td><td> .0</td>

<td> 350 .</td><td> 2 -ASP2 PEE</td><td> SOLFOLAHE</td><td> .0</td><td> .0</td>

<td> 351 351</td><td> 3 -GL02 TYR PHE</td><td> SOLFOLAHE</td><td> .0 IHS3aHT2dHEP3a</td><td> .0</td>

<td> 352</td><td> 5 -GL02 ASP2 TYR2 PHE2 ORH</td><td> SOLFOLAHE</td><td> .0</td><td> .0</td>

<td> 353</td><td> 5 -GL02 ASP2 TYR2 PHE2 ORH'</td><td> .SOLFOLAHE</td><td> .0</td><td> .0</td>

<td> Glossary:</td><td colspan="2"> a=aaorphous o=oil * =varying teiperature</td><td> + =cook tine change</td><td></td>

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PROTEIKOIDJUTCBES

Appendix a page 4 o£ g

<td rowspan="2"> Bt.</td><td rowspan="2"> Ho.</td><td rowspan="2"> /aa</td><td rowspan="2"> COMPOSITION</td><td colspan="3"> ADDITIVE TEMP TIKE</td><td rowspan="2"> SPBERE RATIHG</td><td rowspan="2"> BATCH SIZE »olar</td>

<td colspan="2"> C</td><td> (hr)</td>

<td> 295</td><td> -CP</td><td> 1</td><td> ASP</td><td> SOL-H</td><td> 180'</td><td> . 1.5</td><td> IHS2aHT2aBEP3oa</td><td> 0.3</td>

<td> 297</td><td> -cp</td><td> 4</td><td> GLO ASP-TYR PHE</td><td> SOL-H</td><td> 190</td><td> 1.5</td><td> IHS2a KT4a HEP3</td><td> 0.5</td>

<td> 299</td><td> -cp</td><td> 4</td><td> GLO LYS PHE ASP</td><td> SOL-H</td><td> 190</td><td> 1.7</td><td> IHS5 HT4 HEP2</td><td> 0.6</td>

<td> 300</td><td> -cp</td><td> 5</td><td> GLO ORH ASP LYS PHE</td><td> --</td><td> 180</td><td> 3.0</td><td> INS3 HT3 BEP3</td><td> 0.3</td>

<td> 303</td><td> -cp</td><td> 4</td><td> GLO ASP-TYR PHE</td><td> SOL-H</td><td> 175</td><td> 8.0</td><td> IKS4 KT2 BEP3a</td><td> 2.0</td>

<td> 304</td><td> -cp</td><td> 5</td><td> GLO ASP-TYR PHE ORHO.5</td><td> SOL-H</td><td> 180</td><td> 3.0</td><td> INS4 KT2 HEP3</td><td> 2.0</td>

<td> 305</td><td></td><td> 4</td><td> -PGLO ASP.5TYR PHE</td><td> SOL</td><td></td><td> 0.0</td><td> IHS3 KT2 HEP3</td><td> 0.3</td>

<td> 306</td><td> -cp</td><td> 4</td><td> -GLO ASP .5TYR PHE</td><td> SOL</td><td></td><td> 0.0</td><td> INS3aHT2aBEP2a</td><td> 0.3</td>

<td> 307</td><td> -cp</td><td> 4</td><td> GLH ASP TYR PHE</td><td> SULFOLANE</td><td> 175</td><td> 4.0</td><td> IHS4o HT4 HEP40</td><td> 0.3</td>

<td> -</td><td></td><td></td><td></td><td></td><td></td><td> .0</td><td></td><td> .0</td>

<td> 000</td><td></td><td></td><td></td><td></td><td></td><td> .0</td><td></td><td> .0</td>

<td> 001</td><td></td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 170</td><td> 4.0</td><td></td><td> 0.0</td>

<td> 002</td><td></td><td> 2</td><td> GLO ASP EQO</td><td> —</td><td> 149</td><td> 0.0</td><td></td><td> 0.0</td>

<td> 003</td><td></td><td> 2</td><td> GLO ASP EQO</td><td> —</td><td> 163</td><td> 0.0</td><td></td><td> 0.0</td>

<td> 004</td><td></td><td> 0</td><td></td><td> —</td><td> 204</td><td> 0.0</td><td></td><td> 0.0</td>

<td> 005</td><td></td><td> 2</td><td> GLO ASP EQO</td><td> —</td><td> 176</td><td> 3.0</td><td></td><td> 0.0</td>

<td> 006</td><td></td><td> 2</td><td> GLO ASP EQO</td><td> —</td><td> 154</td><td> 3.0</td><td></td><td> 0.0</td>

<td> 007</td><td></td><td> 2</td><td> GLO ASP EQO</td><td> —</td><td> 196</td><td> 2.0</td><td></td><td> 0.0</td>

<td> 008</td><td></td><td> 2</td><td> GLO ASP EQO</td><td> —</td><td> 154</td><td> 3.6</td><td></td><td> 0.0</td>

<td> 009</td><td></td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 192</td><td> 3.0</td><td></td><td> 0.0</td>

<td> 010</td><td></td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 163</td><td> 4.0</td><td></td><td> 0.0</td>

<td> Oil</td><td></td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 160</td><td> 5.0</td><td></td><td> 0.0</td>

<td> 012</td><td></td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 154</td><td> 4.0</td><td></td><td> 0.0</td>

<td> 013</td><td></td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 176</td><td> 4.0</td><td></td><td> 0.0</td>

<td> 014</td><td></td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 174</td><td> 3.5</td><td></td><td> 0.0</td>

<td> 016</td><td></td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 170</td><td> 3.5</td><td></td><td> 0.0</td>

<td> 017</td><td></td><td> 2</td><td> C-L02 ASP2 EQO</td><td> —</td><td> 170</td><td> 3.5</td><td></td><td> 0.0</td>

<td> 018</td><td></td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 170</td><td> 3.5</td><td></td><td> 0.0</td>

<td> 019</td><td></td><td> 2</td><td> GLO ASP EQO</td><td> —</td><td> 180</td><td> 3.5</td><td></td><td> 0.0</td>

<td> 020</td><td></td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 180</td><td> 4.5</td><td> HT</td><td> 0.0</td>

<td> 021</td><td></td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 180</td><td> 3.5</td><td></td><td> 0.0</td>

<td> 022</td><td></td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 180</td><td> 3.5</td><td></td><td> 0.0</td>

<td> 023</td><td></td><td> 2</td><td> GUJ2 ASP2 EQO</td><td> —</td><td> 180</td><td> • 3.3</td><td></td><td> 0.0</td>

<td> 024</td><td></td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 175</td><td> 3.3</td><td></td><td> 0.0</td>

<td> 025</td><td></td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 175</td><td> 3.0</td><td></td><td> 0.0</td>

<td> 026</td><td></td><td> 3</td><td> GL02 ASP2 ASPG</td><td> —</td><td> 175</td><td> 3.0</td><td></td><td> 0.0</td>

<td> 027</td><td></td><td> 3</td><td> GL02 ASP2 SER</td><td> -;</td><td> 195</td><td> 5.0</td><td></td><td> 0.0</td>

<td> 028</td><td></td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 175</td><td> 3.5</td><td></td><td> 0.0</td>

<td> 029</td><td></td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 175</td><td> 3.5</td><td></td><td> 0.0</td>

<td> 031</td><td></td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 170</td><td> 3.3</td><td></td><td> 0.0</td>

<td> 032</td><td></td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 170</td><td> 3.5</td><td> HTO</td><td> 0.0</td>

<td> 033</td><td></td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 175</td><td> 3.0</td><td> HTO</td><td> 0.0</td>

<td> 034</td><td></td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 180</td><td> 0.0</td><td> HTO</td><td> 0.0</td>

<td> 035</td><td></td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td></td><td> 3.0</td><td> HTO</td><td> 0.0</td>

<td> 036</td><td></td><td> 2</td><td> GL02 ASP2 EQO</td><td> —</td><td> 175</td><td> 3.6</td><td> HTO</td><td> 0.0</td>

<td> 037</td><td></td><td> 2</td><td> GL02 A-SP2 EQO</td><td> —</td><td> 175</td><td> 21.0</td><td></td><td> 0.0</td>

Glossary: a=asorphous o=oil ‘ =varyiag teaperature &gt; =cook tine change

WO 93/25583

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Appendix Λ

PROTE1KOID BATCHES page 5 of 9

<td> Bt. Ho.</td><td> /AA</td><td> COHPOSITIOH</td><td> ADDITIVE</td><td> TEIP</td><td> TIHE SPHERE</td><td> BATCH SIZE</td>

<td></td><td></td><td></td><td></td><td> C</td><td> (hr) RATING</td><td> eolar</td>

<td> 249 &lt;3K</td><td> 4</td><td> GW2LE02LYSH2PGW</td><td> —</td><td> 180</td><td> 3.0 IHSO HTO HEPO</td><td> 0.1</td>

<td> 250 &lt;3K</td><td> 5</td><td> PGWARGH2LYS2LE0ASP2</td><td> —</td><td> 180</td><td> 3.0 IHSO HTO HEPO</td><td> 0.1</td>

<td> 251 &lt;3K</td><td> 4</td><td> GW2ASP2TYR5-PHE5</td><td> SOL-H</td><td> 180</td><td> 3.0 IHS4 KT4 HEP2</td><td> 0.1</td>

<td> 252 -cp</td><td> 4</td><td> (GLWASP)VAL LYS</td><td> —</td><td> 170</td><td> 3.0 IHS1 HT2 HEP1</td><td> 0.0</td>

<td> 253 -cp</td><td> 4</td><td> GW ASP-TYR PHE</td><td> SOL-H</td><td> 180</td><td> 4.5 IHS1 HTO HEPO</td><td> 2.0</td>

<td> 253</td><td> 4</td><td> GW ASP-TYR PHE</td><td> SOL-H</td><td> 180</td><td> 10.0 IHS4 HT4 HEP4</td><td> 1.0</td>

<td> 254 -cp</td><td> 5</td><td> GW2ASP2-TYR2PHE2ORH</td><td> SOL-H</td><td> 180</td><td> 8.5 IHS4 KT4 HEP4</td><td> 0.1</td>

<td> 255 -cp</td><td> 5</td><td> GW ASPTYR-PHE ORH</td><td> SOL-H</td><td> 180</td><td> 3.0 IHS2· HT4 HEP4</td><td> 0.3</td>

<td> 256 -cp</td><td> 4</td><td> GW2LYSH2PHE2PGW</td><td> —</td><td> 180</td><td> 3.0 IHSO HTO HEPO</td><td> .0.1</td>

<td> 257 -cp</td><td> 4</td><td> GW ASP ARGH ORHB</td><td> —</td><td> 180</td><td> 3.0 IHSO HTO HEPO</td><td> 0.0</td>

<td> 258 &lt;3K</td><td> 3</td><td> GW ASP ARGH</td><td> —</td><td> 180</td><td> 3.0 IHS1 HT1 HEPO</td><td> 0.0</td>

