Crystalline anti-human IL-12 antibodies

(54) Title: ANTIBODIES IL-12 ANTI-HUMAN (57) Summary:

CRYSTALLINES (30) Unionist Priority: 29/03/2007 us 60 / 920,608 (73) Holder (s): Abbott Laboratories (72) Inventor (s): Anette Koenigsdorfer, David W. Borhani, Gerhard Winter, Hans-Juergen Krause, Stefan Gottschalk, Wolfgang Fraunhofer (74) Attorney (s): Nellie Anne Daniel-Shores (86) International Application: pct 11Ξ2008004006 of 27/03/2008 (87) International Publication: wo 2oos / i2i3oide 09/10/2008

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IL-12 ANTI-HUMAN CRYSTALLINE ANTIBODIES ”

CROSS REFERENCE TO RELATED ORDERS

This order claims priority over US Provisional Order serial number 60 / 920,608, filed on March 29, 2007.

FIELD OF THE INVENTION

The present invention relates to a batch crystallization method for crystallizing an antibody, which allows the production of the antibody on an industrial scale; antibody crystals, specifically as obtained according to the disclosed method; and compositions containing the crystals, as well as methods of using the crystals and compositions.

BACKGROUND OF THE INVENTION

a) Antibody Crystals

The mAb market is considered to be one of the most promising biopharmaceutical markets, with around 100 monoclonal antibodies (mAbs) currently being evaluated in clinical study phases 2 or 3. Since these groups are released in simple doses often exceeding 100 mg, there is an urgent need to find appropriate formulation strategies that satisfy the stability, safety and suitability of the patient. However, highly concentrated liquid mAb formulations show increased viscosity, which makes it difficult for patients to use thin needle syringes that are less aggressive. In addition, the tendency of mAb molecules to aggregate at such high concentrations increases exponentially when compared to moderately concentrated solutions. This is unacceptable, with regard to security and stability requirements.

Thus, the release of high doses of mAb is reserved for large volumes, which should generally be released through infusion. This dosage mode is expensive and significantly reduces patient suitability.

Therefore, suspensions of low volume mAb crystals, pharmaceutically applicable for subcutaneous injection would be highly desirable. Theoretically, degradation pathways that influence the integrity of mAb would be significantly slowed down due to the rigidity of a crystal grid, where movements in the protein structure are prevented. In addition, an increase in viscosity would be significantly reduced when compared to highly concentrated crystal suspensions with liquid formulations. With regard to prolonged release, it may be possible to generate or alter protein crystals, such that they dissolve slowly when placed in a patient's body. This would be a very effective way to release an extended release formulation, since the extensive use of excipients and processes that damage the mAb structure would not be used.

Despite the great potential for using protein crystals as a drug substance, some attempts have been made to systematically evaluate this strategy.

A well-known example is insulin, which was successively crystallized for decades. Currently, the use of crystal insulin suspensions is well described, offering stable and long-lasting formulations, which are well established in the market. The discrepancy between the development of insulin crystals and the crystallization of all other proteins may be related to the fact that ordered insulin aggregates are formed natively in the pancreas. Thus, insulin crystals are easily obtained when insulin is brought into contact with an excess of zinc ions. Most other proteins tend to form disordered precipitates rather than crystals and, therefore, finding crystallization conditions for a protein is a non-routine and time-consuming task.

Despite great interest in harvesting protein crystals for X-ray diffraction analysis, obtaining appropriate crystallization conditions is still an empirical science, as, in principle, any protein behaves differently. To date, no general rule has been found that can reliably predict a successful crystallization condition for choosing a protein. Thus, obtaining crystals of a given protein is always referred to as "the narrowing" of any intended application that is planned later.

Antibodies are especially difficult to crystallize, due to the flexibility of the molecule. Nevertheless, examples of immunoglobulin crystals have been known for a long time. The first example of immunoglobulin crystals was described 150 years ago by an English physician, Henry Bence Jones; he isolated crystals of an abnormal Ig light chain dimer from the urine of a patient with myeloma (Jones, Η. B. (1848) Philosophical Transactions of the Royal Society, London 138: 55-62). Such abnormal Igs are known as Bence Jones proteins. In 1938, spontaneous crystallization of an abnormal Ig distinct from the serum of a myeloma patient was described (von Bonsdorf, B. et al. (1938) Folia Haematologia 59: 184-208), apparently as an Ig heavy chain oligomer ( MW (molecular weight) of 200 kDa).

Crystalline human immunoglobulins of normal structure (two heavy chains linked to two light chains) have been described in the thirties, again, mostly isolated from myeloma patients (Putnam, FW (1955) Science 122: 275-7). Davies and colleagues were the first to characterize the structure of an intact human myeloma antibody, called “Dob” using x-ray crystallography (Terry, WD and others (1968) Nature 220 (164): 239-41), and determined their three-dimensional structure in 1971 (Sarma, VR et al. (1971) J. Biol. Chem. 246 (11): 3753-9). His pioneering work was followed by others, yielding crystal structures from IgG “Kol” (Huber, R. et al. (1976) Natu3 re 264 (5585): 415-20), to IgG “Mcg” (Rajan, SS et al. (1983) Mol. Immunol. 20 (7): 78799), and a canine lymphoma IgG2a (Harris, LJ et al. (1992 Nature 360 ​​(6402): 369-72).

The immunoglobulin crystals maintain their distinct immunological activities when redissolving. Nisonoff and others reported in 1968 a rabbit anti-pazobenzoate antibody, "X4", which was easily crystallized (Nisonoff, A. et al. (1968) Cold Spring Harbor Symposia on Quantitative Biology 32: 89-93). The X4 antibody was extensively characterized before crystallization, as well as after the crystallization of the crystals. It was found that [¹²⁵l] -p-iodobenzoate binds specifically and potentially to redissolved X4; the redissolved crystals also exhibited multiple specific Ouchterlony immunodiffusion reactions typical of unpurified rabbit serum (Nisonoff et al., 1968). Connell and colleagues described a gamma-immunoglobulin-1-human myeloma layer (IgG-K) called “Tem”, which spontaneously crystallized from serum in cold temperatures (Connell, GE and others (1973) Canad. J. Biochem. 51 ( 8): 1137-41). It was verified that Tem crystals are well formed and have rhombohedral symmetry. Serum containing "Tem" was extensively characterized by agarose immunodiffusion techniques. The electrophoresis and immunodiffusion of a redissolved solution of the “Tem” crystals showed that they are identical to the material obtained from the serum by cryoprecipitation and with the isolated myeloma protein (Connell et al., 1973).

Mills and colleagues reported in 1983 an unusual crystallocrioglobulinemia resulting from human monoclonal antibodies to albumin (Mills, LE et al. (1983) Annals of Internai Med 99 (5): 601-4). Here, very similar cuboid crystals were isolated from two patients. The redissolution of the crystals followed by electrophoresis and immunoelectrophoresis indicated that the crystals were composed of two protein components, a monoclonal IgGlambda and human serum albumin at a ratio of 1: 2 (Jentoft, JE et al. (1982) Biochem. 21 (2 ): 289-294). The components were separated on the preparation scale by dissolving the original crystals, followed by column chromatography. Although no crystallized component has properly separated, when recombining, the original bipartite complex was reformed and then recrystallized. Additional studies of the distinct sedimentation characteristics and immunological reactivity of the redissolved and separated IgG and its Fab fragment with human serum albumin indicated that the reassociation of the two separate redissolved components was of an immunological nature, that is, that the crystalline antibody once redissolved still it had its highly specific binding characteristics (for human serum albumin) (Mills et al. 1983).

Recently, Margolin and colleagues reported the potential therapeutic uses of crystalline antibodies (Yang, MX and others (2003) Proc. Natl. Acad. Sci. 100 (12): 6934-6939). They found that the therapeutic monoclonal antibody trastuzumab (Herceptin (R)) would be crystallized (Shenoy, B. et al. (2002) International PCT Application

WO / 2002/072636, (Altus Biologies Inc., USA), page 173). Crystalline trastuzumab suspensions were therapeutically effective in a mouse tumor model, thus demonstrating retention of biological activity by crystalline trastuzumab (Yang et al., 2003).

b) Crystallization Techniques

Crystallization of several proteins cannot be carried out successively using defined methods or algorithms. Certainly, there have been major technical advances in the past 20-30 years, as noted by world-renowned protein crystallization expert, A. McPherson. McPherson provides extensive details on tactics, strategies, reagents and devices for crystallizing macromolecules. (McPherson, A. (1999) Crystallization of Biological Macromolecules. Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press, p. 159). However, it does not provide a method for ensuring that any macromolecule can, in reality, be crystallized by a person skilled in the art with a reasonable expectation of success. McPherson states, for example: Whatever the procedure, no effort should be made to refine and optimize system parameters, both solvent and suture, to encourage and promote specific binding interactions between molecules and stabilize them once they have if formed. This last aspect of the problem usually depends on the chemical and physical properties of the specific protein or nucleic acid being crystallized ”.

It is widely accepted by those skilled in the protein crystallization technique that there is no algorithm for obtaining a new protein of interest, application of the defined process steps and, therefore, obtaining the desired crystals.

Several classification systems are commercially available (for example, Hampton 1 and 2, Wizzard I and II) that allow, on a micro-liter scale, to classify crystallization conditions potentially appropriate for a specific protein. However, positive results obtained in such a classification system do not necessarily allow successful crystallization on a larger batch scale, industrially applicable. The conversion of crystallization experiments on a micro-liter scale to industrial dimensions is described as a challenging task (see Jen, A., Merkle, Η. P. (2001) Pharm. Res. 18, 11, 1483).

Baldock and others reported a comparison of micro batch and vapor diffusion for initial classification of crystallization conditions (Baldock, P. et al. (1996) J. Crystal Growth 168 (1-4): 170-174. Six commercially available proteins were classified using a set of crystallization solutions. The classifications were carried out using the most common vapor diffusion method and three variants of a micro batch crystallization method including a new evaporation technique. Of the 58 crystallization conditions identified, 43 (74%) were identified by micro batch, while 41 (71%) were identified by vapor diffusion. Twenty-six conditions were verified by both methods and 17 (29%) would have been lost if the micro batch had not been used. This shows that the vapor diffusion technique, which is the most commonly used in the initial crystallization classifications, does not guarantee positive results.

c) IL-12 Anti-Human Antibody Crystals

Human IL-12 plays an important role in pathology associated with various diseases involving immune and inflammatory responses, for example, multiple sclerosis, Crohn's disease and psoriasis. There is, therefore, a great need for appropriate methods for the treatment of such disorders related to human IL-12. A promising therapeutic approach comprises the administration of pharmaceutically effective doses of human IL-12 antibodies.