<td> 259 &lt;3K</td><td> 4</td><td> GW ASP-TYR PHE</td><td> SOL-H</td><td> 180</td><td> 3.0 IHS3 KT3 HEP3</td><td> 0.3</td>

<td> 260 &lt;3K</td><td> 4</td><td> GW ASP-TYR PHE</td><td> SOL-H</td><td> 180</td><td> 2.5 IHS2 HT3 HEP2</td><td> 0.3</td>

<td> 261 -cp</td><td> 4</td><td> GW ASP-TYR PHE</td><td> SOL-K</td><td> 180</td><td> 3.0 IHSO HTO HEPO</td><td> 0.3</td>

<td> 262 -cp</td><td> 4</td><td> GW LORH ASP LYSFB</td><td> SOL-H</td><td> 180</td><td> 3.5 IHSO HT1 HEP4</td><td> 0.3</td>

<td> 263 -cp</td><td> 4</td><td> GW2 LYSH2 PHE2 ASP</td><td> —</td><td> ' 190</td><td> 3.0 IHS3 HT3 HEPO</td><td> 0.3</td>

<td> 264 -cp</td><td> 4</td><td> GW2 LYSH2 PHE2 ASP</td><td> —</td><td> 180</td><td> 3.2 IHS5 HT3 HEP4</td><td> 0.3</td>

<td> 266 -cp</td><td> 4</td><td> GW2 LYSH2 PHE2 ASP</td><td> —</td><td> 180</td><td> 3.0 IHS4 HT4 HEP4</td><td> 0.3</td>

<td> 267 -cp</td><td> 3</td><td> GW LYSFB ASP LYSFB</td><td> SOL-H</td><td> 180⁴</td><td> 3.0 IHSa HTc HEPc</td><td> 0.3</td>

<td> 268</td><td> 4</td><td> GW ASP-TYR PHE</td><td> SOL-H</td><td> 190</td><td> 2.5 IHSO HTO HEPO</td><td> 0.3</td>

<td> 269 -cp</td><td> 4</td><td> GW ORHB ASP-LYSFB</td><td> SOL-H</td><td> 180</td><td> 4.0 IKSc HTC HEPc</td><td> 0.1</td>

<td> 270 -cp</td><td> 4</td><td> GW ASP-TYR PHE</td><td> SOL-H</td><td> 180</td><td> 1.5 IHS5 HT4 HEPO</td><td> 1.5</td>

<td> 271</td><td> 3</td><td> GW LYSFB-PHE</td><td> SUL-H</td><td> 190</td><td> 1.5 IHS3aHT4oHEP4o</td><td> 0.0</td>

<td> 272 -cp</td><td> 4</td><td> GW2 LE02 LYSH2 TYR1</td><td> —</td><td> 180</td><td> 3.0 IHSc HT1 HEP4</td><td> 0.1</td>

<td> 273 -cp</td><td> 4</td><td> GW2 LEU2 LYSH2 PHE1</td><td> —</td><td> 180</td><td> 3.0 IHS2aHT2 HEP2,a</td><td> 0.1</td>

<td> 274 -cp</td><td> 4</td><td> GW LEO ARG TYR</td><td> —</td><td> 180</td><td> 3.0 IHSc HTc HEPc</td><td> 0.1</td>

<td> 275 -cp</td><td> 4</td><td> GW ARGH-TYR</td><td> SUL ,</td><td> 190</td><td> 1.5 IHSc HTc HEPc</td><td> 0.3</td>

<td> 276 -cp</td><td> 4</td><td> GW2 LE02 ARG2 PHE</td><td> — .</td><td> 180</td><td> 3.0 IHS3 KT3 HEP4</td><td> 0.1</td>

<td> 277 -cp</td><td> 3</td><td> GW LYS TYR '</td><td> SUL-H</td><td> 190</td><td> 1.5 IHSc HTc H£P4o</td><td> 0.3</td>

<td> 278 -cp</td><td> 3</td><td> GW LYS PHE</td><td> SOL-H</td><td> 190</td><td> 1.5 IHSc HTc HEP4</td><td> 0.3</td>

<td> 279 -cp</td><td> 3</td><td> GW LYS ALA</td><td> —</td><td> 190</td><td> 1.5 IHSc HTc HEPc</td><td> 0.3</td>

<td> 280 -cp</td><td> 4</td><td> GWGWASPGLOTYRGLPHE</td><td> SOL-H</td><td> 190</td><td> 1.5 IHS4 KT3 HEP4</td><td> 0.4</td>

<td> 281 -cp</td><td> 4</td><td colspan="2"> GL01 ASP1 TYR2.5-PHE2.5 SOL-H</td><td> 180</td><td> 3.0 IHS4 HTa HEP2a</td><td> 1.0</td>

<td> 282 -cp</td><td> 3</td><td> GW2 LYS5 PHE2</td><td> — _</td><td> 190</td><td> 1.5 IHSO HTO HEP2</td><td> 0.3</td>

<td> 283 -cp</td><td> 4</td><td> GW2 LYS5 ΡΗΣ5 TYR2</td><td> —</td><td> 190</td><td> 1.5 IHSO HTO HEP3</td><td> 0-1 .</td>

<td> 284 -</td><td> 5</td><td> GW2ASP2-TYR2PHE20RH</td><td> SOL-H</td><td> 180</td><td> 3.0 IHS4aHT4oHEP2a</td><td> 1.0</td>

<td> 285 -cp</td><td> 2</td><td> GW(2X) ASP(2I)</td><td> — .</td><td> 180</td><td> 3.0 IHSc HTc HEPc</td><td> ’ 0.3</td>

<td> 286 -cp</td><td> 2</td><td> GW ASP(2X)</td><td> —</td><td> 180</td><td> 2.5 IHSc HTc HEPc</td><td> ’ 0.3</td>

<td> 287 -cp</td><td> 2</td><td> GW PHE</td><td> —</td><td> 180</td><td> 3.5 IRS3 KT2 HEP3</td><td> 0.3</td>

<td> 288 -cp</td><td> 3</td><td> GW ORH PHE</td><td> —</td><td> 180</td><td> 3.0 IHSc HTc HEPc</td><td> 0.3</td>

<td> 289</td><td> 2</td><td> GW ARG</td><td> —</td><td> 180</td><td> 1.0</td><td> 0.3</td>

<td> 290 -cp</td><td> 3</td><td> GW ARG PHE</td><td> —</td><td> 180</td><td> 3.0 IHS2 HT2 HEP2</td><td> 0.3</td>

<td> 291 -CP</td><td> 3</td><td> GW LYS PHE</td><td> SOL-H</td><td> 190</td><td> 1.5 IHS4 KT3o HEP4o</td><td> 0.3</td>

<td> 292 -cp</td><td> 5</td><td> GW ASP ARG ORH PHE</td><td> SUL-H</td><td> 180</td><td> 3.0 IHSO HTO HEPO</td><td> 0.3</td>

<td> 293 -cp</td><td> 4</td><td> GW ASP ARG ORH PHE</td><td> SOL-H</td><td> 180</td><td> 3.0 IHS3 HT3 HEP3</td><td> 0.3</td>

<td> 294 -cp</td><td> 4</td><td> GW2 LYSH2 PHE2 ASP</td><td></td><td> 180</td><td> 3.0 IHS3 KT4 HEP4</td><td> 0.3</td>

<td> Glossary:</td><td colspan="2"> a=anorphous o=oil ⁴</td><td colspan="2"> =varying tenperature</td><td> + =cook tine change</td><td></td>

WO 93/25583

PCT/US93/05723

Appendix

Page 6 of q

PROTEIHOID BATCHES

<td> Bt. Ho.</td><td> /λλ COHPOSITiOH</td><td> ADDITIVE</td><td> TEKP</td><td> TIHE</td><td> SPHERE</td><td> BATCH SIZE</td>

<td></td><td></td><td></td><td> C</td><td> (hr)</td><td> RATING</td><td> tolar</td>

<td> 192 -cp</td><td> 3 GLO LYSFB ASP</td><td> —</td><td> 195</td><td> 3.0</td><td> IHS4 HTO</td><td> 0.3</td>

<td> 193 &gt;6R</td><td> 4 (GLO+ASP) TYP PHE</td><td> PA</td><td> 175</td><td> 4.0</td><td> HTO HEPO</td><td> 0.3</td>

<td> 194 -cp</td><td> 3 GLO LYSFB ASP</td><td> TRIGL</td><td> 195</td><td> 3.0</td><td> MSI HTO HEP2</td><td> 0.3</td>

<td> 195 -cp</td><td> 3 GLO ASP VAL2</td><td> —</td><td> 170</td><td> 3.2</td><td> IHS2 KT1 HEPO</td><td> 0.3</td>

<td> 196 -cp</td><td> 4 GLO ASP TYR PHE</td><td> PA</td><td> 175</td><td> 4.2</td><td> IHS2 HTS HEP4</td><td> 1.0</td>

<td> 197 -cp</td><td> 4 GLO ASP TYR PHE</td><td> SOL</td><td> 175</td><td> 2.7</td><td> IHS2 HT5 HEP5</td><td> 0.3</td>

<td> 198 -cp</td><td> 3 GLO LYSFB ASP</td><td> —</td><td> 195</td><td> 3.2</td><td> IHS3</td><td> 1.0</td>

<td> 200 -cp</td><td> 3 GLO LYSH ASP</td><td> PA</td><td> 185</td><td> 3.0</td><td> IKSi HTO HEPO</td><td> 0.3</td>

<td> 201 -cp</td><td> 3 GLO LYSFB ASP</td><td> SOLFA</td><td> 195</td><td> 3.0</td><td> IHS4 HTO HEP3</td><td> 0.3</td>

<td> 203 -cp</td><td> 4 GLO ASP VAL LYS</td><td> —</td><td> 170</td><td> 3.0</td><td> IHS5 HT5 HEP</td><td> 0.3</td>

<td> 204 -cp</td><td> 3 GLO LYS ASP</td><td> —</td><td> 185</td><td> 3.0</td><td> IHS4 HTO HEPO</td><td> 0.3</td>

<td> 205 -cp</td><td> 4 GLO ASP-ra PHE</td><td> SOLFA</td><td> 175</td><td> 3.7</td><td> IHS4 HTO</td><td> 0.6</td>

<td> 208</td><td> 3 GLOH LYSH ASPH</td><td colspan="2"> HaBCOAHeOH 80</td><td> 8.0</td><td> IHSO HTO HEPO</td><td> 0.0</td>

<td> 209 -cp</td><td> 4 GLO ASP-VAL LYS'</td><td> SOLFA</td><td> 170</td><td> 3.2</td><td> IHS4 HT4 HEP3 H</td><td> 0.3</td>

<td> 210 -cp</td><td> 4 GLO ASP-VAL LYSFB</td><td> SOLFA</td><td> 170</td><td> 3.0</td><td> IHS4 KT4 HEP3</td><td> 2.0</td>