Due to the role of human IL-12 in a wide variety of human disorders, therapeutic strategies have been designed to inhibit or disable IL-12 activity. Specifically, antibodies that bind and neutralize IL-12 have been sought as a means to inhibit IL-12 activity. Some of the previous antibodies were murine monoclonal antibodies (mAbs), secreted by hybridomas prepared by lymphocytes from mice immunized with IL-12 (see, for example, WO 97/15327). These murine IL-12 antibodies are, however, limited in their use in vivo due to the problems associated with the administration of murine antibodies in humans, such as, short serum half-life and inability to trigger certain human effector functions and promote a unwanted immune response against the mouse antibody in a human (the “anti-mouse antibody in humans” (HAMA) reaction.

In general, attempts to overcome the problems associated with the use of full murine antibodies in humans have involved genetic engineering of the antibodies to be more "human-like". For example, chimeric antibodies have been prepared, in which the variable regions of the antibody chains are derived from murine and the constant regions of the antibody chains are derived from humans. However, since these chimeric and humanized antibodies still maintain some murine sequences, they can still promote an unwanted immune reaction, the human anti-chimeric antibody (HACA) reaction, especially when administered for prolonged periods.

US Patent 6,914,128 discloses human antibodies, preferably recombinant human antibodies, which specifically bind to human interleukin-12 (hlL-12). Preferred antibodies disclosed in the patent have high affinity for hlL-12 and neutralize hlL-12 activity in vitro and in vivo. Antibodies or antibody portions are useful for detecting hlL-12 and for inhibiting hlL-12 activity, for example, in a human individual suffering from a disorder in which hlL-12 activity is harmful. Nucleic acids, vectors and host cells for expression of the recombinant human antibodies of the invention and methods of synthesizing the recombinant human antibodies are also presented. Crystalline forms of anti hlL-12 antibodies or methods of preparing them are not specifically described in the '128 patent.

The problem to be solved according to the present invention, therefore, is to develop appropriate crystallization conditions, specifically batch crystallization conditions, for anti-IL-12 antibodies, and to establish crystallization process conditions applicable to the relevant volumes for industrial production. of the antibody crystal. At the same time, a crystallization process is established, which does not use toxic agents that can negatively affect the pharmaceutical applicability of such antibodies.

SUMMARY OF THE INVENTION

The above-mentioned problem was surprisingly solved by the verification that it is possible to obtain crystals of an integral anti-human IL-12 antibody in batch crystallization volumes above the micro-liter scale, by applying physiologically polyalkylene polyols acceptable as the crystallization inducing agent. In a first aspect, the invention provides a batch crystallization method for crystallizing an anti-human IL-12 antibody, comprising the steps of:

(a) providing an aqueous solution of the IL-12 antibody in admixture with at least one polyalkylene polyol crystallizing agent, as defined in more detail below, for example polyalkylene glycol; for example, by mixing an aqueous solution of the antibody, where the antibody is present, preferably, in dissolved form, with an aqueous crystallization solution comprising at least one polyalkylene glycol as the crystallizing agent in dissolved form, or alternatively, by adding the crystallizing agent in solid form;

(b) and incubation of the aqueous crystallization mixture until antibody crystals are formed.

According to a further embodiment, the method of the present invention can also be performed, such that the crystallization mixture obtained in step a) can be supplemented with an appropriate amount of pre-existing anti-human IL-12 antibody crystals as crystals in seed to initiate or assist crystallization.

The crystallization method of the invention is generally carried out at a pH of the aqueous crystallization mixture in the range of about pH 4 to about 6.5, specifically about 4.5 to about 6.0, about 5.0 to about 5.8 or about 5.3 to about 5.7, such as, for example, 5.4, 5.5 or 5.6.

In addition, the aqueous crystallization mixture can contain at least one buffer. The buffer may comprise an acetate component as a major component, especially an alkali metal salt thereof, for example a sodium or potassium salt, such as sodium acetate. The salt is adjusted by adding an acid, specifically acetic acid, to the required pH. In a preferred embodiment of the crystallization method, the concentration of the buffer (total acetate) in the aqueous crystallization mixture is about 0 to about 0.5 M, or about 0.02 to about 0.5 M, such as, for example, about 0.05 to about 0.3 M, or about 0.07 to about 0.2 M, or about 0.09 to about 0.12 M.

A polyalkylene polyol crystallizing agent is defined in more detail below:

One skilled in the art will recognize that the term is to be understood widely and comprises polyalkylene polyols as well as derivatives thereof.

I0 A polyalkylene polyol as used according to the invention is a polyalkylene polyol C₂-Ç₆ straight or branched chain, specifically straight chain. The polyether is formed from at least one type of a polyfunctional aliphatic alcohol carrying 2 to 6, 2 to 4 and specifically 2 or 3, preferably vicinal groups, hydroxy and having 2 to 6, specifically 2, 3 or 4 carbon atoms, preferably forming a structure of

I5 linear carbon. Non-limiting examples are ethylene-1 2-diol (glycol), propylene-1,2-diol, propylene-1-3-diol, and n-butylene-1,3-diol and n-butylene-1,4-diol. A specifically preferred diol is glycol.

The polyalkylene polyols of the invention can be composed of a simple type of polyol or mixtures of at least two different polyols, which can be randomly polymerized or can be present as block copolymers.

In addition, the term "polyalkylene polyol also includes derivatives thereof. Non-limiting examples are esters and alkyl ethers, specifically monoalkyl ethers and dialkyl ethers. “Alkyl” is defined, specifically as CiC alkyl residue₆straight or branched chain, specifically, methyl, ethyl, n or i-propyl, n, i, sec, or t'5 butyl or i-pentyl; and n-hexyl.

Polyalkylene polyols, specifically polyalkylene glycols, as used according to the invention are further characterized by a wide range of molecular weights. The molecular weight range, declared as average molecular weight by weight or numerical average molecular weight is typically in the range of 400 to 10,000, such as 1,000 to 8,000 or 2,000 to 6,000, 3,000 to 6,000 or 3,200 to 6,000, such as for example, 3,350 to 6,000, 3,350 to 5000 or 3,800 to 4,200, specifically about 4,000.

Specific polyalkylene polyols are polyethylene glycols (PEGs) and polypropylene glycols (PPGs) and corresponding block and random copolymers. Specific examples of suitable polyols are PEG 2,000, PEG 3,000, PEG 3,350, PEG 4,000, PEG 5,000 and PEG

6.000.

Specifically, the concentration of polyalkylene polyol, specifically the concentration of polyethylene glycol, in the crystallization mixture is in the range of about 5 to about

30% (weight / volume), such as about 7 to about 15% (weight / volume) or about 9 to about 16% (weight / volume) or about 10 to about 14% ( weight / volume) or about 11 to about 13% (weight / volume). Preferably, polyethylene glycol with an average molecular weight of about 4,000 is employed at a concentration in the crystallization mixture of about 11 to about 13% (weight / volume).

In a preferred embodiment of the invention, the antibody protein solution and the crystallization solution are combined in a ratio of about 1: 1. Thus, molarities of the buffering agents / crystallizing agents in the original crystallization solution are as high as double in the crystallization mixture.

Typically, the crystallization method is carried out in a batch volume in the range of about 1 mL to about 20,000 L or 1 mL to about 15,000 L or 1 mL to about 12,000 L, or about 1 mL to about 10,000 L, or 1 mL to about 6,000 L or 1 mL to about 3,000 L or 1 mL to about 1,000 L, or 1 mL to about 100 L such as, for example, about 50 mL to about 8,000 mL , or about 100 ml to about 5,000 ml, or about 1,000 ml to about 3,000 ml; or about 1 L to about 1,000 L; or about 10 L to about 500 L.

In addition, the crystallization method of the invention can be performed, so that at least one of the following additional crystallization conditions can be obtained:

a) incubation is carried out between about 1 hour to about 250 days, or 1 to 250 days or 13 to 250 days, for example about 1 to about 30 days, or about 2 to 10 days;

b) incubation is carried out at a temperature between about 0 ° C and about 50 ° C, for example about 4 ° C and about 37 ° C or about 15 ° C and about 25 ° C;

c) the concentration of the antibody (that is, the concentration of the protein) in the crystallization mixture is in the range of about 0.5 to 280 mg / ml or about 1 to 200 mg / ml or 1 to 100 mg / ml, for example 1.5 to 20 mg / ml, specifically in the range of about 2 to 15 mg / ml, or 5 to 10 mg / ml. The protein concentration can be determined according to standard protein determination procedures.

In a preferred embodiment, the crystallization method, for example, with polyethylene glycol as the crystallizing agent, is carried out, such that the incubation is carried out between about 13 to 60 days at a temperature of about 20 ° C and at an antibody concentration of about 5 to 10 mg / ml.

According to a specifically preferred method, crystallization is carried out under the following crystallization mixing conditions:

Polyalkylene glycol: Buffer: pH:

Concentration of anti-hlL-12

PEG 4000 10 to 15% (weight / volume) sodium acetate, 0 to 0.3 M, (total acetate) 5.3 to 5.8 to 10 mg / mL

Temperature: Batch volume: Agitation: Duration:

at 24 ° C to 100 L

None about 1 to 60 days

Crystallization mixtures as mentioned above are generally obtained by adding a crystallizing agent to the solution or as a solid to the protein solution. Both solutions can be, but do not need to be buffered. The concentration of the crystallizing agent and the molarity of the buffer in the original crystallization solution are generally higher in the crystallization mixture, since they are "diluted" with the protein solution.

In a further embodiment, the crystallization method of the invention can additionally comprise the step of drying the obtained crystals. Additional drying methods include evaporative drying, spray drying, lyophilization, vacuum drying, fluid bed drying, spray freeze drying, close to critical drying, supercritical drying and nitrogen gas drying.

In a further embodiment, the crystallization method of the invention may additionally comprise the step of exchanging the main crystallization liquor for a different liquid or a buffered buffer, for example, a liquid or buffer containing a different polyalkylene polyol than that used for crystallization with a molar mass in the range of about 300 to 8,000 Daltons or mixtures of such polyols, for example, by centrifugation, diafiltration, ultrafiltration or other commonly used buffer exchange techniques. The different liquid or buffer can also be designated as an "artificial main liquid" which differs from the "natural" crystallization main liquid of the crystals and prevents the crystals from dissolving.

The present invention also relates to an anti-hlL-12 crystal or antibody that can be obtained by a method of crystallization as defined above and, in general, to the crystals of an anti-hlL-12 antibody.

The crystals of the invention may have a different shape. The shape is often referred to as "swordlike". Specifically, the term also includes "platelets", "needles" or "bunches of needles" (see, similar to sea urchin). For example, the crystals of the invention can be characterized by a needle-like morphology with a maximum length (I) of about 2 - 500 / zm or about 100 - 300 / zm and a length / diameter ratio (l / d) from about 1 to 100. The height of such needle-like crystals is roughly in the diameter dimension.

The platelets of the invention can have the following dimensions: A maximum length (I) of about 2 - 500 µm or about 100 - 300 µm and a length / diameter ratio (l / d) of about 1 to 100. The height of such platelets is considerably less than the diameter.

The bunches of needles of the invention can have the following dimensions. A maximum length I of about 2 - 200 pm or about 10 - 100 pm and a length / diameter (l / d) ratio of about 1 to 3.