<td> 213 -cp</td><td> 3 GLO-LYS HIS</td><td> SOLFA</td><td> 180</td><td> 3.0</td><td> IKSO HTO HEPO</td><td> 0.3</td>

<td> 215 -cp</td><td> 3 GLO ASP GLY2 .-</td><td> —</td><td> 180</td><td> 5.5</td><td> IHS3 HTO HEPO</td><td> 0.3</td>

<td> 216 -cp</td><td> 4 GLO ASP-TYR PHE</td><td> SOLFA</td><td> 175</td><td> 3.0</td><td> IHS4 KT4 BEP4</td><td> 2.0</td>

<td> 217</td><td> 3 GLOASPLYS(DIETESTER)</td><td> HE0B/Et3H</td><td> 75</td><td> 29.0</td><td></td><td> 0.0</td>

<td> 218 -cp</td><td> 4 GLO ASP-TYR PHE</td><td> SOLFA</td><td> 175</td><td> .0</td><td> IKSO HTO HEPO A</td><td> 0.3</td>

<td> 219 -cp</td><td> 3 GLO-LYS-LEO</td><td> SU1/POC13</td><td> 180*</td><td> 8.5</td><td> IHS2 KTO HEP2</td><td> 0.3</td>

<td> 220 -cp</td><td> 4 GLO ASP-TYR PHE</td><td> SOLFA</td><td> 180</td><td> 20.5</td><td> IHS4 KT4 HEP5</td><td> 0.3</td>

<td> 221 -cp</td><td> 3 -ASP2 TYR F3E</td><td> SOLFA</td><td> 180</td><td> 22.0</td><td> IHS2 HTO HEPO</td><td> 0.3</td>

<td> 222 -cp</td><td> 3 -LYSFB2 ARG2 LEO</td><td> SOLFA</td><td> 180</td><td> 4.0</td><td> IHSO HTO HEP2</td><td> 0.3</td>

<td> 224 -cp</td><td> 4 GLU ASP-TYR PHE-</td><td> SO/PA</td><td> ISO</td><td> 6.0</td><td> IHS3 HTO HEPO</td><td> 0.3</td>

<td> 225 -cp</td><td> 3 PRO-SER TO</td><td> SOLE</td><td> 180</td><td> 3.5</td><td> IKS2 HTO HEPO</td><td> 0.3</td>

<td> 226 &lt;3K</td><td> 4 GLU ASP TYR PHE</td><td> SOLF</td><td> 180</td><td> 4.0</td><td> IHS3 HT4 HEP3</td><td> 0.3</td>

<td> 229 &lt;3K</td><td> 4 -GLO ASP -TYR PHE</td><td> SOLF</td><td> 180</td><td> 5.5</td><td> IHS3 HTO HEPO</td><td> 0.3</td>

<td> 230 -cp</td><td> 2 GLO TYR -</td><td> —</td><td> 180</td><td> 4.0</td><td> IHS4 HTO HEPO</td><td> 0.3</td>

<td> 231 -cp</td><td> 3 GLO LYSPB PHE</td><td> SOLF</td><td> 180</td><td> 3.5</td><td> IHS2 HTO HEPO</td><td> 0.3</td>

<td> 232 -cp</td><td> 3 GLO LEO ARG</td><td> —</td><td> 180</td><td> 4.0</td><td> IHSO HTO HEPO</td><td> 0.3</td>

<td> 233 -cp</td><td> 3 GLO LEO LYSH</td><td> —</td><td> 180</td><td> 4.0</td><td> IHSO HTO HEPO</td><td> 0.3</td>

<td> 234 -cp</td><td> 4 -(GLO ASP TYR PHE)</td><td> SOLF</td><td> 150</td><td> 27.0</td><td> IHS3 HTO HEPO</td><td> 0.3</td>

<td> 235 -cp</td><td> 4 -(GLO ASP)TYR10PHE10</td><td> SOLF</td><td> 180</td><td> 22.0</td><td> IHSO KTO HEPO</td><td> 0.0'</td>

<td> 236 -cp</td><td> 3 GLO TYR LYSHCL</td><td> —.</td><td> 180</td><td> 2.0</td><td> IHSO HTO HEPO</td><td> 0.3</td>

<td> 237 &lt;3K</td><td> 4 GL02 LE02 LYSH2 ASP</td><td> —</td><td> 180</td><td> 3.0</td><td> IHSO HTO HEPO</td><td> 0.1</td>

<td> 238 &lt;3K</td><td> 4 GLO ASP TYR5 PHE5</td><td> SOLF</td><td> 180</td><td> 3.0</td><td> IHSO HTO HEPO ’</td><td> 0.1</td>

<td> 239 -cp .</td><td> 3 -GLO ASP LEO</td><td> SOL-H</td><td> 190</td><td> 1.0</td><td> IHS3 HTO HEPO -</td><td> 0.0</td>

<td> 240 -cp</td><td> 3 -(GLO ASP) LEO</td><td> SOL-H</td><td> 170</td><td> 4.0</td><td> IHS4 HTO HEPO</td><td> 0.0</td>

<td> 241 &lt;3R</td><td> 3 -(GLO ASP) LEO</td><td> SOL-H</td><td> 190</td><td> 5.0</td><td> IHS3 HTO HEPO</td><td> 0.0</td>

<td> 242 -cp</td><td> 3 (GLO ASP LEO)</td><td> SOL-H</td><td> 170</td><td> 2.5</td><td> IHSO HTO HEPO</td><td> 0.0</td>

<td> 243 -cp</td><td> 5 PGL02ASPARG2LYS2LE0</td><td> —</td><td> 180</td><td> 3.0</td><td> IHSO KTO HEPO</td><td> 0.1</td>

<td> 244 -cp</td><td> 3 (GLO ASP) LEO</td><td> —</td><td> 190</td><td> 2.5</td><td> IHSO HTO HEPO</td><td> 0.0</td>

<td> 245 -cp</td><td> 3 (GLO ASP) LEO</td><td> —</td><td> 170</td><td> 1.0</td><td> IHS3 HT1 HEPO</td><td> 0.0</td>

<td> 246 &lt;3K</td><td> 5 GL02 LYSH2 PHE2 ASP</td><td> —</td><td> 180</td><td> 6.0</td><td> IHS4 KT4 HEP4</td><td> 0.0</td>

<td> 247 -cp</td><td> 3 GLO ASP LEO</td><td> —</td><td> 170</td><td> 5.0</td><td> IHSO HTO HEPO</td><td> 0.0</td>

Glossary: a=asorphous o=oil * =varying teeperature + =cook tise change

WO 93/25583

PCT/US93/05723

Appendix A

Page 7 of 9

PROTEIKOID BATCHES

<td> Bt. Ho.</td><td> /AA COHPOSITIOH</td><td> ADDITIVE</td><td> TEKP</td><td> TIKE</td><td> SPHERE</td><td> BATCH SIZE Operator Date</td>

<td></td><td></td><td></td><td> C</td><td> (hr)</td><td> RATIHG</td><td> tolar</td>

<td> 145</td><td> 3 GLO ASP LYSFB</td><td> PPA</td><td> 185</td><td> 6.0</td><td> KTO</td><td> 0.0</td>

<td> 146</td><td> 3 a' ASP VAL2</td><td> PPA</td><td> 170</td><td> 3.5</td><td> IHS2 HTO</td><td> 0.0</td>

<td> 147</td><td> 4 GLO ASP PBE ALA</td><td> —</td><td> 170</td><td> 3.0</td><td> IHS4 KT3 HEP4</td><td> 0.0</td>

<td> 14S</td><td> 4 GLO ASP TYR PHE</td><td> PA</td><td> 170</td><td> 3.0</td><td> HTO HEP4</td><td> 0.0</td>

<td> 149</td><td> 3 GLO ASP PHE2</td><td> —</td><td> 170</td><td> 3.0</td><td></td><td> 0.0</td>

<td> 150</td><td> 4 GLO ASP LEO PHE</td><td> PA</td><td> 170</td><td> 24.0</td><td> KTO ΒΕΡΟ</td><td> 0.0</td>

<td> 151</td><td> 4 GLO ASP TYR PHE</td><td> PA</td><td> 170</td><td> 6.0</td><td> IHS4 HT4 HEP4</td><td> 0.0</td>

<td> 152</td><td> 4 GLO ASP TYR PHE</td><td> PA</td><td> 170</td><td> 5.0.</td><td></td><td> 0.0</td>

<td> 153</td><td> 3 GLO LYSFB PHE</td><td> PA</td><td> 170</td><td> 24.0</td><td></td><td> 0.0</td>

<td> 154</td><td> 4 GLO ASP TYR PBE</td><td> PA</td><td> 170</td><td> 4.0</td><td></td><td> 0.0</td>

<td> •155</td><td> 3 GL02 TYR PHE</td><td> PA</td><td> 170</td><td> 4.0</td><td> IHS4 HT5 HEP3</td><td> 0.0</td>

<td> '456</td><td> 3 GL04 LYS2 PHE</td><td> —</td><td> 170</td><td> 6.0</td><td> IHSO HTO HEPc</td><td> 0.0</td>

<td> '157</td><td> 3 GL02 TYR LEO</td><td> PA</td><td> 170</td><td> 5.0</td><td> IHS2 KT1 HEPO</td><td> 0.0</td>

<td> 158</td><td> 3 GL02 PHE LEO</td><td> PA</td><td> 175</td><td> 5.0</td><td> IHS4 KTO HEP4</td><td> 0,0</td>

<td> 159</td><td> 3 GL03 PHE TYR</td><td> PA</td><td> 175</td><td> 5.0</td><td> IHS4 HT4 HEP4</td><td> 0.0</td>

<td> 160</td><td> 4 GL06 LYS2 PHE TYR</td><td> PA</td><td> 170</td><td> 6.0</td><td> IKSa KTc HEPc</td><td> 0.0</td>

<td> 161</td><td> 4 GL04 PHE2 TYR2 CYS</td><td> PA</td><td> 170</td><td> 4.0</td><td> IHS4 KT HEP</td><td> 0.0</td>

<td> 162</td><td> 3 GL02 TYR PHE</td><td> PA</td><td> 170</td><td> 5.5</td><td> IHS3 KTO HEP2</td><td> 0.0</td>

<td> 163</td><td> 3 GL02 PHE TYR</td><td> PA</td><td> 170</td><td> 5.0</td><td> IKS3 KT2 HEP3</td><td> 0.0</td>

<td> 164</td><td> 3 GL02 PHE TYR</td><td> PA</td><td> 170</td><td> 5.0</td><td> IHS4 HT4 HEP4</td><td> 0.0</td>

<td> 165</td><td> 4 GL03 ASP PHE2 TYR2</td><td> PA</td><td> 170</td><td> 3.0</td><td> IHS3 HTO HEPO</td><td> 0.0</td>

<td> 166 '</td><td> 3 GLO LYSFB PGLO</td><td> PA</td><td> 170</td><td> 7.0</td><td></td><td> 0.0</td>

<td> h</td><td> 4 GLO ASP TYR PHE</td><td> PA</td><td> 170</td><td> 6.5</td><td></td><td> 0.0</td>

<td> Ibc</td><td> 3 GLO ASP LYSFB</td><td> PPA</td><td> 185*</td><td> 72.0</td><td> KTO</td><td> 0.0</td>

<td> 169</td><td> 3 GLO ASP LYSFB</td><td> PPA</td><td> 185</td><td> 72.0</td><td> KTO</td><td> 0.0</td>

<td> 170</td><td> 3 GLO ASP LYSFB</td><td> —</td><td> 195</td><td> 7.0</td><td> KT5</td><td> 0.0</td>

<td> 171</td><td> 3 GLO LSYHCL ASP</td><td> H.OIL</td><td> 180</td><td> 7.0</td><td> KTO</td><td> 0.0</td>

<td> 172</td><td> 4 GLO ASP TYR PHE</td><td> PA</td><td> 170</td><td> 6.0</td><td> KT1</td><td> 0.0</td>

<td> 173</td><td> 3 GLO LYS ASP</td><td> tineral o.</td><td> 185</td><td> 3.0</td><td> ABORT</td><td> 0.3</td>

<td> 174 &gt;6R</td><td> 3 GLO LYS ASP</td><td> GLycerin</td><td> 185</td><td> 3.0</td><td> IHS2 HT1 HEP3</td><td> 0.3</td>

<td> 175 &gt;6K</td><td> 4 GLO ASP TYR PEE</td><td> PA</td><td> 172</td><td> 3.5</td><td></td><td> 1.0</td>

<td> 176 &gt;6K</td><td> 3 GL02 LYS2 LYS</td><td> —</td><td> 180 .</td><td> 3.0</td><td> IHSO KTO HEPO</td><td> 0.3</td>

<td> 177 &gt;6K</td><td> 3 GLO ARG ASP</td><td> —</td><td> 180 ·</td><td> 3.2</td><td> IHSO KT2 HEPO</td><td> 0.3</td>

<td> 178 &gt;6K</td><td> 3 GLO LYS ASP</td><td> —</td><td> 190</td><td> 3.2</td><td> IHSO KTO HEP1</td><td> 0.3</td>

<td> 179 &gt;6K</td><td> 4 GLO ASP TYR PHE</td><td> PA</td><td> 175</td><td> 4.0</td><td></td><td> 1.0</td>

<td> 180 &gt;6K</td><td> 4 GLO ASP TYR PHE</td><td> PA</td><td> 175</td><td> 7.0</td><td> See Notes.</td><td> 1.0</td>

<td> 181 &gt;6K</td><td> 3 GLO LYS ASP</td><td> —</td><td> 185</td><td> 3.0</td><td> IHSO KTO</td><td> 0.3</td>

<td> 182 &gt;6K</td><td> 4 GLO ASP TYR PHE</td><td> PA</td><td> 175</td><td> 3.7</td><td> KT1 HEP1</td><td> 0.3</td>

<td> 183</td><td> 4 PGLO ASP TYR PHE</td><td> PA</td><td> 175</td><td> 4.0 '</td><td> ABORT-RETRY</td><td> 0.3</td>

<td> 184 -cp</td><td> 4 GLO ASP TYR PHE</td><td> PA</td><td> 175</td><td> 3.5</td><td> HT2 HEP4</td><td> 0.3</td>

<td> 185 -cp</td><td> 4 GLO ASP TYR PHE</td><td> PA</td><td> 176</td><td> 4.2</td><td> IKS HT4 HEP4</td><td> 1.0</td>

<td> 187</td><td> 3 ASP TYR PHE</td><td> PA</td><td> 170</td><td> .0</td><td> ABORT</td><td> 0.3</td>

<td> '188 -cp</td><td> 3 ASP TYR PHE</td><td> PA</td><td> 150</td><td> 21.2</td><td> IHSO HTO HEPO</td><td> 0.3</td>

<td> 189 -cp</td><td> 4 GLO ASP TYR PHE</td><td> PA</td><td> 176</td><td> 4.0</td><td> KT4 HEP5</td><td> 1.0</td>

<td> 190 -cp</td><td> 4 GLO ASP TYR PHE</td><td> PA</td><td> 175</td><td> 4.0</td><td> HT5 HEP4</td><td> 1.0</td>

<td> 191 -cp</td><td> 3 ASP2 TYR PHE</td><td> PA</td><td> 150</td><td> 24.0</td><td> HTO HEPO</td><td> 0.3</td>

Glossary: a=anorphous o=oil ¹ =varying teEperatere * =cook tine change

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Appendix λ Pace ft _oi

PROTEIHOID BATCHES

<td> Bt. Ho.</td><td> 111 COHPOSITIQK</td><td> ADDITIVE</td><td> TEHP</td><td> TIKE</td><td> SPHERE</td><td> BATCH SIZE</td>

<td></td><td></td><td></td><td> C</td><td> (hr)</td><td> RATIHG</td><td> »olar</td>

<td> 015</td><td> 2 GL02 .'3P2 EQO</td><td> —</td><td> 170</td><td> 2.5</td><td></td><td> 0.0</td>

<td> 100</td><td> 3 GLO ASP VAL2</td><td> —</td><td> 170</td><td> 3.0</td><td></td><td> 0.0</td>

<td> 101</td><td> 3 GLO ASP VAL2</td><td> —</td><td> 170</td><td> 3.0</td><td></td><td> 0.0</td>

<td> 102</td><td> 3 GLO ASP VAL2</td><td> —</td><td> 170</td><td> 3.0</td><td></td><td> 0.0</td>

<td> 103</td><td> 4' GLO ASP GLY VAL</td><td> —</td><td> 170</td><td> 3.5</td><td> IHS4</td><td> 0.0</td>

<td> 104</td><td> 4 GLO ASP VAL LEO</td><td> —</td><td> 170</td><td> 3.5</td><td> IHS4 HT2 HEPS</td><td> 0.0</td>

<td> 105</td><td> 3 GLO ASP GLY2</td><td> —</td><td> 180</td><td> 4.0</td><td> IKS4 KT2</td><td> 0.0</td>

<td> 106</td><td> 4 GLO ASP VAL LEO</td><td> —</td><td> 170</td><td> 5.0</td><td></td><td> 0.0</td>

<td> 107</td><td> 4 GL02 ASP2 GLY VAL2</td><td> •—</td><td> 170</td><td> 3.0'</td><td> IHS5</td><td> 0.0</td>

<td> IOS</td><td> 4 GL02 ASP2 GLY VAL2</td><td> —</td><td> 170</td><td> 4.0</td><td> IHS4 KO AHORPHO</td><td> 0.0</td>

<td> 109</td><td> 4 GL02 ASP2 GLY VAL2</td><td> —</td><td> 170</td><td> 4.0</td><td> IHS4 HT1</td><td> 0.0</td>

<td> 110</td><td> 4 GL02 ASP2 GLY2 VAL</td><td> —</td><td> 170</td><td> 3.5</td><td></td><td> 0.0</td>

<td> 111</td><td> 5 GLO ASP GLY VAL CYS</td><td> —</td><td> 170</td><td> 3.0</td><td></td><td> 0.0</td>

<td> 112</td><td> 4 GLO ASP GLY PHE</td><td> —</td><td> 170</td><td> 4.0</td><td> IHS4 HT3 HEP4</td><td> 0.0</td>

<td> 113</td><td> 4 GLO ASP· VAL2 GLY</td><td> —</td><td> 170</td><td> 3.0</td><td> IHS2 KTO</td><td> 0.0</td>

<td> 114</td><td> 3 GLO ASP VAL</td><td> —</td><td> 170</td><td> 3.0</td><td> IHS4 HTO</td><td> 0.0</td>

<td> 115</td><td> 3 GLO VAL TYR</td><td> —</td><td> 170</td><td> 4.0</td><td></td><td> 0.0</td>

<td> 116</td><td> 4 GLO ASP VAL LYS</td><td> —</td><td> 170</td><td> 4.0</td><td></td><td> 0.0</td>

<td> 117</td><td> 3 GLO VAL TYR</td><td> —</td><td> 170</td><td> 3.0</td><td> IHS5</td><td> 0.0</td>

<td> ns</td><td> 2 GLO TYR</td><td> —</td><td> 170</td><td> 3.5</td><td> IHS5 HTO ΒΕΡΟ</td><td> 0.0</td>

<td> 119</td><td> 2 GL02 ASP2 EQO</td><td> —</td><td> 170</td><td> 3.5</td><td> IHS5 HT1</td><td> 0.0</td>

<td> 120</td><td> 3 GLO ASP TYR</td><td> —</td><td> 170</td><td> 4.5</td><td> IHSO HTO HEPO</td><td> 0.0</td>

<td> 121</td><td> 4 GLO ASP TYR PHE</td><td> —</td><td> 170</td><td> 4.0</td><td> IHS5 HT3 HEP4</td><td> 0.0</td>

<td> 122</td><td> 4 GLO ASP VAL TYR</td><td> —</td><td> 170</td><td> 3.0</td><td> IHS3 HTO HEPO</td><td> 0.0</td>

<td> 123</td><td> 1 GLO</td><td> —</td><td> 170</td><td> 4.5</td><td> CAH'T DRY</td><td> 0.0</td>

<td> 124</td><td> 3 GLO TYR VAL</td><td> —</td><td> 170</td><td> 3.5</td><td> IHS4 KT3 HEP3</td><td> 0.0</td>

<td> 125</td><td> 3 PGLO VAL TYR</td><td> —</td><td> 170</td><td> 3.5</td><td> IKS3 HT2</td><td> 0.0</td>

<td> 126</td><td> 4 GLO ASP VAL2 GLY</td><td> —</td><td> 170</td><td> • 4.0</td><td> IHS1</td><td> 0.0</td>

<td> 127</td><td> 4 GL02 ASP2 VAL2 PHE</td><td> —</td><td> 170</td><td> 4.0</td><td> IHS3 HT2 HEP4</td><td> 0.0</td>

<td> 128</td><td> 2 GL02 ASP2 EQO</td><td> —</td><td> 170</td><td> 3.5</td><td> HTO</td><td> 0.0</td>

<td> 129</td><td> 2 GL02 ASP2 EQO</td><td> —</td><td> VARY</td><td> 4.0</td><td> IHS3 HTO</td><td> 0.0</td>

<td> 130</td><td> 2 GL02ASP2 EQO</td><td> —</td><td> 220</td><td> 3.0</td><td> IHS5 HTO</td><td> 0.0</td>

<td> 131</td><td> 2 GUJ2 LYSFB</td><td> —</td><td> 185</td><td> 3.0</td><td> IHS1 HTO</td><td> 0.0</td>