The crystal can be obtained from a polyclonal antibody or preferably a monoclonal antibody.

Specifically, the antibody is selected from the group consisting of non-chimeric or chimeric antibodies, humanized antibodies, non-glycosylated antibodies, human antibodies and antibodies from mice. Specifically, the antibody to be crystallized is an optional non-chimeric, human antibody and further processed to improve antigen binding and / or effectiveness.

Preferably, the crystals are obtained from an IgG antibody, such as, for example, an IgGI, IgG2, IgG3 or IgG4 antibody. Specifically, the antibody is an integral anti-human IL12 antibody of the IgGL group.

In a preferred embodiment, the crystals are prepared from an isolated human antibody, which dissociates from hlL-12 with Kd of 1 χ10^1θ M or less and a rate constant k_Off 1 x 10 '³ s'¹ or less, as determined by surface plasmon resonance.

Specifically, the crystals can be prepared from an isolated human antibody with a light chain variable region (LCVR) compared to the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region (HCVR) compared to the amino acid sequence SEQ ID NO: 1.

Preferred human antibodies are described, for example, in US Patent No. 6,914,128.

The most preferred crystals are prepared from the ABT-874 antibody.

In a further embodiment, the invention relates to a solid, liquid or semi-solid pharmaceutical composition, comprising: (a) crystals of an anti-hLL-12 antibody as defined above and (b) at least one stable, pharmaceutically acceptable excipient the antibody crystals.

Another aspect of this invention relates to a solid, liquid or semi-solid pharmaceutical composition comprising: (a) crystals of an anti-hLL-12 antibody as defined herein and (b) at least one pharmaceutically acceptable excipient encapsulating or embedding the crystals of antibody. The composition can additionally comprise (c) at least one stable, pharmaceutically acceptable excipient, maintaining the antibody crystals. In addition, encapsulation and embedding can be implemented together.

Specifically, the compositions of the invention may have an antibody crystal concentration greater than about 1 mg / ml, specifically about 200 mg / ml or more, for example about 200 to about 600 mg / ml, or about 300 to about 500 mg / ml.

Excipients can comprise at least one polymeric vehicle, optionally biodegradable or at least one oil or lipid vehicle.

The polymeric carrier can be one or more polymers selected from the group consisting of: poly (acrylic acid), poly (cyanoacrylates), poly (amino acids), poly (anhydrides), poly (despsipetide), poly (esters), poly (lactic acid) ), poly (lactic-co-glycolic acid) or PLGA, poly (jff-hydroxybutyrate), poly (caprolactone), poly (dioxanone); poly (ethylene glycol), poly (hydroxypropyl) methacrylamide, poly (organo) phosphazene, poly (ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone), alkyl vinyl ether copolymers, maleic anhydride, pluronic polyols, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polysaccharides, combinations and copolymers thereof.

The oil (or oily liquid) can be one or more oils (or oily liquid) selected from the group consisting of oilseed almond oil, corn oil, corn seed oil, ethyl oleate, isopropyl myristate, isopropyl palmitate, mineral oil, light mineral oil, octyldodecanol, olive oil, peanut oil, persian oil, castor oil, soybean oil, squalene, liquid triglycerides, liquid waxes and higher alcohols.

The lipid carrier can be one or more lipids selected from the group consisting of fatty acids and salts of fatty acids, fatty alcohols, fatty amines, mono, di and triglycerides of fatty acids, phospholipids, glycolipids, sterols and waxes and similar related substances. Waxes are further classified into natural and synthetic products. Natural materials include waxes obtained from plant, animal or mineral sources, such as beeswax, carnauba or mountain wax. Chlorinated naphthalenes and ethylene polymers are examples of synthetic wax products.

In a preferred embodiment, the composition is an injectable composition comprising anti-hLL-12 antibody crystals as defined above and having an antibody crystal concentration in the range of about 10 to about 400 mg / ml or about 50 to about 300 mg / ml.

In a further aspect the invention relates to a crystal paste comprising anti-hLL-12 antibody crystals as defined above having an antibody crystal concentration greater than about 100 mg / ml, for example about 150 to about 600 mg / ml, or about 200 to about 400 mg / ml.

The present invention also relates to a method for treating a mammal comprising the step of administering to the mammal an effective amount of integral anti-hLL-12 antibody crystals as defined above or an effective amount of a composition as defined above. Preferably, the composition is administered parenterally, orally or by injection.

In addition, the present invention relates to a method for treating a hlL-12 related disorder in an individual which comprises administering a therapeutically effective amount of antibody crystals as defined above.

Specifically, the hlL-12-related disorder is selected from: rheumatoid arthritis, osteoarthritis, chronic juvenile arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflamed bowel disease, insulin-dependent diabetes mellitus, thyroiditis, thyroiditis, asthma, illness scleroderma dermatitis, atopic dermatitis, graft versus host disease, organ transplant rejection, acute or chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki disease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome, Wegener granulomatosis, Henoch-Schoenlein purpura, microscopic kidney vasculitis, chronic active hepatitis, uveitis, septic shock, syndrome of toxic shock, septicemia syndrome, cachexia, infectious diseases, parasitic diseases, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignancies, heart failure, myocardial infarction, Addison's disease, type I polyglandular deficiency and sporadic type II polyglandular deficiency, Schmidt syndrome, adult respiratory distress syndrome (acute), alopecia, alopecia in areas, seronegative arthropathy, arthropathy, Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy, enteropathic synovitis, chlamydia-associated arthropathy, yersinia and salmonella, spondyloatropathy, atheromatous disease / arteriosclerosis, atopic allergy, bullous autoimmune disease, common pemphigus, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune hemolytic anemia, positive hemolytic anemia, acquired Coombs juvenile pernicious anemia, myalgic encephalitis / Royal Free disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing hepatitis, autoimmune cryptogenic hepatitis, AIDS, diseases related to acquired immunodeficiency, hepatitis C, varied common immunodeficiency (common variable hypogammaglobulinemia), dilated cardiomyopathy, female infertility, ovarian failure, premature ovarian failure, fibrotic pulmonary disease, cryptogenic fibrosing alveolitis, post-pulmonary interstitial lung disease inflammatory disease, interstitial pneumonitis, interstitial lung disease associated with connective tissue disease, lung disease associated with mixed connective tissue disease, interstitial lung disease associated with systemic sclerosis, interstitial lung disease associated with rheumatoid arthritis, pulmonary disease associated with systemic lupus erythematosus, pulmonary disease associated with dermatomyositis / polymyositis, lung disease associated with Sjodgren's disease , lung disease blew13 associated with ankylosing spondylitis, diffuse vasculitic lung disease, pulmonary disease associated with hemosiderosis, drug-induced interstitial lung disease, radiation fibrosis, obliterating bronchiolitis, eosinophilic pneumonia, infiltrative lymphocytic lung disease, post-infectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type 1 autoimmune hepatitis (classic or lupoid autoimmune hepatitis), autoimmune hepatitis type 2 (anti-LKM antibody hepatitis), autoimmune mediated hypoglycemia, type B insulin resistance with acanthose nigricans, hypoparathyroidism, acute immune disease associated with organ transplantation, chronic immune disease associated with organ transplantation, osteoarthritis, primary sclerosing cholangitis, idiopathic leukopenia, autoimmune neutropenia, NOS kidney disease, glomerulonephritis, microscopic kidney vasculitis, Lyme disease, discoid lupus erythematosus, infertility idiopathic male or NOS, sperm autoimmunity, multiple sclerosis (all subtypes), insulin-dependent diabetes mellitus, sympathetic ophthalmia, secondary pulmonary hypertension in connective tissue disease, Goodpasture syndrome, pulmonary manifestation of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Takayasu's disease / arteritis, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroid disease , hyperthyroidism, bocioso autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxoedema, facogenic uveitis, primary vasculitis and vitiligo. The human antibodies and antibody portions of the invention can be used to treat autoimmune diseases, specifically those associated with inflammation, including, rheumatoid spondylitis, allergy, autoimmune diabetes, autoimmune uveitis.

In addition, the present invention relates to the use of integral anti-hLL12 antibody crystals as defined above for the preparation of a pharmaceutical composition for the treatment of a hlL-12 related disease as defined above.

Finally, the present invention provides anti-hLL-12 antibody crystals as defined above for use in medicine.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objectives, aspects and advantages of the present invention, as well as the invention itself will be more fully understood from the description of the preferred modalities that follow, when reading in conjunction with the attached drawings, where:

Figure 1 shows a light micrograph of the crystals of ABT-874 at crystallization.

Figures 2-5 show SEMs of the ABT-874 crystals in different increments; figure 2 shows 1,250x; figure 3 shows 10,000x; figure 4 shows 3,227x and figure 5 shows 15,000x.

Figure 6 shows the results of the Isoelectric Focusing Experiments

Capillary (CIEF) with ABT-874; A) ABT-874 crystal buffer and pl markers 8.4, 8.5, 10.1 and 10.4; B) ABT-874 crystals; some pl marker and characteristic ABT-874 signal at pl = 9.29; C) Reference Standard, the same marker pl and ABT-874 signal characteristic at pl = 9.29.

Figure 7 shows microscopic crystal photos in light (bunches of needles) obtained according to Example 28 (crystallization with agitation).

Figure 8 shows microscopic crystal photos in light (needles) obtained according to Example 32 (crystallization without agitation).

Figure 9 shows microscopic crystal photos in light (needles) obtained according to Example 33 (crystallization without agitation).

Figure 10 shows microscopic crystal photos in light (needles) obtained according to Example 34 (crystallization without agitation).

Figure 11 shows second IR derived spectra from ABT874 samples. Figure 11A shows crystal suspension spectra recorded with a BioATR cell. Figure 11B shows spectra of redissolved crystals recorded with an AquaSpec cell. Solid lines represent samples of crystalline ABT-874, dotted lines represent liquid pattern. A deviation between the sample and the standard was inserted for better illustration.

Figure 12 shows second spectra derived from IR from ABT874 samples, 50 mg / mL of crystalline protein in PEG 4,000 to 22% buffer in 0.1 M sodium acetate buffer, pH 5.5, stored for 3 months at 25 ° C. Figure 11A shows spectra of the crystal suspension registered with a BioATR cell. Figure 11B shows spectra of redissolved crystals recorded with an AquaSpec cell. A deviation between the sample and the standard was inserted for better illustration.

Figure 13: 40 mL batch crystallization of ABT-874 with and without sowing (for example, using 3.25% crystallized protein as seeding material in relation to the mass of ABT-874 in the batch). R² is 0.9711 for not sown and 0.9763 for sown batch, respectively.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

A batch method of crystallization ”comprises the step of adding the crystallization solution comprising the crystallizing agent, preferably in dissolved form, to the antibody solution to be crystallized.

A micro-scale crystallization method, which may be based, for example, on vapor diffusion, comprises the steps of mixing a small volume of the antibody solution in the micro-liter range with a reservoir buffer containing a crystallizing agent; placing a droplet of the mixture in a sealed container adjacent to an aliquot of the reservoir plug; allowing the exchange of solvent between the droplet and the reservoir by vapor diffusion, during which time the content of the solvent in the droplet changes and crystallization can be observed if the appropriate crystallization conditions are achieved.