<td> 132</td><td> 3 GLO ASP LYSFB</td><td> —</td><td> 185</td><td> 3.0</td><td> IHS3 HT2 HEP2</td><td> 0.0 .</td>

<td> 133</td><td> 3 GLO ASP LYSFB</td><td> PA</td><td> 180</td><td> 6.2</td><td> INS5 HT1 HEP2</td><td> 0.0</td>

<td> 134</td><td> 4 GLO ASP LYS VAL</td><td> „PA ’</td><td> 185</td><td> 3.0</td><td></td><td> 0.0</td>

<td> 135</td><td> 3 GLO ASP LYSFB</td><td> - ' GLYC</td><td> 185</td><td> 6.5</td><td> IHS1 HT1</td><td> 0.0</td>

<td> 136</td><td> 2 GL02 ASP2 EQO</td><td> —</td><td> 155</td><td> 3.0</td><td> KTO</td><td> 0.0</td>

<td> 137</td><td> 5 GL02ASP2LE0 THY VAL</td><td> —</td><td> 185</td><td> 4.0 ·</td><td> IHSO HTO</td><td> 0.0</td>

<td> 13S .</td><td> 4 GLO ASP VAL TYR</td><td> —</td><td> 185</td><td> 4.5</td><td> IHS1 HT3</td><td> 0.0</td>

<td> 139</td><td> 4 GLO ASP VAL TYR</td><td> PPA</td><td> 160*</td><td> 72.0</td><td> IHS2 HT3</td><td> 0.0</td>

<td> 140</td><td> 3' GLO ASP LYSFB</td><td> PPA</td><td> 120*</td><td> 72.0</td><td> IHS5 HT2 HEP4</td><td> 0.0</td>

<td> 141</td><td> 3 GLO LYSFBSYHPEPagqp</td><td> —</td><td> 185</td><td> 6.0</td><td></td><td> 0.0</td>

<td> 142</td><td> 3 GLO ASP LYSFB</td><td> PPA</td><td> 120*</td><td> 72.0</td><td> IHS1 HT1</td><td> 0.0</td>

<td> 143</td><td> 3 GLO VAL TYR</td><td> —</td><td> 170</td><td> 3.0</td><td> IHS2 HT2</td><td> 0.0</td>

<td> 144</td><td> 4 GL02 ASP2 GLY VAL2</td><td> —</td><td> 170</td><td> 3.0</td><td> IKS1 HT1</td><td> 0.0</td>

Glossary: amorphous o=oil * =varying tenperature -t =cook tine change

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Appendix A page 9 of «

PROTEIKOID BATCHES

Bt. Ho. /AA COMPOSITION

ADDITIVE TEHP TIHE SPHERE C (hr) R”%G

- BATCH SIZE «olar

354

-(GLU ASP TTR PHE)2 ORH SULFOLANE .0 .0

Sphere rating: . 0= worst

5= best

INT = insulin

MT = empty microsphere HEP = heparin

Sul-M = sulfolane, medical grade Sulfa = Sulf = Sul = Sulfolane PA = phosphoric acid

Equ = equilents

GLYC = glycerol

TRIGL = triglyme

PFA = polyphosphoric acid M.Oil = mineral o. = mineral oil

Glossary: a=asorphous o=oil * varying teaperature + =cook tine change

WO 93/25583

IEF TABLES

Proteinoid sorting, pKa and ««position Chenical basis for eicrosphere ODS

PCT/US93/05723 . .ppendix B pane 1 of 2 . Haterial ID Ho. CoEposition Sphere frac Sphere pH Sphere IEF Sphere- Max OV Rax OV —&gt;(*no asp) nujiber range rating Matrix rating Frac.Ho. pB

<td> 202B</td><td> Glu2.4 Asp2Val2GLy *</td><td> no spher</td><td> —</td><td> —</td><td colspan="2"> IHS4 HTO HEP3 -</td><td> —</td>

<td> 210&gt;lR</td><td> Glu Asp Val Lys</td><td> 14-20</td><td> 2.3-4.4</td><td> 2-3</td><td> IHS4 HT4 HEP3</td><td> 14-19</td><td> 4.4-3.0</td>

<td> 213&gt;lk</td><td> Glu LysFB HisFB</td><td> 16-19</td><td> 1.7-2.1</td><td> 2</td><td> IHSO KTO ΒΕΡΟ</td><td> 11-16</td><td> 7.6-2.1</td>

<td> 218&lt;3k</td><td> Glu Asp Tyr Phe</td><td> 18-20</td><td> 3.2-2.7</td><td> 2</td><td> IHSO HTO HEPO</td><td> 1-7</td><td> 12.2-9.3</td>

<td> 129</td><td> Glu Asp Egu</td><td> 10-18</td><td> 2.5-4.4</td><td> 3</td><td> IHS3 KTO</td><td> 15-18</td><td> 2.9-2.5</td>

<td> 21 Oik</td><td> Glu LysFB Arg</td><td> no spher</td><td> —</td><td> —</td><td> IHSO KTO ΒΕΡΟ</td><td> 1-4</td><td> 11.8-9.2</td>

<td> 176</td><td> Glu2 LysFB2 LysFB</td><td> no spher</td><td> —</td><td> - .</td><td> IHSO HTO HEPO</td><td> 5-6</td><td> 9-8.6</td>

<td> 222-cp</td><td> Arg2 LysFB2 Leu</td><td> ?l-4?</td><td> 9.9-11.7</td><td> 2?</td><td> IHSO HTO HEP2</td><td> 2-6</td><td> 8.5-11.5</td>

<td> 202B</td><td> Glu2.4 Asp2 Val2 Gly</td><td> 8-12</td><td> 3.3-5</td><td> 2,1-2</td><td> IKS4 KTO HEP3</td><td> 8-12</td><td> 5-3.3</td>

<td> 223-cp</td><td> Arg2 LysFB2 Leu pGlu</td><td> I</td><td> 10.3</td><td> 2-3</td><td> IHSO HTO HEP2</td><td> 1</td><td> 10.3</td>

<td> 223-cp</td><td> Arg2 LysFB2 Leu pGlu</td><td> 1-5</td><td> 9.0-12</td><td> 2</td><td> IHSO KTO BEP2</td><td> 3</td><td> 10.7</td>

<td> 170a</td><td> Glu Asp LysFB</td><td> 16-20</td><td> 2.3-5.0</td><td> 2-3</td><td> KT5</td><td> 16-20</td><td> 5-2.3</td>

<td> 216&lt;3K</td><td> Glu Asp Tyr Pbe(sul)</td><td> 14-20</td><td> 2.4-3.9</td><td> 2</td><td> IHS4 HT4 HEP4</td><td> 14-27</td><td> 3.9-2.8</td>

<td> 125</td><td> pGlu Val Tyr</td><td> 13-20</td><td> 2.7-3.6</td><td> 1-2</td><td> IHS3 HT2</td><td> 14-20</td><td> 3.2-2.7</td>

<td> 228-E20</td><td> sul Val2 Gly2 pGlu</td><td> no spher</td><td> —</td><td> —</td><td> IHSO HTO HEPO</td><td> 9-13</td><td> 5-3.4</td>

<td> 228-AB</td><td> sul Val2 Gly2 pGlu</td><td> no spher</td><td> —</td><td> —</td><td> IHSO HTO HEPO</td><td> 16-18</td><td> 3.8-3.3</td>

<td> 177</td><td> Glu Asp Arg</td><td> 14-19</td><td> 5.2-3.9</td><td> 2-3</td><td> IHSO KT2 HEPO</td><td> 14-19</td><td> 5.2-3.9</td>

<td> 118</td><td> Glu Tyr</td><td> 12-14</td><td> 5.2-6.0</td><td> 1-2</td><td> IHS5 HTO HEPO</td><td> 12-19</td><td> 6-4.2</td>

<td> 153</td><td> Glu LysFB Phe</td><td> no spher</td><td> —</td><td> —</td><td></td><td> 8-9</td><td> 9.1-10.2</td>

<td> 131</td><td> Glu2 Lys</td><td> no spher</td><td> —</td><td> —</td><td> IHS1 KTO</td><td> 14-17</td><td> 4.3-3.6</td>

<td> 162</td><td> Glu2 Tyr Phe</td><td> 16-17</td><td> 4.1-3.7</td><td> 2-3</td><td> IHS3 HTO HEP2</td><td> 16-17</td><td> 4.1-3.7</td>

<td> 156</td><td> C-lu4 Lys2 Pbe</td><td> no spher</td><td> —</td><td> —</td><td> IHSO</td><td> 20</td><td> 4.5</td>

<td> 124</td><td> Glu Val Tyr</td><td> no spher</td><td> —</td><td> —</td><td> IHS4 HT3</td><td> 13-14</td><td> 3.7-3.S</td>

<td> 21O1K</td><td> Glu Asp Lys Val ¹</td><td> 12-20</td><td> 3.6-3.1</td><td> 1-2</td><td> IHS4 HT4 HEP3</td><td> 12-19</td><td> 3.6-3.2</td>

<td> 156</td><td> Glu4 Lys2 Phe</td><td> 14-17</td><td> 3.6-3.1</td><td> 2</td><td> IHSO</td><td> 14</td><td> 3.6</td>

<td> 231</td><td> Glu Lys Phe sul</td><td> no spher</td><td> —</td><td> —</td><td> IHS2 KTO HEP3</td><td> IS</td><td> 9.1</td>

<td> 232</td><td> Glu Leu Lys</td><td> no spher</td><td> —</td><td> —</td><td> IHSO HTO BEP2</td><td> 6</td><td> 10.9</td>

<td> 233</td><td> Glu Leu Arg</td><td> no spber</td><td> —</td><td> —</td><td> IHSO HTO HEP2</td><td> 9</td><td> 10</td>

<td> blank</td><td> 2&lt; anpholytes</td><td> —</td><td> —</td><td> —</td><td> —</td><td> —</td><td> —</td>

<td> 216&lt;3k</td><td> Glu Asp sul Tyr Phe</td><td> 14-20</td><td> 4.1-2.6</td><td> 2</td><td> IHS4 HT4 HEP4 -</td><td> 18-20</td><td> 3-2.6</td>

<td> 230&gt;lk</td><td> Glu Tyr</td><td> 13-20</td><td> 3.9-3</td><td> 3</td><td> IHS4 HT4 HEPO</td><td> 15-20</td><td> 3.3-3</td>

<td> 170a</td><td> Glu Asp LysFB</td><td> 15-20</td><td> 3.9-2.2</td><td> 2</td><td> HT5</td><td> 18-19</td><td> 3.3-2.6</td>

<td> 236-cp</td><td> Glu Tyr Lys-HCl</td><td> 19-20</td><td> 3.0</td><td> 0-1</td><td> IKS2 HTO HEPO</td><td> 19-20</td><td> 3</td>

<td> 216&lt;3k</td><td> Glu Asp sul TyrPhe *</td><td> 14-20</td><td> 3.3-2.2</td><td> 2</td><td> IHS4 HT4 HEP4</td><td> 20</td><td> 2.2</td>