A crystallizing agent, for example, a polyethylene glycol, favors the formation of the antibody crystal to be crystallized.

A crystallization solution contains a crystallizing agent in dissolved form. Preferably the solution is an aqueous system, that is, its liquid constituents consist, predominantly, of water. For example, 80 to 100% by weight or 95 to 100% by weight or 98 to 100% by weight can be water.

Antibody "crystals" are a form of the solid state of the protein matter, which is distinct from a second solid form, that is, the amorphous state, which exists essentially as a heterogeneous, unorganized solid. The crystals have a regular three-dimensional structure, typically referred to as a grid. An antibody crystal comprises a regular three-dimensional row of antibody molecules (see, Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2^The edition, pages 1-16, Oxford University Press, New York (1999)).

An "integral" or "intact" anti-hLL-12 antibody when crystallized according to this invention is a functional antibody that is capable of recognizing and binding to its human IL-12 antigen in vitro and / or in vivo. The antibody can initiate subsequent reactions from a patient's immune system associated with the binding of the antibody to its antigen, specifically Direct Cytotoxicity, Complement-Dependent Cytotoxicity (CDC) and Antibody-Dependent Cytotoxicity (ADCC). The antibody molecule has a structure composed of two identical heavy chains (MW each about 50 kDa) covalently linked to each other and two identical light chains (MW each about 25 kDa), each covalently linked to one of the heavy chains. The four chains are arranged in a classic Ύ ”pattern. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a variable light chain region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of a domain, CL. The regions of VH and VL can be further subdivided into regions of hypervariability, called regions of complementarity determination (CDR), dispersed with regions that are more conserved, called regions of structure (FR). Each VH and VL is composed of three CDRs and four FRs, with amino termini for carboxy termini in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The complete antibody molecule has two antigen binding sites, that is, "bivalent". The two antigen binding sites are specific for an hlL-12 antigen, that is, the antibody is "specific for mono".

Monoclonal antibodies are antibodies that are derived from a simple clone of B lymphocytes (B cells), and recognize the same antigenic determinant. Integral monoclonal antibodies are those that have the same classic molecular structure mentioned above that includes two complete heavy chains and two complete light chains. Monoclonal antibodies are routinely produced by targeting the antibody-producing B cell with an immortal myeloma cell to generate cell hybridomas

B that continuously produce monoclonal antibodies in the cell culture. Other production methods are available, for example, expression of monoclonal antibodies in bacterial, yeast, insect or mammalian cell culture using phage display technology; in vivo production in genetically modified animals, such as cows, goats, pigs, rabbits, chickens or in transgenic mice that have been modified to contain and express the entire human B cell genome; or production in genetically modified plants, such as tobacco and corn. Anti-hLL-12 antibodies from all such sources can be crystallized according to this invention.

Monoclonal antibodies to be crystallized according to the invention include "chimeric" anti-hLL-12 antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequences in the antibodies derived from a specific species or belonging to a specific class or subclass of antibody, while the rest of the chain (s) is identical or homologous with respect to sequences in antibodies derived from other species or belonging to another class of antibodies or subclass. For example, a mouse / human chimera contains the variable portions of antigen binding genomics of a murine antibody and the constant portions derived from a human antibody.

"Humanized" forms of non-human (e.g., murine) anti-hLL-12 antibodies are also encompassed by the invention. Humanized antibodies are chimeric antibodies that contain minimal sequence derived from a non-human immunoglobulin. For most, humanized antibodies are human immunoglobulins, in which residues from one or more complementarity determining regions (CDRs) or hypervariable bonds (HVLs) from human immunoglobulin are replaced by residues from a CDR or HVL of non-human species, such as mouse, rat, rabbit or non-human primate, having the desired functionality. Residues from the framework region (FR) of human immunoglobulin5 can be replaced by corresponding non-human residues to improve the binding affinity of the antigen. In addition, humanized antibodies can comprise residues that are not found in the human or the corresponding non-human antibody portions. These modifications may be necessary to improve the additional effectiveness of the antibody.

A "human antibody" or "fully human antibody" is one that has an amino acid sequence that corresponds to that of an antibody produced by a human or that is produced recombinantly. The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., randomly introduced mutations or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example, in CDRs and specifically CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which the CDR sequences derived from the germ of other mammalian species, such as mice, have been grafted into the sequences of the human structure.

The term "recombinant human antibody" as used herein is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected in a host cell, antibodies isolated from a recombinant human combinatorial library, antibodies isolated from an animal (eg, mouse) that is transgenic to human immunoglobulin genes (see, for example, Taylor, LD and others (1992) Nucl. Acids Res. 20: 6287-6295) or antibodies prepared, expressed, raised or isolated by any other means involving recomposition of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have constant variables and regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or when Ig sequences from a transgenic to human animal are used, in somatic mutagenesis in vivo) and thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although derived and related to human germline VH and VL sequences, they cannot naturally exist within the human antibody germline repertoire in vivo.

A "neutralizing antibody" as used herein (or an "antibody that neutralized hlL-12 activity") refers to an antibody whose binding to hlL-12 results in inhibition of the biological activity of hlL-12. This inhibition of hlL-12 biological activity can be assessed in vitro or in vivo by measuring one or more indicators of hlL-12 biological activity, such as hlL-12-induced cell proliferation and hlL-12 binding to hlL-12 receptors or hlL-12 induced decrease in leukocytes in vivo.

These hlL-12 biological activity indicators can be evaluated by one or more of the severe standard in vitro or in vivo assays known in the art. Preferably, the ability of an antibody to neutralize hLL-12 activity is assessed by inhibiting hlL-12-induced cell proliferation in the phytomagglutinin blasts and murine 2D6 cells.

An "matured affinity" anti-hlL-12 antibody is one with one or more changes in one or more hypervariable regions, which results in an improvement in the affinity of the antibody to antigen compared to the parent antibody. Antibodies of mature affinity will show nanomolar or even picomolar affinity values ​​for the target antigen. Antibodies of mature affinity are produced by procedures known in the art. Marks et al. (1992) Bio / Technology 10: 779-783 describes affinity matured by scrambling the VH and VL domains. The random mutagenesis of structure residues and / or CDR is described by Barbas et al (1994) Proc. Nat. Acad. Know. USA 91: 3809-3813 (1994); Scier et al (1995) Gene 169: 147-155; Yelton et al. (1995) J. Immunol. 155: 1994-2004; Jackson et al. (1995) J. Immunol. 154 (7): 3310-9; and Hawkins et al. (1992) J. Mol Biol. 226: 889-896.

An "isolated antibody" as used herein refers to an antibody that is substantially free of other antibodies having different antigen specificities (for example, an isolated antibody that specifically binds hlL-12 is substantially free of antibodies that specifically bind antigens that do not hlL12). An isolated antibody that specifically binds hlL-12 may, however, cross-react with other antigens, such as hlL-12 molecules from other species. In addition, an isolated antibody can be substantially free of other cellular material and / or chemicals.

The phrase "human interleukin 12" (abbreviated herein as hlL-12 or IL-12), as used herein, includes a human cytokine that is secreted primarily by macrophages and dendritic cells. The term includes a heterodimeric protein comprising a 35 kD (p35) subunit and a 40 kD (p40) subunit that are both linked together with a disulfide bridge. The heterodimeric protein is referred to as a "p70 subunit". The structure of human IL-12 is further described, for example, in Kobayashi, et al. (1989) J. Exp Med. 170: 827- 845; Seder, et al. (1993) Proc. Natl. Acad. Know. 90: 10188-10192; Ling, et al. (1995) J. Exp Med. 154: 116-127; Podlaski, et al. (1992) Arch. Biochem. Biophys. 294: 230-237. The term IL-12 is intended to include recombinant human IL-12 (rh IL-12), which can be prepared by standard recombinant expression methods.

The term k_Off, as used herein, is intended to refer to the constant rate for dissociation of an antibody from the antibody / antigen complex.

The term k_d, as used in this document, refers to the dissociation constant of a specific antibody-antigen interaction.

A "functional equivalent" of an anti-hlL-12 antibody of specific "origin" as crystallized according to the invention is one that shows the same specificity for antigen, but differs with respect to the molecular composition of the "source" antibody in amino acid level or glycosylation level. The differences can be few, such that the crystallization conditions do not deviate from the parameter ranges as revealed in this document.

Encapsulation of antibody crystals refers to a formulation where the embedded crystals are individually coated by at least one layer of a coating material. In a preferred embodiment, such coated crystals can have a prolonged dissolution rate.

Inlay of antibody crystals refers to a formulation where the crystals, which can be encapsulated or not, are incorporated into a solid, liquid or semi-solid vehicle in a dispersed manner. Such embedded crystallized antibody molecules can be released or dissolved in a controlled, extended manner from the vehicle.

B. Crystallization method

The crystallization method of the invention is a principle applicable to any anti-hLL-12 antibody. The antibody can be a polyclonal antibody or, preferably, a monoclonal antibody. The antibody may consist of chimeric antibodies, humanized antibodies, human or non-human antibodies, each in glycosylated or non-glycosylated form. Specifically, the method is applicable to ABT-874 and its functional equivalents.

Preferably the anti-hLL-12 antibody is an IgG antibody, specifically an anti-human IL-12 antibody of the lgG1 group.

Unless otherwise stated, the crystallization method of the invention makes use of technical equipment, chemicals and methodologies well known in the art. However, as explained above, the present invention is based on the surprising finding that the selection of specific crystallization conditions, especially the selection of specific crystallization agents, is optional and additionally combined with specific pH conditions and / or concentration ranges of the agents (buffer, antibody, crystallizing agent) allows for the first time, reproducible and large-scale preparation of stable antibody crystals, specifically non-chimeric human antibodies, directed against hlL-12, which can be further processed to form an active ingredient of superior pharmaceutical composition, highly advantageous.

The starting material for carrying out the crystallization method normally comprises a concentrated antibody solution to be crystallized. The protein concentration can be, for example, in the range of about 5 to about 300 mg / ml, preferably about 5 to about 200 mg / ml, preferably about 5 to about 75 mg / ml. The solution may contain additives stabilizing the dissolved antibody and it may be advisable to remove the additives in advance. This can be achieved by carrying out a buffer exchange step.

Preferably, the starting material for carrying out the crystallization contains the antibody in an aqueous solution, having a pH adjusted in the range of about 3.2 to about 8.2 or about 4.0 to about 8.0, specifically about 4.5 to about 6.5, preferably about 5.0 to about 5.5. The pH can be adjusted by means of an appropriate buffer applied in a final concentration of about 1 to about 500 mM, specifically about 1 to about 100 mM or 1 to about 10 mM. The solution can contain additives, for example, in a proportion of about 0.01 to about 15, or about 0.1 to about 5, or about 0.1 to about 2% by weight, based on the total weight of the solution, such as salts, sugars, sugar alcohols and surfactants, in order to further stabilize the solution. The excipients are preferably selected from the physiologically acceptable compounds, routinely applied in pharmaceutical preparations. As non-limiting examples, excipients include salts, such as, NaCl; surfactants, such as polysorbate 80 (Tween 80), polysorbate 20 (Tween 20); sugars such as sucrose, trehalose, sugar alcohols such as mannitol, sorbitol and buffering agents such as phosphate-based buffer systems, sodium and potassium hydrogenated phosphate buffers as defined above, acetate buffer, phosphate buffer , citrate buffer, TRIS buffer, maleate buffer or succinate buffer, histidine buffer; amino acids, such as histidine, arginine and glycin.

The buffer exchange can be carried out using routine methods, for example, dialysis, diafiltration or ultrafiltration.

The initial protein concentration of the aqueous solution used as the starting material would be in the range of about 0.5 to about 200 or about 1 to about 50 mg / ml.

Depending on the size of the intended initial batch (which can be in the range of 1 mL to 20,000 liters) an initial volume of the aqueous antibody solution is placed in an appropriate container (such as a container, bottle or tank) made of material inert, such as glass, polymer or metal. The initial volume of the aqueous solution can correspond to about 30 to 80%, usually about 50% of the final batch size.

If necessary, the solution after being filled into the container will be brought to standard conditions. Specifically, the temperature will be adjusted in the range of about 4 ° C to about 37 ° C.

Then, the crystallization solution containing the crystallizing agent in an appropriate concentration, optionally preconditioned in the same way as the antibody solution is added to the antibody solution.

The addition of the crystallization solution is carried out continuously or discontinuously, optionally under gentle agitation, in order to facilitate the mixing of the two liquids. Preferably, the addition is carried out under conditions where the protein solution is provided under stirring and the crystallization solution (or agents in solid form) is / are added in a controlled manner.

The formation of the antibody crystals is initiated by applying a polyalkylene polyol as defined above, specifically a polyalkylene glycol and preferably a polyethylene glycol (PEG), or a mixture of at least two different polyalkylene glycols, as defined above, as the crystallization. The crystallization solution contains the agent in a concentration, which is sufficient to provide a final concentration of polyalkylene polyol in the crystallization mixture in the range of about 5 to 30% (weight / volume).

Preferably, the crystallization solution additionally contains an acidic buffer, for example, different from that of the antibody solution, in an appropriate concentration to allow adjustment of the pH of the crystallization mixture in the range of about 4 to 6.

After completing the addition of the crystallization solution, the obtained mixture can be further incubated for about 1 hour to about 250 days in order to obtain a maximum yield of the antibody crystals. If appropriate, the mixture can be agitated, gently rotated, laminated or otherwise moved.

Finally, the obtained crystals can be separated by known methods, for example, filtration or centrifugation, such as, for example, by centrifugation at about 200 - 20,000 rpm, preferably 500 - 2,000 rpm, at room temperature or 4 ° C. The remaining main liquid can be discarded or further processed.

If necessary, the isolated crystals can be washed and subsequently dried or the main liquor can be exchanged for a different solvent system suitable for storage and / or end use of the antibodies suspended therein.

The antibody crystals formed in accordance with the present invention can vary in their shape, as already explained above. For therapeutic administration, the size of the crystals will vary depending on the route of administration, for example, for subcutaneous administration, the size of the crystals may be larger than for intravenous administration.

The shape of the crystals can be changed by adding additives to the crystallization mixture, as previously described for both protein crystals and crystals of low molecular weight organic and inorganic molecules.

If necessary, it can be verified that the crystals are indeed crystals of the antibody. The crystals of an antibody can be analyzed microscopically by birefringence. In general, crystals, unless the ones with internal cubic symmetry will rotate the polarization plane of the polarized light. In yet another method, the crystals can be isolated, washed, resolubilized and analyzed by SDS-PAGE and, optionally, stained with an anti-Fc receptor antibody. Optionally, the resolubilized antibody can also be tested for binding to its hlL-12 using standard assays.

The crystals as obtained according to the invention can also be cross-linked together. Such crosslinking can improve the stability of the crystals. Methods for crosslinking the crystals are described, for example, in US Patent No. 5,849,296. The crystals can be cross-linked using a bifunctional reagent, such as glutaraldehyde. Once cross-linked, the crystals can be lyophilized and stored for use, for example, in diagnostic or therapeutic applications.

In some cases, it may be desirable to dry the crystal. Crystals can be dried using inert gases, such as nitrogen gas, vacuum oven drying, freeze drying, evaporation, tray drying, fluid bed drying, spray drying, vacuum drying or laminator drying. Appropriate methods are well known.

The crystals formed according to the invention can be kept in the original crystallization solution or they can be washed and combined with other substances, such as inert vehicles or ingredients to form compositions or formulations comprising crystals of the invention. Such compositions or formulations can be used, for example, in therapeutic and diagnostic applications.

A preferred embodiment is to combine an appropriate vehicle or ingredient with crystals of the invention, such that the crystals of the formulation are embedded or encapsulated by an excipient. Suitable vehicles can be taken from the non-limiting group of: poly (acrylic acid), poly (cyanoacrylates), poly (amino acids), poly (anhydrides), poly (depsipeptide), poly (esters), poly (lactic acid), poly ( lactic-co-glycolic acid) or PLGA, poly (βhydroxybutyrate), poly (caprolactone), poly (dioxanone); poly (ethylene glycol), poly (hydroxypropyl) methacrylamide, poly (organo) phosphazene, poly (ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone), vinyl ether alkyl copolymers, maleic anhydride, pluronic polyols, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polysaccharides, combinations and copolymers thereof, SAIB, fatty acids and fatty acid salts, fatty alcohols, fatty amines, mono-, di- and triglycerides of fatty acids, phospholipids, glycolipids, sterols and waxes and similar related substances. Waxes are further classified into natural and synthetic products. Natural materials include waxes obtained from plant, animal or mineral sources, such as beeswax, carnauba and mountain wax. Naphthalenes chlorates and ethylene polymers are examples of synthetic waxy products.

C. Compositions

Another aspect of the invention relates to compositions / formulations comprising anti-hLL-12 antibody crystals, in combination with at least one vehicle / excipient.

The formulations can be solid, semi-solid or liquid.

The formulations of the invention are prepared in a form suitable for storage and / or use, by mixing the antibody having the necessary degree of purity with a physiologically acceptable additive, such as vehicle, excipient and / or stabilizer (see, for example, Remington's Pharmaceutical Sciences , 16^The edition, Osol, A. Ed. (1980)), in the form of suspensions, lyophilized or otherwise dried. Optionally, additional active ingredients, such as different antibodies, biomolecules, chemically or enzymatically synthesized low molecular weight molecules can be incorporated.

Acceptable additives are non-toxic to containers at the dosages and concentrations used. Non-limiting examples of these include:

- Acidifying agents, such as acetic acid, citric acid, fumaric acid, hydrochloric acid, maleic acid, phosphoric acid, diluted phosphoric acid, sulfuric acid, tartaric acid.

- Aerosol propellants such as butane, dichlorodifluoromethane, dichlorotetrafluoroethane, isobutane, propane, trichloromonofluoromethane.

- Air displacements, such as carbon dioxide, nitrogen;

- Alcoholic denaturants, such as methyl isobutyl ketone, sucrose octacetate;

- Alkalizing agents, such as ammonia solution, ammonium carbonate, diethanolamine, diisopropanolamine, potassium hydroxide, sodium bicarbonate, sodium borate, sodium carbonate, sodium hydroxide, trolamine;

- Defoaming agents, such as dimethicone, simethicone.

- Antimicrobial preservatives, such as benzalkonium chloride, benzalkonium chloride solution, benzeltonium chloride, benzoic acid, benzyl alcohol, butylparaben, cetylpyridinium chloride, chlorobutanol, chlorocresol, cresol, dehydroacetic acid, ethylparaben, methylparaben, methylparaben, methylparaben, methylparaben, methylparaben phenylethyl alcohol, phenylmercuric acetate, phenylmercuric nitrate, potassium benzoate, potassium sorbate, propylparaben, sodium propylparaben, sodium benzoate, dehydroacetate, sodium propionate, sorbic acid, thimerosal, thymol.

- Antioxidants, such as ascorbic acid, ascorbyl palmitate, butylated hydroxyanisol, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium formic aldehyde sulfoxylate, sodium metiabysulfite, sodium thiosulfate, sulfur dioxide, tocopherol dioxide, sulfur dioxide ;

- Buffering agents, such as, acetic acid, ammonium carbonate, ammonium phosphate, boric acid, citric acid, lactic acid, phosphoric acid, potassium citrate, meta24 potassium phosphate, monobasic potassium phosphate, sodium acetate, citrate sodium, sodium lactate solution, dibasic sodium phosphate, monobasic sodium phosphate, histidine.

- Chelating agents, such as disodium edetate, ethylenediamine tetraacetic acid and salts, edetic acid;

- Coating agents such as sodium carboxymethyl cellulose, cellulose acetate, cellulose acetate phthalate, ethyl cellulose, gelatin, pharmaceutical enamel, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose phthalate, methacrylic acid copolymer, methyl cellulose, polyethylene glycol, polyethylene glycol, polyvinyl, phthalate, gum, sucrose, titanium dioxide, carnauba wax, microcrystalline wax, zein, poly amino acids, other polymers like PLGA, etc., and SAIB.

- Coloring agents, such as ferric oxide.

- Complexing agents such as ethylenediamine tetraacetic acid and salts (EDTA), edetic acid, gentisic acid ethanolamide, oxyquinoline sulfate.

- Desiccants, such as calcium chloride, calcium sulfate, silicon dioxide.

- Emulsifying and / or solubilizing agents, such as acacia, cholesterol, diethanolamine (adjuvant), glyceryl monostearate, lanolin alcohols, lecithin, mono and diglycerides, monoethanolamine (adjuvant), oleic acid (adjuvant), oleyl alcohol (stabilizer), poloxamer, polyoxyethylene stearate 50, polyoxyl 35 castor oil, polyoxyl hydrogenated castor oil 40, polyoxyl oleyl ether 10, polyoxyl keto stearyl ether 20, polyoxyl 40 stearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, propylene glycol diacetate, propylene glycol monostearate, sodium lauryl sulfate, sodium stearate, sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, stearic acid, trolamine.

- Filtration aids, such as cellulose powder, purified silica soil.

- Flavors and perfumes, such as anethole, benzaldehyde, ethyl vanillin, menthol, methyl salicylate, monosodium glutamate, orange blossom oil, peppermint, peppermint oil, rose oil, stronger rose water, thymol, tincture of balsam-detolu, vanilla, tincture of vanilla, vanillin.

- Glidants and / or anti-cake agents, such as calcium silicate, magnesium silicate, colloidal silicon dioxide, talc.