<td> 216&lt;3k</td><td> GLu Asp sul Tyr Phe</td><td> 16-20</td><td> 3.9-2.3</td><td> 1-2</td><td> IKS4 KT4 HEP4</td><td> 20</td><td> 2-3</td>

<td> 237-cp</td><td> Glu2 Leu2 Lys2 Asp</td><td> no spber</td><td> —</td><td> —</td><td> IHSO HTO· HEPO</td><td> 9-12</td><td> 4.6-4</td>

<td> 243-cs</td><td> pGluArg2Lys2LeuAsp</td><td> no spher</td><td> —</td><td> —</td><td> IHSO KTO HEPO</td><td> 15-17</td><td> 8.5-7</td>

<td> 246-cs</td><td> Glu2 LysH2 Phe2 Asp</td><td> 17-20</td><td> 4.1-2.2</td><td> 2-3</td><td> IHS4 HT4 HEP4</td><td> 17-20</td><td> 4.1-2.2</td>

<td> 250-cs</td><td> pGluArg2LysH2LeuAsp2</td><td> no spher</td><td> —</td><td> —</td><td> IHSO HTO HEPO</td><td> 11-14</td><td> 8-6.8 ·</td>

<td> 249&lt;3K</td><td> Glu2 Leu2 LysH2 pGlu</td><td> no spher</td><td> ---</td><td> —</td><td> IHSO HTO HEPO</td><td></td><td></td>

<td> 254-cp</td><td> GluAspsulTyrPheOm. 5</td><td> 18</td><td> 5.4-2.5</td><td> 2</td><td> IHS3 HT4 HEP4</td><td> 14-20</td><td> 3.7-2.1</td>

<td> 253-cp</td><td> Glu Asp sul Tyr Phe</td><td> 18-20</td><td> 3-4</td><td> 2</td><td> IHSO HTO HEPO</td><td> 1-4,19-20</td><td> 11.5,3-3.5</td>

<td> 235&lt;3k</td><td> Gl uAspsulTyrlOPbelO</td><td> 18-20P</td><td> 2.6-3.2</td><td> 1-2</td><td> IHSO HTO HEPO</td><td> 1-3</td><td> 11</td>

<td> 256-cp</td><td> Glu2 LysH2 Phe2 pGlu</td><td> 13-20</td><td> 3.7-4.0</td><td> 1-2</td><td> IHSO HTO HEPO</td><td> 2-5,14-20</td><td> 2.7-4,7-10</td>

<td> 238&lt;3K</td><td> C-luAspsulTyr5Phe5</td><td> no .spher</td><td> —</td><td> —</td><td> IHS1 HT3 HEP4</td><td> 2</td><td> 11.4</td>

<td> 255-CP</td><td> Glu AspTyrsulPheOrn</td><td> 13-20</td><td> 5.3-3.1</td><td> 2</td><td> IHS1 KT3 HEP4</td><td> 1-6,19-20</td><td> 11-9.3.6-3</td>

<td> 25K3R</td><td> Glu2Asp2sulTyr5Phe5</td><td> 16-20</td><td> 5.5-3.3</td><td> 2</td><td> IHS4 HT4 ΕΓΕΡ4</td><td> 17-20</td><td> 5.8-3.8</td>

PCT/US93/05723

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ADoennix B oaoe 2 of 2

TEF TABLES

Proteinoid sorting, pRa and coDposition Cheaical basis for aicrosphere ODS

Run Ho. Material ID Ho. Coaposition Sphere frac Sphere pH Sphere IEF Sphere Wax (JV Max ov

<td colspan="2"></td><td> —&gt;(*no^ap)</td><td> nuEber</td><td> range</td><td> rating</td><td> Hatrix rating</td><td> Frac.Ho.</td><td> PH -</td>

<td> 47</td><td> 257-CP</td><td> Glu Asp ArgH OmB</td><td> no spher</td><td></td><td></td><td> IKSO HTO HEPO</td><td> 19-20</td><td> 5-3</td>

<td> 48</td><td> 257-CP</td><td> Glu Asp ArgH OmB*</td><td> no spher</td><td> —</td><td> —</td><td> IHSO HTO HEPO</td><td> 15-17</td><td> 8.9-8.5</td>

<td> 49</td><td> 258&lt;3K</td><td> Glu Asp ArgH</td><td> no spher</td><td> —</td><td> —</td><td> IHSO HTO HEPO</td><td> 1,17-20</td><td> 9.8,4-2.5</td>

<td> 50</td><td> 262-CP</td><td> Glu Ora Asp LysFB</td><td> 11-18</td><td> 6.8-3.5</td><td> 2</td><td> IHSO KT1 HEP4</td><td> 15</td><td> 4.8</td>

<td> 51</td><td> 262-FILT</td><td> Glu Ora Asp LysFB</td><td> 4-11</td><td> 7.7-5.4</td><td> 1-2</td><td> insO atl hep4</td><td> 1-2,12-20</td><td> 9.4.6-1.8</td>

<td> 52</td><td> 267-cp</td><td> Glu LysFB Asp LysFB</td><td> no spher</td><td> —</td><td> —</td><td> IHSa HTc HEPc</td><td> 14-20</td><td> 6.3-3.8</td>

<td> x3</td><td> 268-cp C</td><td> Glu Asp sul Tyr Phe</td><td> 15-20</td><td> 4.5-2.34</td><td> 2-3</td><td> IHS4oHT4oHEP4a</td><td colspan="2"> 1-10,18-20 12-2.5</td>

<td> 4</td><td> 269-cp</td><td> Glu OmH Asp LysFB</td><td> 17-20</td><td> 2/91-1.4</td><td> 1</td><td> IHSc HTc HEPc</td><td> 4,7,9</td><td> 9-7.5</td>

<td> 5</td><td> 273-cp</td><td> Glu Leu LysB Phe</td><td> 17-20</td><td> 3-1.2</td><td> 1-2</td><td> IHS2aHT2 HEP2a</td><td> 19</td><td> 2</td>

<td> 6</td><td> 272/273</td><td> Glu Leu LysB</td><td> no spher</td><td> —</td><td> —</td><td> —</td><td> 3-9,13-15</td><td> 9.8.5,8-8</td>

<td> 7</td><td> 276</td><td> Glu Leu Arg Phe</td><td> 12-18</td><td> 3.57-1.4</td><td> 1-2,2</td><td> IHS2 HT2 HEP3</td><td> 1-7,17-20</td><td> 9-6,1.5-1</td>

<td> &gt;8</td><td> 27.4</td><td> Glu Leu Arg Tyr</td><td> no spher</td><td> —</td><td> —</td><td> IHSc HTc Hepc</td><td> 16-20</td><td> 4.14-1.4</td>

<td> 9</td><td> 272</td><td> Glu Leu LysB Tyr</td><td> no spher</td><td> —</td><td> —</td><td> IHSc HTC HEPc</td><td> 1-2</td><td> 9.4-9.3</td>

<td> &gt;0</td><td> 274A.</td><td> Glu Leu Arg</td><td> no spher</td><td> —</td><td> —</td><td> —</td><td> all frac.</td><td> —</td>

<td> .1</td><td> 278</td><td> Glu Lys Phe sul</td><td> 16-20</td><td> 4.8-3.5</td><td> 1-2</td><td> IHSc HTc HEP4</td><td> all frac.</td><td> —</td>

<td> .2</td><td> 284E</td><td> GluAspTyrPhesulOra</td><td> 14-20</td><td> 3.8-2.1</td><td> 1-2</td><td> IHS4oHT4oHEP3</td><td> 15-20</td><td> 3.3-2.1</td>

<td> •3</td><td> 287-cp</td><td> Glu Phe</td><td> 10-20</td><td> 3.55-2.3</td><td> 2</td><td> IHS3 HT2 HEP3</td><td> 18-20,1-7</td><td> 2.4-2.3,8</td>

<td> 1 1</td><td> 284-E</td><td> Glu2Asp2Tyr2Phe20m</td><td> 14-20</td><td> 3.95-1.6</td><td> 2 v/oil</td><td> IHS4a KT4aHEP2a</td><td> 3-8</td><td> 12.3-7.76</td>

<td> 5</td><td> 288-cp</td><td> Glu Ora Phe</td><td> no spher</td><td> —</td><td> —</td><td> IHSc HTc HEPc</td><td> 17-20</td><td> 2.7-1.02</td>

<td> 6</td><td> 293-cp</td><td> Glu Asp sul Tyr Phe</td><td> 1-8</td><td> 1.9-3.9</td><td> 1-2</td><td> IHS1 HT2 HEPla</td><td></td><td></td>

<td> 7</td><td> 290-cp</td><td> Glu Arg Phe</td><td> 1-7</td><td> 1.05-3.8</td><td> 1-2</td><td> IHS1 HT1 HEP1</td><td> 18-20</td><td> 12.1-12.6</td>

<td> 8</td><td> 292-cp</td><td> Glu Asp Arg</td><td> no spher</td><td> —</td><td> —</td><td> IKS HT HEP</td><td> 16-20</td><td> 3.19-1.5</td>

<td> 9</td><td> 300-cp</td><td> Glu Ora Asp Lys Phe</td><td> 15-19</td><td> 4.05-1.5</td><td> 1-2</td><td> IHS3 HT3 HEP3?</td><td> all frac.</td><td> —</td>

<td> 0</td><td> 297-cp</td><td> GLU ASP SUL TOR PHE</td><td> 1-7</td><td> 2.38-4.15</td><td> 2-3</td><td> IHS2 HT4 HEP</td><td> 1-2</td><td> 2.38-2</td>

<td> 1</td><td> 301&lt;3K</td><td> GLU ASP SUL TYR PHE</td><td> —</td><td> —</td><td> —</td><td> IHS4 HT2 HEP3</td><td> 1-20</td><td> —</td>

<td> 2</td><td> 303</td><td> GLU ASP SUL TYR PBE</td><td> 1-8</td><td> 2.83-3.76</td><td> 2-3</td><td> IHS4 HT2 HEP3a</td><td> 1-3,18-20</td><td> 2.88/1</td>

<td> 3</td><td> 299</td><td> GLU2 LYS2 PHE2 ASP</td><td> 1-7</td><td> 1.13-3.82</td><td> 1</td><td> IKS4a HT't HEP2a</td><td> 3-7</td><td> 1.58-3</td>

<td> 4</td><td> 305</td><td> PGLU ASP.5 TOR PHE</td><td> 1-12</td><td> 2.12-4.20</td><td> 2-3</td><td> IHS3 HT2 HEP3</td><td> 11-20</td><td> 5.54-1</td>

<td> 5</td><td> 307-CP</td><td> GLH ASP TYR PHE</td><td> 1-9</td><td> 2.43-4. ‘3</td><td> 2-3</td><td> IHS4O HT4 HEP4a</td><td> 1-13</td><td> 2.43-7.0</td>