- Humectants, such as, glycerin, hexylene glycol, propylene glycol, sorbitol;

- Ointment bases, such as lanolin, anhydrous lanolin, hydrophilic ointment, white ointment, yellow ointment, polyethylene glycol ointment, petrolatum, hydrophilic petrolatum, white petroleum, rose-based ointment, squalene.

- Plasticizers, such as castor oil, lanolin, mineral oil, petrolatum, benzyl benzyl formate, chlorobutanol, diethyl phthalate, sorbitol, diacetylated monoglycerides, diethyl phthalate, glycerin, glycerol, mono and diacetylated monoglycerides, polyethylene glycol, propyl25 in glycol, triacetin, triethyl citrate, ethanol.

- Polypeptides, such as those with low molecular weight (less than about 10 residues);

Proteins, such as serum albumin, gelatin or immunoglobulins;

- Polymeric membranes, such as cellulose acetate membranes.

- Solvents such as acetone, alcohol, diluted alcohol, amylene hydrate, benzyl benzoate, butyl alcohol, carbon tetrachloride, chloroform, corn oil, cottonseed oil, ethyl acetate, glycerin, hexylene glycol, alcohol isopropyl, methyl alcohol, methylene chloride, methyl isobutyl ketone, mineral oil, peanut oil, polyethylene glycol, propylene carbonate, propylene glycol, sesame oil, water for injection, sterile water for irrigation, purified water, liquid triglycerides, liquid waxes, higher alcohols.

- Sorbents, such as powdered cellulose, coal, purified silica soil, carbon dioxide sorbents, barium hydroxide lime, soda lime.

- Stiffening agents, such as hydrogenated castor oil, cetostearyl alcohol, cetyl alcohol, cetyl esters wax, hard fat, paraffin, polyethylene excipient, stearyl alcohol, emulsifying wax, white wax, yellow wax.

- Suppository bases, such as cocoa butter, hard fat, polyethylene glycol;

- Agents for suspending and / or increasing viscosity, such as acacia, agar, alginic acid, aluminum monostearate, bentonite, purified bentonite, magma bentonite, carbomer 934p, calcium carboxymethylcellulose, sodium carboxymethylcellulose, sodium carboxymethylcellulose 12, carrageenan, carboxymethyl cellulose cellulose and microcrystalline, dextrin, gelatin, guar gum, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, aluminum magnesium silicate, methylcellulose, pectin, polyethylene oxide, polyvinyl alcohol, povidone, propylene glycol alginate, silicon dioxide, colloidal silicon dioxide, sodium alginate, tragacanth, xanthan gum;

- Sweetening agents, such as aspartame, dextrates, dextrose, excipient dextrose, fructose, mannitol, saccharin, saccharin calcium, saccharin sodium, sorbitol, sorbitol in solution, sucrose, compressible sugar, powdered sugar, syrup;

- Binders for tablets, such as acacia, alginic acid, sodium carboxymethylcellulose, microcrystalline cellulose, dextrin, ethylcellulose, gelatin, liquid glucose, guar gum, hydroxypropyl methylcellulose, methylcellulose, polyethylene oxide, povidone, pregelatinized starch, syrup.

- Diluents for tablets and / or capsules, such as calcium carbonate, dibasic calcium phosphate, tribasic calcium phosphate, calcium sulfate, microcrystalline cellulose, powdered cellulose, dextrans, dextrin, excipient dextrose, fructose, kaolin, lactose, mannitol , sorbi26 tol, starch, pregelatinized starch, sucrose, compressible sugar, powdered sugar;

- Tablet disintegrants such as alginic acid, microcrystalline cellulose, croscarmellose sodium, corspovidone, potassium polacrylin, sodium starch glycolate, starch, pregelatinized starch.

- Tablet and / or capsule lubricants, such as calcium stearate, glyceryl behenate, magnesium stearate, light mineral oil, polyethylene glycol, sodium stearyl fumarate, stearic acid, purified stearic acid, talc, hydrogenated vegetable oil, stearate zinc;

- Tonicity agent, such as dextrose, glycerin, mannitol, potassium chloride, sodium chloride carrier: aromatic aromatic and / or sweetened elixir, compound benzaldehyde elixir, isoalcoholic elixir, peppermint water, sorbitol solution, syrup, syrup of tolu balm.

- Vehicles such as oilseed almond oil, corn oil, cottonseed oil, ethyl oleate, isopropyl myristate, isopropyl palmitate, mineral oil, light mineral oil, myristyl alcohol, octyldodecanol, olive oil, oil peanut, persian oil, sesame oil, soy oil, squalene; sugar sphere in a solid vehicle; sterile bacteriostatic water for injection, water for bacteriostatic injection of sodium chloride; liquid triglycerides, liquid waxes, higher alcohols.

- Water repellent agents, such as cyclomethicone, dimethicone, simethicone;

- Wetting and / or solubilizing agents, such as benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, sodium docusate, nonoxynol 9, nonoxynol 10, octoxynol 9, poloxamer, polyoxyl 35 castor oil, polyoxyl 40, hydrogenated castor oil , polyoxyl 50 stearate, polyoxyl 10 ether, polyoxyl 20, keto stearyl ether, polyoxyl 40 stearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, sodium lauryl sulfate, sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate and tiloxapol.

The crystals can be combined with a polymeric vehicle to provide stability and / or prolonged release. Such polymers include biocompatible and biodegradable polymers. A polymeric vehicle can be a simple polymer type or it can be composed of a mixture of polymer types. Non-limiting examples of polymeric vehicles have already been stated above.

Examples of preferred ingredients or excipients include:

- amino acid salts, such as, glycine, arginine, aspartic acid, glutamic acid, lysine, asparagine, glutamine, proline, histidine;

- monosaccharides, such as, glucose, fructose, galactose, mannose, arabinose, xylose, ribose;

- disaccharides, such as, lactose, trehalose, maltose, sucrose;

- polysaccharides, such as maltodextrins, dextrans, starch, glycogen;

aiditols, such as mannitol, xylitol, lactitol, sorbitol;

- glucuronic acid, galacturonic acid;

- cyclodextrins, such as methyl cyclodextrin, hydroxypropyl- (3-cyclodextrin);

- inorganic salts, such as sodium chloride, potassium chloride, magnesium chloride, sodium and potassium phosphates, boric acid, ammonium carbonate and ammonium phosphate;

- organic salts, such as acetates, citrate, ascorbate, lactate;

- emulsifying or solubilizing agents, such as acacia, diethanolamine, glyceryl monostearate, lecithin, monoethanolamine, oleic acid, oleyl alcohol, poloxamer, polysorbates, sodium lauryl sulfate, stearic acid, sorbitan monolaurate, sorbitan monostearate and other sorbitan monostearate sorbitan, polyoxyl derivatives, wax, polyoxyethylene derivatives, sorbitan derivatives; and

- reagents to increase viscosity, such as agar, alginic acid and its salts, guar gum, pectin, polyvinyl alcohol, polyethylene oxide, cellulose and its derivatives of propylene carbonate, polyethylene glycol, hexylene glycol and tiloxapol.

The formulations described in this document also comprise an effective amount of the crystalline antibody. Specifically, the formulations of the invention can include a "therapeutically effective amount" or a "prophylactically effective amount" of antibody crystals of the invention. A "therapeutically effective amount" refers to an effective amount, in dosages and for periods of time necessary to obtain the desired therapeutic result. A "therapeutically effective amount" of the antibody crystals can vary according to factors, such as the condition of the disease, age, sex and weight of the individual and the ability of the antibody to promote a desired response in the individual. A therapeutically effective amount is also one in which any toxic or harmful effects of the antibody are counterbalanced by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an effective amount, in dosages and for periods of time necessary, to obtain the desired prophylactic result. Typically, since a prophylactic dose is used in individuals before or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

Appropriate dosages can be readily determined using standard methodology. The antibody is appropriately administered to the patient at once or in a series of treatments. Depending on the factors mentioned above, about 1 / g / kg to about 50 mg / kg, such as, for example, 0.1-20 mg / kg of antibody is an initial candidate dosage for administration to the patient, if, for example, example, by one or more separate administrations or by continuous infusion. A typical daily or weekly dosage can vary from about 1 µg / kg to about 20 mg / kg or more, depending on the condition, treatment will be repeated until a desired suppression of the symptoms of the disease occurs. However, other dosage regimens can be useful. In some cases, the formulations comprise an antibody concentration of at least 1 g / L or more, when resolubilized. In other embodiments, the concentration of antibodies is at least about 1 g / L to about 100 g / L, when resolubilized.

Crystals of an antibody or formulations comprising such crystals can be administered alone or as part of a pharmaceutical preparation. They can be administered parenterally, orally or topically. For example, they can be administered orally, pulmonary, nasal, aural, anal, dermal, ocular, intravenous, intramuscular, intraarterial, intraperitoneal, mucosal, sublingual, subcutaneous, transdermal, topical or intracranially or in the oral cavity. Specific examples of administration techniques include pulmonary inhalation, intralesional application, needle injection, dry powder inhalation, electroporation into the skin, aerosol delivery and needle-free injection technologies, including needle-free subcutaneous administration.

The present invention will now be explained in more detail by means of the following non-limiting illustrative examples. Guided by the general part of the description and based on his general knowledge, one skilled in the art will be able to provide additional modalities to the invention, without undue experimentation.

Exemplification

A. Materials

a) Protein

ABT-874 frozen monoclonal antibody (mAb) was obtained from Abbott Laboratories. All experiments were carried out on a product batch, where the original mAb concentration was 64 mg / mL.

b) Fine Chemistry

Sodium acetate was obtained from Grüssing GmbH, Filsum. Polyethylene glycols of different polymerization classifications were obtained from Clariant GmbH, Sulzbach. In addition, commercial crystallization screens and reagents (Hampton Research, Nextal Biotechnologies) were used for certain micro-scale experiments. All other chemicals were obtained from Sigma-Aldrich, Steinheim or Merck, Darmstadt.

B. General Methods

a) Thawing of the drug substance ABT-874

ABT-874 was thawed at 25 ° C in shaking water baths.

b) Buffer exchange - Method A

An aliquot of ABT-874 solution was pipetted into a 30 KDa MWCO Vivaspin 20 concentrator (Vivascience). The protein sample was diluted with the new buffer at a rate of 1:10 and by centrifugation at 5,000 xga 4 ° C (Sigma 4 k 15 laboratory centrifuge), the sample volume was brought back to the sample volume initial. The dilution / centrifugation steps were repeated once, resulting in a 1: 100 dilution of the original sample buffer. After adjusting the protein concentration, the solution was filtered and sterilized through a 0.2 pm syringe-driven filter unit.

b) Changing the Buffer - Method B

An aliquot of the ABT-874 solution was placed in a SLIDEA-LYZER dialysis cassette (Pierce Biotechnology Inc.). The dialysis cassette was placed in a beaker containing the chosen buffer and the buffer exchange was carried out at 4 ° C all night with agitation. After adjusting the protein concentration, the solution was filtered and sterilized through a 0.2 pm syringe-driven filter unit.

c) OD280 - measurements of protein concentration

A ThermoSpectronics UV1 device was used to assess the protein concentration at a wavelength of 280 nm, applying a 1.42 cm extinction coefficient² mg '¹. For this purpose, aliquots of crystallization were centrifuged at 14,000 rpm and the concentration of the residual protein was determined in the supernatant.

d) pH measurements

Measurements were conducted using a Mettler Toledo MP220 pH meter. 413 Inlab electrodes and 423 Inlad micro electrodes were used.

e) Crystallization Methods e1) Micro scale crystallization - Drop Steam Diffusion at Rest Hydra II

Initial crystallization filtrations were performed using a Hydra II crystallization robot and Greiner 96-well plates (three-drop wells, Hampton Research). After adjusting the plates, the wells were sealed with Clearseal film (Hampton Research).

e2) Micro-scale crystallization - Pending Drop Steam Diffusion

Drop droplet vapor diffusion experiments were conducted using VDX plates (with sealant, Hampton Research) and OptiClear plastic plating sheets (square, Hamp-ton Research) or siliconized glass plating sheets (circular, Hampton Research), respectively. After preparing the reservoir solutions, a drop of the reservoir solution was mixed with a drop of the protein solution on a coating slide and the well was sealed with the inverted coating slide, so that the drop was hanging above the reservoir.

e3) Batch Crystallization - Method A (24-well plate)

Batch crystallization was performed by mixing the protein solution with an equal amount of the crystallization buffer (500 / L) in a well. The well was subsequently sealed with adhesive tape to prevent water evaporation.

e4) Batch crystallization - Method B (Eppendorff Reaction Tube)

Batch crystallization was performed by mixing the protein solution with an equal amount of the crystallization buffer in a 1.5 ml or 2 ml Eppendorff reaction tube.

e5) Batch crystallization - Method C (Falcon tubes, without stirring)

Batch crystallization was performed by mixing the protein solution with an equal amount of the crystallization buffer in a 15 ml or 50 ml Falcon tube.

e6) Batch crystallization - Method D (Falcon tubes, with agitation)

Batch crystallization was performed by mixing the protein solution with an equal amount of the crystallization buffer in a 15 ml or 50 ml Falcon tube. Immediately after closing, the tube was placed on a laboratory stirrer (GFL 3013 or GFL 3015) or alternately shaken by tipping. By applying these methods, the introduction of agitators in the sample was avoided.

f) SDS-PAGE

Samples were prepared by adjusting the protein concentration to 8 / rg / 20 χ / L. The samples were diluted with an SDS / Tris / glycerin buffer containing bromophenol blue. Qualitative SDS PAGE analysis was performed using Invitrogen NuPage BisTris 10% Gels, NuPae SDS Operation Buffer and Mark12 Wide Range Protein Standard. Twenty micro liters of the sample were pipetted into a gel pouch. After operating the gel and fixing with acetic acid / methane reagent, staining was performed using the Novex Colloidal Blue Stain kit. The gels were dried using Invitrogen GelDry drying solution.

g) Light microscopy

The crystals were observed using a Zeiss 25 or Nikon Labophot microscope. The latter was equipped with a set of polarization filters and a color JVC TK C1380 video camera.

h) SE-HPLC

Aggregation levels of ABT-874 samples were assessed by SE-HPLC. A Dionex P680 pump, ASI-100 autosampler and UVD170U device were used. Aggregate species were separated from the monomer by an Amersham Bioscience Superdex 200 10/300 GL gel filtration column, applying a validated Abbott standard protocol (A-796874.0 - ABT 874, J 695).

C. Vapor diffusion crystallization experiments

The concentration values ​​provided in the following examples are initial values ​​with reference to the antibody solution and the reservoir solution before mixing the two solutions.

All pH values, if not described otherwise, refer to the pH of a master acetate buffer solution before it is combined with other substances, such as the crystallizing agent.

All buffer molarities, if not otherwise described, refer to the concentrations of sodium acetate in a master solution prior to pH adjustment, typically performed using glacial acetic acid.

Example 1 - Filtration by PEG 4000 / Sodium Acetate Grid in Drop Drop Steam Diffusion Mode

A pendant drop vapor diffusion crystallization method was performed on ABT-874. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.2. The protein concentration was adjusted to 10 mg / ml.

A greased VDX plate and square OptiClear plastic cladding sheets were used. 500 pL of a specific reservoir solution was prepared by mixing acetate buffer, 50% w / v of PEG 4,000 solution and Milli Q water (completely desalted and optionally pre-distilled) in each well. In this example, the molarity of the acetate buffer was kept constant at about 0.1 M and PEG 4,000 ranged from about 6% weight / volume to about 28% weight / volume in 2% steps. The pH was around 5.2 from start to finish. Each condition was evaluated in duplicate. About 1 μΥ. of the protein solution was mixed with about 1 pL of a specific reservoir solution on a square OptiClear plastic coating slide and the well was sealed with the inverted slide generating a pendant drop experiment. The plates were stored at room temperature. Droplet microscopy was performed several times during the next thirty days. The conditions were classified as clear drops, drops containing random precipitation, drops containing crystals and drops containing mixtures of species and precipitated crystals.

RESULTS: Crystals were not observed in the 24 evaluated wells.

Example 2 - PEG 4000 / Filtration by Sodium Acetate in Drop Droplet Steam Diffusion Mode, Different protein concentration

A pendant drop vapor diffusion crystallization method was performed on ABT-874 at a different protein concentration. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.2. The protein concentration was adjusted to 50 mg / ml.

A greased VDX plate and square OptiClear plastic cladding sheets were used. 500 μΥ. of a specific reservoir solution were prepared by mixing acetate buffer, 50% weight / volume of PEG 4,000 solution and Milli Q water in each well. In this example, the molarity of the acetate buffer was kept constant at about 0.1 M and PEG 4,000 ranged from about 6% weight / volume to about 28% weight / volume in 2% steps. The pH was around 5.2 from start to finish. Each condition was evaluated in duplicate. About 1 μΥ. of the protein solution was mixed with about 1 μΥ. of a specific reservoir solution on a square OptiClear plastic plating slide and the well was sealed with the inverted slide generating a pending drop experiment. The plates were stored at room temperature. Droplet microscopy was performed several times during the next thirty days. The conditions were classified as clear drops, drops containing random precipitation, drops containing crystals and drops containing mixtures of species and precipitated crystals.

RESULTS: In the 24 wells evaluated, the crystals were observed at a PEG 4,000 concentration of about 16%. The crystals showed a morphology similar to the needle or bunch of needles.

Example 3 - PEG 400 / Sodium Acetate Grade Filtration in Drop Droplet Steam Diffusion Mode

A pendant drop vapor diffusion crystallization method was performed on ABT-874 using PEG 400. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.2. The protein concentration was adjusted to 10 mg / ml.

A greased VDX plate and square OptiClear plastic cladding sheets were used. 500 / vL of a specific reservoir solution was prepared by mixing acetate buffer, 50% weight / volume PEG using PEG 400 solution and Mil li Q water in each well. In this example, the molarity of the acetate buffer was kept constant at about 0.1 M and PEG 400 ranged from about 30% weight / volume to about 40% weight / volume in 2% steps. The pH was around 5.2 from start to finish. Each condition was evaluated in duplicate. About 1 pL of the protein solution was mixed with about 1 μΥ. of a specific reservoir solution on a square OptiClear plastic plating slide and the well was sealed with the inverted slide generating a pending drop experiment. The plates were stored at room temperature. Droplet microscopy was performed several times during the next thirty days. The conditions were classified as clear drops, drops containing random precipitation, drops containing crystals and drops containing mixtures of species and precipitated crystals.

RESULTS: Crystals were not observed in the 12 wells evaluated.

Example 4 - PEG 400 / Sodium Acetate Grid Filtration in Drop Diffusion Steam Diffusion Mode, Different Protein Concentration

A pendant drop vapor diffusion crystallization method was performed on ABT-874 at a different protein concentration. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.2. The protein concentration was adjusted to 50 mg / ml.

A greased VDX plate and square OptiClear plastic cladding sheets were used. 500 pL of a specific reservoir solution was prepared by mixing acetate buffer, 50% weight / volume PEG using PEG 400 solution and Milli Q water in each well. In this example, the molarity of the acetate buffer was kept constant at about 0.1 M and PEG 400 ranged from about 30% weight / volume to about 40% weight / volume in 2% steps. The pH was around 5.2 from start to finish. Each condition was evaluated in duplicate. About 1 μϊ. of the protein solution was mixed with about 1 μϊ- of a specific reservoir solution on a square OptiClear plastic coating slide and the well was sealed with the inverted slide generating a pendant drop experiment. The plates were stored at room temperature. Droplet microscopy was performed several times during the next thirty days. The conditions were classified as clear drops, drops containing random precipitation, drops containing crystals and drops containing mixtures of species and precipitated crystals.

RESULTS: Crystals were not observed in the 12 wells evaluated.

Example 5 - PEG 400 / Sodium Acetate Grid Filtration in Drop Drop Steam Diffusion Mode, Different Protein Concentration and Adjustment

A pendant drop vapor diffusion crystallization method was performed on ABT-874 employing a different protein concentration and a different fit. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.2. The protein concentration was adjusted to 50 mg / ml.

A greased VDX plate and square OptiClear plastic cladding sheets were used. 500 pL of a specific reservoir solution was prepared by mixing acetate buffer, 50% weight / volume of PEG 400 solution and Milli Q water in each well. In this example, the molarity of the acetate buffer was kept constant at about 0.1 M1 and PEG 400 ranged from about 30% weight / volume to about 40% weight / volume in 2% steps. The pH was about 5.7 or 6.7, respectively. Each condition was evaluated in duplicate. About 1 μϊ- of the protein solution was mixed with about 1 μϊ. of a specific reservoir solution on a square OptiClear plastic plating slide and the well was sealed with the inverted slide generating a pending drop experiment. The plates were stored at room temperature. Droplet microscopy was performed several times during the next twenty-one days. The conditions were classified as clear drops, drops containing random precipitation, drops containing crystals and drops containing mixtures of species and precipitated crystals.

RESULTS: Crystals were not observed in the 24 evaluated wells.

Example 6 - PEG 10,000 / Sodium Acetate Grade Filtration in Drop Droplet Steam Diffusion Mode

A pendant drop vapor diffusion crystallization method was performed on ABT-874 employing PEG 10,000. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.2. The protein concentration was adjusted to 10 mg / ml.

A greased VDX plate and square OptiClear plastic cladding sheets were used. 500 / vL of a specific reservoir solution was prepared by mixing acetate buffer, 50% weight / volume of PEG 10,000 solution and Milli Q water in each well. In this example, the molarity of the acetate buffer was kept constant at about 0.1 M and PEG 10,000 ranged from about 4% weight / volume to about 14% weight / volume in 2% steps. The pH was around 5.2 from start to finish. Each condition was evaluated in duplicate. About 1 // L of the protein solution was mixed with about 1 // L of a specific reservoir solution on a square OptiClear plastic coating slide and the well was sealed with the inverted slide generating a drop drop experiment. The plates were stored at room temperature. Droplet microscopy was performed several times during the next thirty days. The conditions were classified as clear drops, drops containing random precipitation, drops containing crystals and drops containing mixtures of species and precipitated crystals.