<td> 6</td><td> 305</td><td> PGLU ASP.5 TYR PHE</td><td> 1*6</td><td> 2.05-5.56</td><td> 2-3</td><td> IHS3 HT2 HEP3</td><td> 4-7</td><td> 3.3-7.0</td>

<td> 7</td><td> 124/156</td><td> GLU TYR VAL/GLU2 LYS2 PHE</td><td> —</td><td> —</td><td> —</td><td> 14 H3 H3/I0H0B0</td><td> 1</td><td> 10.58</td>

<td> 3</td><td> 223-CP</td><td> LYS ARG LEU PGLU</td><td></td><td></td><td></td><td> IHSO KTO HEP2</td><td></td><td></td>

<td> )</td><td> 319-CP</td><td> SUL-U TYR PHE ASP PGLU</td><td> 1-10</td><td> 2.28-5.3</td><td> 2-3o</td><td> IHS4aHT4hEP4a</td><td> 2-8,19-20</td><td> 2.3-4,12</td>

<td> )</td><td> 314—CP</td><td> SUL TYR PHE ASP PGLU</td><td> 1-11</td><td> 1.93-5.30</td><td> 2</td><td> IHS2aHT4aBEP4a</td><td> 18</td><td> 8.95</td>

<td> .</td><td> 320-CP</td><td> SOL TOR PHE PGLU ASP</td><td> 1-7</td><td> 2.12-4.4</td><td> 2a</td><td> IHS4aHT4HEP4</td><td> 16-20</td><td> 9.3-10.25</td>

<td> )</td><td> 188&gt;6K</td><td> ASP2 TYR PHE</td><td> 1-6</td><td> 1.85-5</td><td> Oa</td><td> IHSOHTOHEPO</td><td> 18-20</td><td> 12-12.5</td>

<td> (</td><td> 286-CP</td><td> GLU ASP</td><td> 14-18</td><td> 2.38-2.02</td><td> PARTICLES</td><td> IHSCHTCHEPC</td><td> 14-18</td><td> 2.38</td>

<td></td><td> 288/188</td><td> ASP2 TYR PHE/GLU ORH PHE</td><td> 14-17</td><td> 4.9-3.1</td><td> 1-2 '</td><td> IHSCHTCHEPC</td><td> 17-19</td><td> 3.1-1.65</td>

<td> 1</td><td> 66</td><td> GLU2 GLY</td><td> —</td><td> —</td><td> —</td><td> •HTO</td><td> 16-19</td><td> 2.85-2.55</td>

<td> •</td><td> O112-2A.</td><td> GLU ASP TYR PBE</td><td> 1-10</td><td> 3.17-13.48</td><td> 1,0-1</td><td> IHS5aHT3aBEP3a</td><td> 14,2</td><td> 9.20,3.32</td>

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Sphere Testing of Externally Prepared Proteinoids

Appendix C Page 1 of 3

<td> SOI Ko.</td><td> Cotpostion pB</td><td colspan="2"> 0.85 CA 51 AA</td><td> 0.85 CA 4GH</td><td> 51 AA +GK</td><td> INS/CA GH/GL/CD</td><td> IHS/AA GH/GL/&lt;</td><td> EEP/CA 3D 1 GH</td>

<td> Si£HJPOOlF2OBL5A0°</td><td> GLO ASP TYR HE -</td><td> • Rating 1-2</td><td> 1-2</td><td> 3</td><td> 2-3</td><td> 2-3</td><td> 0</td><td> 2-3</td>

<td></td><td></td><td> Desc. a</td><td> a</td><td> —</td><td> a</td><td> a</td><td> a</td><td> a</td>

<td></td><td></td><td> pB —-</td><td> —----</td><td> .....</td><td> ---</td><td> —</td><td> —</td><td> —</td>

<td> 91EHIP001F21B1SA07</td><td> GLU ASP TYR PEE ---</td><td> Rating 2-3</td><td> 2-3</td><td> 3-4</td><td> 3-4</td><td> 2-3</td><td> 4</td><td> 4-5</td>

<td></td><td></td><td> Desc. a,ag</td><td> a,ag</td><td> •a</td><td> a</td><td> a,p</td><td> a,ag</td><td> a,p</td>

<td></td><td></td><td> pB--</td><td></td><td> — — -</td><td></td><td> ——</td><td> -------</td><td> -.</td>

<td> 91EMP00ir21BlSA07</td><td> GLU ASP TYR PHE --</td><td> Rating 3</td><td> ' 3</td><td> 4-5</td><td> 3</td><td> 3-4</td><td> 3-4 '</td><td> 4-5</td>

<td></td><td></td><td> Desc. ag,a</td><td> a</td><td> a</td><td> ag,p</td><td> ag,p·</td><td> ag,p</td><td> a,p</td>

<td></td><td></td><td> pH ---</td><td> ——</td><td>¹</td><td></td><td> —-</td><td></td><td> —.—</td>

<td> 91EKIP001F22B1SA.7</td><td> GLU ASP TYR PEE --</td><td> Rating 2-3</td><td> 2-3</td><td> 3</td><td> 4-5</td><td> 0</td><td> 3-4</td><td> 4-5</td>

<td></td><td></td><td> Desc. -—</td><td> a</td><td> —</td><td> a,o</td><td> a</td><td> 0</td><td> a</td>

<td></td><td></td><td> pB--</td><td></td><td> —</td><td> —-</td><td> —</td><td> —</td><td> —</td>

<td> 91EHIP011F23B1SA8</td><td> GLU ASP TYR PEE ORH--</td><td> Rating 2-3</td><td> 3</td><td> 4</td><td> 4</td><td> 0-1</td><td> 4</td><td> 3-4</td>

<td></td><td></td><td> Desc. a,p</td><td> a</td><td> —</td><td> —</td><td> a</td><td> a,o</td><td> a,o</td>

<td></td><td></td><td> pB ---</td><td> “--</td><td> —</td><td> ——</td><td> ----</td><td> —</td><td> —</td>

<td> 91EKIP011F24SA7</td><td> GLU ASP TYR PEE ORH ---</td><td> Rating 2</td><td> 2</td><td> 2-3</td><td> 3</td><td> 4</td><td> 0-1</td><td> 5</td>

<td></td><td></td><td> Desc. a</td><td> a</td><td> 0</td><td> —</td><td> a</td><td> a,o</td><td> P</td>

<td></td><td></td><td> pH--</td><td> __</td><td></td><td> —</td><td> —-</td><td> —</td><td> —</td>

<td> 91EHIP001F25B1SA2A</td><td> GLO ASP TYR PEE ----</td><td> Rating 0-1</td><td> 0</td><td> 3-4</td><td> 0</td><td> 3-4</td><td> 4-5</td><td> 4-5</td>

<td></td><td></td><td> Desc. ---</td><td> a,p</td><td> a</td><td> a</td><td> a</td><td> a</td><td> a</td>

<td></td><td></td><td> pH ---</td><td> —</td><td> —</td><td> —</td><td> —</td><td> —</td><td> —</td>

<td> 91EHIP001F25B1SA5</td><td> GLU ASP TYR PEE ---</td><td> Rating 0-1</td><td> 1</td><td> 2-3</td><td> 2-3</td><td> 0-1</td><td> 3-4</td><td> 2-3</td>

<td></td><td></td><td> Desc. a</td><td> a</td><td> a</td><td> —</td><td> a</td><td> a,o</td><td> a</td>

<td></td><td></td><td> pE--</td><td> ---</td><td> -----</td><td> —</td><td> —-</td><td> —</td><td> —</td>

<td> 91DiTP001F26BlSA2A</td><td> GLU ASP TYR PHE -</td><td> Rating 2-3</td><td> 2-3</td><td> 3</td><td> 3-4</td><td> 2</td><td> 2-3</td><td> 3-4</td>

<td></td><td></td><td> Desc. --</td><td> a</td><td> —</td><td> —</td><td> a</td><td> —</td><td> 0</td>

<td></td><td></td><td> pH · -</td><td> —</td><td> ---¹¹</td><td> ----‘</td><td> —</td><td></td><td> •</td>

<td></td><td></td><td> Rating</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> Desc.</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> pB</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> CA= citric acid</td><td> INS=</td><td colspan="2"> insulin</td><td></td><td></td><td></td><td></td>

<td></td><td> ¢3-1= gum acacia</td><td> HEP =</td><td colspan="2"> = heparin</td><td></td><td></td><td></td><td></td>

<td></td><td> GL= gelatin '</td><td> CD =</td><td colspan="2"> cyclodextri:</td><td> n</td><td></td><td></td><td></td>

<td></td><td> a= amorphous</td><td> o =</td><td> oil</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> p= particulate</td><td> ag=</td><td colspan="2"> aggregate</td><td></td><td></td><td></td><td></td>

O— i.irhrq f = best.

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Sphere Testing of External)y I'repared Proteinoid:

ADpendix c pace 2 of 3

<td> SOI Ho.</td><td> Conpostion</td><td> pB</td><td colspan="2"> 0.85 CA 51 AA</td><td colspan="2"> 0.85 CA 51 λλ 4GH +GH</td><td> IHS/CA GH/GL/CD</td><td colspan="2"> IHS/AA HEP/CA GH/GL/CD 4 GH</td>

<td> 91CTAPOO1FO14BO2</td><td> GLO ASP TYR PHE</td><td></td><td> Rating 2-3</td><td> 3</td><td> 5</td><td> 4-5</td><td> 4-5</td><td> 2-3</td><td> 3 ·</td>

<td></td><td></td><td></td><td> Desc. a</td><td> a</td><td> a</td><td> —</td><td> a</td><td> a</td><td> a</td>

<td></td><td></td><td></td><td> pH ----</td><td> ——</td><td> ——</td><td></td><td></td><td> —-</td><td> —</td>

<td> 91CTAPOO1FO14BO2</td><td> GLU ASP TYR PHE</td><td> —</td><td> Rating 2</td><td> 4-5</td><td> 5</td><td> 5</td><td> 5</td><td> 5</td><td> 5</td>

<td></td><td></td><td></td><td> Desc. ----</td><td> a</td><td> —</td><td> —</td><td> —</td><td> —</td><td> —</td>

<td></td><td></td><td></td><td> pH -</td><td> --.</td><td></td><td></td><td> ' ¹</td><td> *'—</td><td> —</td>

<td> 91CTAP001F014B02</td><td> GLU ASP TYR PHE</td><td> —</td><td> Rating 2-3 ’</td><td> 3</td><td> 5</td><td> 4-5</td><td> 4-5</td><td> 2-3</td><td> 3</td>

<td></td><td></td><td></td><td> Desc. a</td><td> a</td><td> a</td><td> —</td><td> a</td><td> a</td><td> a</td>

<td></td><td></td><td></td><td> pH--</td><td> ——</td><td> ——</td><td> ——</td><td> —-</td><td> -^,n '· ·</td><td></td>

<td> 91CTAP001F014B03</td><td> GLU ASP TYR PHE</td><td> —</td><td> Rating 2</td><td> 2</td><td> 3-4</td><td> 3-4</td><td> 5</td><td> 3</td><td> 3-4</td>

<td></td><td></td><td></td><td> Desc. a</td><td> a</td><td> a</td><td> a</td><td> a</td><td> a</td><td> a</td>

<td></td><td></td><td></td><td> pH ---</td><td></td><td> —</td><td> ——</td><td> - —</td><td> ““—</td><td> —</td>

<td> 91CTAPOOirO14BG3</td><td> GLU ASP TYR PHE</td><td> —</td><td> Rating 0-1</td><td> 0-1</td><td> 5</td><td> 5</td><td> 5</td><td> 2-3</td><td> 4</td>

<td></td><td></td><td></td><td> Desc. a,ag</td><td> a,ag</td><td> a,ag</td><td> a,ag</td><td> a,ag</td><td> a</td><td> —</td>

<td></td><td></td><td></td><td> pH ---</td><td></td><td> ——</td><td> “ ¹</td><td> ———</td><td> ——</td><td> ——</td>

<td> 91CTAP001F014B03</td><td> GLU ASP TYR PHE</td><td> —</td><td> Rating 2-3</td><td> 2-3</td><td> 5</td><td> 5</td><td> 5</td><td> 3</td><td> 5</td>

<td></td><td></td><td></td><td> Desc. aa</td><td> ag</td><td> a,ag</td><td> —</td><td> a,ag,o</td><td> ag,a</td><td> —</td>

<td></td><td></td><td></td><td> pB--</td><td> ——</td><td></td><td></td><td> —</td><td>¹ -</td><td> ———-</td>

<td> 91CTAP001F014B03</td><td> GLU ASP TYR PHE</td><td> —</td><td> Rating 2</td><td> 2</td><td> 3-4</td><td> 3-4</td><td> 5</td><td> 3</td><td> 3-4</td>

<td></td><td></td><td></td><td> Desc. a</td><td> a</td><td> a</td><td> a</td><td> a</td><td> a</td><td> a</td>

<td></td><td></td><td></td><td> pH ----</td><td> —</td><td></td><td> ——</td><td></td><td></td><td> ----</td>

<td> 91CTAPOOIF014EO4</td><td> GLU ASP TYR PHE</td><td> ——</td><td> Rating 0-1</td><td> 0-1</td><td> 4</td><td> 5</td><td> 2-3</td><td> 0</td><td> 5</td>

<td></td><td></td><td></td><td> Desc. a</td><td> a</td><td> a</td><td> a</td><td> a</td><td> a</td><td> c ·</td>

<td></td><td></td><td></td><td> PH —*</td><td> —— .</td><td> ——</td><td></td><td></td><td></td><td></td>

<td> 91CTAP001F014B05</td><td> GLU ASP TYR PHE</td><td> —</td><td> Rating 3</td><td> 3-4</td><td> 5</td><td> 4-5</td><td> 4</td><td> 4</td><td> 4</td>

<td></td><td></td><td></td><td> Desc. --</td><td> a</td><td> a</td><td> —</td><td> a</td><td> a</td><td> a</td>

<td></td><td></td><td></td><td> pH -</td><td> -----</td><td> ——</td><td></td><td> —-</td><td> ——</td><td> ——</td>

<td> 91CTAP001F014B04</td><td> GLU ASP TYR PHE</td><td> -___</td><td> Rating 3</td><td> 3-4 .</td><td> 5</td><td> 4-5</td><td> 4</td><td> 4</td><td> 4</td>

<td></td><td></td><td></td><td> Desc. ----</td><td> a</td><td> a</td><td> —</td><td> a</td><td> a</td><td> a</td>

<td></td><td></td><td></td><td> pH ---</td><td> ——</td><td></td><td> ----</td><td></td><td> ----</td><td> ----</td>

<td> 91EHIP011F15B1</td><td> GL02ASP2TYR2PHE20RH</td><td> —</td><td> Rating 2-3</td><td> 3 ·</td><td> 4</td><td> . 3-4</td><td> 0</td><td> 3-4</td><td> • 4</td>

<td></td><td></td><td></td><td> Desc. a,ag</td><td> ag</td><td> —</td><td> a,o</td><td> a</td><td> —</td><td> ---</td>

<td></td><td></td><td></td><td> pH--</td><td> ——</td><td></td><td> ”· '</td><td></td><td> -</td><td> ·</td>

<td> 91EHIP001F20B1SA07</td><td> GLU ASP TYR PHE</td><td> -—</td><td> Rating 2</td><td> 2-3</td><td> 3</td><td> 2-3</td><td> 4</td><td> 3</td><td> 4</td>

<td></td><td></td><td></td><td> Desc. ag</td><td> ag</td><td> ---</td><td> -----</td><td> a,o</td><td> a,o</td><td> —</td>

<td></td><td></td><td></td><td> pB ---</td><td> —</td><td> —</td><td> —.......</td><td> —</td><td> —</td><td> ——</td>

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Sphere Testing ol Externally Prepared Proteinojcs

SOI Ho.

Coapostion pB

Appendix c page 3 of :

0.85 a 51 A3. 0.85 CA 51 AA. IHS/CA IHS/AJ. HEP/CA 4GH +GH GH/GL/CD GH/GL/CD 4 GH

Rating

Desc.

PH

<td> 91D(lP00IF25BlSA3a</td><td></td><td> 6.52 Rating 2-3</td><td> 2-3</td><td> 3-4</td><td> 4</td><td> 3-4</td><td> 4</td><td> 5</td>

<td></td><td></td><td> Desc. ag,a</td><td> ag</td><td> —</td><td> —</td><td> a,p</td><td> 0</td><td> —</td>

<td></td><td></td><td> pB -</td><td> ——</td><td>¹¹</td><td> - </td><td></td><td> —</td><td> —</td>

<td> 91CTAP001P012B01</td><td> GLO ASP TYR PHE</td><td> 7.5 Rating 2-3</td><td> 2</td><td> 5</td><td> 5 ·</td><td> 4</td><td> 5</td><td> 5</td>

<td></td><td></td><td> Desc. a</td><td> a</td><td> —</td><td> —</td><td> a</td><td> —</td><td> a</td>

<td></td><td></td><td> pH -</td><td> —</td><td> -—</td><td> —</td><td> ---</td><td> —</td><td> — -</td>

<td> F005-B01</td><td> GL02 LYSH2 PHE2 ASP</td><td> 9.0 Rating -</td><td> —</td><td> 3-1</td><td> —</td><td> 4</td><td> 3</td><td> 3</td>

<td></td><td></td><td> Desc. ---</td><td> -—</td><td> a,o</td><td> —</td><td> a,o</td><td> a,o</td><td> a,o</td>

<td></td><td></td><td> pH--</td><td> —,—</td><td> —</td><td> —</td><td> -—</td><td> —</td><td></td>

<td> 91CTAPR001F010B01</td><td> GL02 LYSH2 PHE2 ASP</td><td> — Rating 2-3</td><td> 2</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td>

<td></td><td></td><td> Desc. a</td><td> a</td><td> —</td><td> ——</td><td> a</td><td> a</td><td> a</td>

<td></td><td></td><td> pH -</td><td> —</td><td> -—</td><td> —</td><td> —</td><td> —</td><td> —--</td>

<td> Γ003-Β01</td><td> GL02 LYSH2 PSE2 ASP</td><td> -- Rating --</td><td> —</td><td> 4</td><td> 3-4</td><td> 3</td><td> 4-5</td><td> 3</td>

<td></td><td></td><td> Desc. ---</td><td> —</td><td> a</td><td> 0</td><td> a</td><td> a</td><td> a,o</td>

<td></td><td></td><td> pH -</td><td> —</td><td> —</td><td> — </td><td> _—</td><td> —</td><td> ---</td>

<td> F004-B01</td><td> GL02 LYSH2 PHE2 ASP</td><td> — Rating —</td><td> —</td><td> 3-4</td><td> 2-3</td><td> 2-3</td><td> 4</td><td> 2-3</td>

<td></td><td></td><td> Desc. —</td><td> —</td><td> a,o</td><td> a</td><td> a</td><td> a</td><td> a</td>

<td></td><td></td><td> pH ---</td><td> —</td><td> —</td><td></td><td> —</td><td> —</td><td> ---</td>

<td> 91CTAP001F011B01</td><td> GLO ASP TYR PHE ORH</td><td> — Rating -</td><td> —</td><td> —</td><td> —</td><td> —</td><td> —</td><td> —</td>

<td></td><td></td><td> Desc. ---</td><td> —</td><td> —</td><td> —</td><td> —</td><td> —</td><td> ---</td>

<td></td><td></td><td> pB -</td><td> - ......</td><td></td><td></td><td> —</td><td> —</td><td> — '</td>

<td> 91CTAP001F013B01</td><td> GLO ASP TYR PHE</td><td> 7.9 Rating 2-3</td><td> 2-3</td><td> 4-5</td><td> 4-5</td><td> 4-5</td><td> 4</td><td> 5</td>

<td></td><td></td><td> Desc. -</td><td> a</td><td> —</td><td> a,o</td><td> a,o</td><td> a,o</td><td> a</td>

<td></td><td></td><td> pH -</td><td> -—</td><td>¹</td><td> ----</td><td> —</td><td> —</td><td></td>

<td> 91CTAPOO1FO14BO1</td><td> GLO ASP TYR PHE</td><td> 8.0 Rating 0</td><td> 0-1</td><td> 4-5</td><td> 4-5</td><td> 4-5</td><td> 3-4</td><td> 4</td>

<td></td><td></td><td> Desc. a</td><td> a</td><td> a</td><td> —</td><td> a</td><td> a</td><td> a</td>

<td></td><td></td><td> pH -</td><td></td><td>¹</td><td></td><td> —</td><td> —</td><td> —-</td>

<td> 91CTAP001F014B01</td><td> GLO ASP TYR PHE</td><td> — Rating 0-1</td><td> 0-1</td><td> 5</td><td> 4</td><td> 4</td><td> 3</td><td> 3-4</td>

<td></td><td></td><td> Desc. a</td><td> a</td><td> a,o</td><td> a,o</td><td> a,o</td><td> a,o</td><td> a,o</td>

<td></td><td></td><td> pB -</td><td> —</td><td> —</td><td></td><td> —</td><td> —</td><td> —</td>

<td> 91CTAP001F014B01</td><td> GLO ASP TYR PHE</td><td> - Rating 2</td><td> 2</td><td> 5</td><td> 5</td><td> 5</td><td> 3</td><td> 5</td>

<td></td><td></td><td> - Desc. ag</td><td> ag,a</td><td> ag</td><td> —</td><td> ag,a</td><td> ag,a</td><td> —</td>

<td></td><td></td><td> pH -</td><td> —</td><td> —</td><td> —</td><td> —.</td><td> —</td><td> —</td>

93/25583

PCT/US93/05723