RESULTS: Crystals were not observed in the 12 wells evaluated.

Example 7 - PEG 10,000 / Sodium Acetate Grade Filtration in Drop Droplet Steam Diffusion Mode, Different protein concentration

A pendant drop vapor diffusion crystallization method was performed on ABT-874 employing PEG 10,000 and at a different protein concentration. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.2. The protein concentration was adjusted to 50 mg / ml.

A greased VDX plate and square OptiClear plastic cladding sheets were used. 500 μΥ. of a specific reservoir solution were prepared by mixing acetate buffer, 50% weight / volume of PEG 10,000 solution and Milli Q water in each well. In this example, the molarity of the acetate buffer was kept constant at about 0.1 M and PEG 10,000 ranged from about 4% weight / volume to about 14% weight / volume in 2% steps. The pH was around 5.2 from start to finish. Each condition was evaluated in duplicate. About 1 μΥ. of the protein solution was mixed with about 1 μΥ. of a specific reservoir solution on a square OptiClear plastic plating slide and the well was sealed with the inverted slide generating a pending drop experiment. The plates were stored at room temperature. Droplet microscopy was performed several times during the next thirty days. The conditions were classified as clear drops, drops containing random precipitation, drops containing crystals and drops containing mixtures of species and precipitated crystals.

RESULTS: Crystals were not observed in the 12 wells evaluated.

Example 8 - PEG 4000 / Sodium Acetate Grade Filtration in Drop Droplet Steam Diffusion Mode. Different Fit

A pendant drop vapor diffusion crystallization method was performed on ABT-874 employing PEG 4,000 and a different fit. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.2. The protein concentration was adjusted to 10 mg / ml.

A greased VDX plate and square OptiClear plastic cladding sheets were used. 500 pL of a specific reservoir solution was prepared by mixing acetate buffer, 50% weight / volume of PEG 4,000 solution and Milli Q water in each well. In this example, the molarity of the acetate buffer was kept constant at about 0.1 M and PEG 4,000 ranged from about 22% weight / volume to about 28% weight / volume in 2% steps. The pH was about 4.2, 4.7, 5.2, 5.7, 6.2 and 6.7, respectively. Each condition was evaluated in duplicate. About 1 pL of the protein solution was mixed with about 1 pL of a specific reservoir solution on a square OptiClear plastic coating slide and the well was sealed with the inverted slide generating a drop-drop experiment. The plates were stored at room temperature. Droplet microscopy was performed several times during the next thirty days. The conditions were classified as clear drops, drops containing random precipitation, drops containing crystals and drops containing mixtures of species and precipitated crystals.

RESULTS: Crystals were not observed in the 48 evaluated wells.

Example 9 - PEG 4000 / Sodium Acetate Grade Filtration in Drop Droplet Steam Diffusion Mode, Different Adjustment

A pendant drop vapor diffusion crystallization method was performed on ABT-874 using PEG 4,000 and another adjustment. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.2. The protein concentration was adjusted to 10 mg / ml.

A greased VDX plate and square OptiClear plastic cladding sheets were used. 500 pL of a specific reservoir solution was prepared by mixing acetate buffer, 50% weight / volume of PEG 4,000 solution and Milli Q water in each well. In this example, the molarity of the acetate buffer was kept constant at about 0.1 M and PEG 4,000 ranged from about 8% weight / volume to about 14% weight / volume in 2% steps. The pH was about 5.7, 6.2 and 6.7, respectively. Each condition was evaluated in duplicate. About 1 pL of the protein solution was mixed with about 1 pL of a specific reservoir solution on a square OptiClear plastic coating slide and the well was sealed with the inverted slide generating a drop-drop experiment. The plates were stored at room temperature. Droplet microscopy was performed several times during the next thirty days. The conditions were classified as clear drops, drops containing random precipitation, drops containing crystals and drops containing mixtures of species and precipitated crystals.

RESULTS: In the 24 evaluated wells, the crystals were observed at a PEG 4,000 concentration of about 10 to 14% at all pHs included in this example. The crystals showed a morphology similar to the needle or bunch of needles.

Example 10 - PEG 400 Grid Filtration Combined with PEG 4,000 / Sodium Acetate in Drop Droplet Steam Diffusion Mode

A pendant drop vapor diffusion crystallization method was performed on ABT-874 employing PEG 400 with 4,000 / sodium acetate. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.2. The protein concentration was adjusted to 10 mg / ml.

A greased VDX plate and square OptiClear plastic cladding sheets were used. 500 // L of a specific reservoir solution was prepared by mixing acetate buffer, 50% weight / volume of PEG 4,000 solution and Milli Q water in each well. In this example, the molarity of the acetate buffer was kept constant at about 0.1 M and PEG 4,000 ranged from about 8% weight / volume to about 12% weight / volume in 2% steps. Simultaneously, PEG 400 was brought to PEG 4,000 / acetate solutions in concentrations of about 26% weight / volume, 28% weight / volume, 30% weight / volume and 32% weight / volume, respectively. The pH was around 5.2 from start to finish. Each condition was evaluated in duplicate. About 1 μΥ. of the protein solution was mixed with about 1 μΥ. of a specific reservoir solution on a square OptiClear plastic plating slide and the well was sealed with the inverted slide generating a pending drop experiment. The plates were stored at room temperature. Droplet microscopy was performed several times during the next thirty days. The conditions were classified as clear drops, drops containing random precipitation, drops containing crystals and drops containing mixtures of species and precipitated crystals.

RESULTS: Crystals were not observed in the 24 evaluated wells.

Example 11 - PEG 400 Grade Filtration Combined with PEG 4,000 / Sodium Acetate in Drop Droplet Steam Diffusion Mode, Different Protein Concentration

A pendant drop vapor diffusion crystallization method was performed on ABT-874 employing PEG 400 with 4,000 / sodium acetate with different protein concentrations. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.2. The protein concentration was adjusted to 50 mg / mL

A greased VDX plate and square OptiClear plastic cladding sheets were used. 500 μΥ. of a specific reservoir solution were prepared by mixing acetate buffer, 50% weight / volume of PEG 4,000 solution and Milli Q water in each well. In this example, the molarity of the acetate buffer was kept constant at about 0.1 M and PEG 4,000 ranged from about 4% weight / volume to about 8% weight / volume in 2% steps. Simultaneously, PEG 400 was brought to PEG 4,000 / acetate solutions and concentrations of about 30% weight / volume, 32% weight / volume, 34% weight / volume and 36% weight / volume, respectively. The pH was around 5.2 from start to finish. Each condition was evaluated in duplicate. About 1 μΥ. of the protein solution was mixed with about 1 // L of a specific reservoir solution on a square OptiClear plastic coating slide and the well was sealed with the inverted slide generating a drop drop experiment. The plates were stored at room temperature. Droplet microscopy was performed several times during the next thirty days. The conditions were classified as clear drops, drops containing random precipitation, drops containing crystals and drops containing mixtures of species and precipitated crystals.

RESULTS: In the 24 evaluated wells, no crystals were observed.

Example 12 - PEG 4000 / Sodium Acetate Grid Filtration in Drop Drop Steam Diffusion Mode, Different Protein Buffer

A pendant drop vapor diffusion crystallization method was performed on ABT-874 employing PEG 4,000 with different protein buffers. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.5. The protein concentration was adjusted to 10 mg / ml.

A greased VDX plate and square OptiClear plastic cladding sheets were used. 500 μΥ. of a specific reservoir solution were prepared by mixing acetate buffer, 50% weight / volume of PEG 4,000 solution and Milli Q water in each well. In this example, the molarity of the acetate buffer was kept constant at about 0.1 M and PEG 4,000 ranged from about 4% weight / volume to about 26% weight / volume in 2% steps. The pH was 5.5 from start to finish. Each condition was evaluated in duplicate. About 1 μΥ. of the protein solution was mixed with about 1 μΥ. of a specific reservoir solution on a square OptiClear plastic plating slide and the well was sealed with the inverted slide generating a pending drop experiment. The plates were stored at room temperature. Droplet microscopy was performed several times during the next five days. The conditions were classified as clear drops, drops containing random precipitation, drops containing crystals and drops containing mixtures of species and precipitated crystals.

RESULTS: In the 24 evaluated wells, the crystals were observed in a PEG 4,000 concentration of about 12% weight / volume, 18% weight / volume, 20% weight / volume, 22% weight / volume and 24% weight / volume, respectively. The crystals showed a morphology similar to the needle or bunch of needles.

Example 13 - PEG 4000 / Sodium Acetate Grid Filtration in Drop Droplet Steam Diffusion Mode, Different protein concentration

A method of crystallization of vapor diffusion in a droplet was performed in

ABT-874 employing PEG 4,000 with different protein concentrations. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.5. The protein concentration was adjusted to 5 mg / ml.

A greased VDX plate and square OptiClear plastic cladding sheets were used. 500 // L of a specific reservoir solution was prepared by mixing acetate buffer, 50% weight / volume of PEG 4,000 solution and Milli Q water in each well. In this example, the molarity of the acetate buffer was kept constant at about 0.1 M and PEG 4,000 ranged from about 4% weight / volume to about 26% weight / volume in 2% steps. The pH was 5.5 from start to finish. Each condition was evaluated in duplicate. About 1 // L of the protein solution was mixed with about 1 μL of a specific reservoir solution on a square OptiClear plastic coating slide and the well was sealed with the inverted slide generating a drop drop experiment. The plates were stored at room temperature. Droplet microscopy was performed several times during the next five days. The conditions were classified as clear drops, drops containing random precipitation, drops containing crystals and drops containing mixtures of species and precipitated crystals.

RESULTS: In the 24 evaluated wells, the crystals were observed at a PEG 4,000 concentration of about 10% weight / volume and 14% weight / volume, respectively. The crystals showed morphology similar to a cluster of needles.

Example 14 - PEG 4000 / Sodium Acetate Grid Filtration in Drop Drop Steam Diffusion Mode, Different Protein Buffer

A pendant drop vapor diffusion crystallization method was performed on ABT-874 employing PEG 4,000 / Sodium acetate with different protein buffer. ABT874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.5. The protein concentration was adjusted to 20 mg / ml.

A greased VDX plate and square OptiClear plastic cladding sheets were used. 500 μϊ. of a specific reservoir solution were prepared by mixing acetate buffer, 50% weight / volume of PEG 4,000 solution and Milli Q water in each well. In this example, the molarity of the acetate buffer was kept constant at about 0.1 M and PEG 4,000 ranged from about 4% weight / volume to about 26% weight / volume in 2% steps. The pH was 5.5 from start to finish. Each condition was evaluated in duplicate. About 1 μϊ. of the protein solution was mixed with about 1 μϊ. of a specific reservoir solution on a square OptiClear plastic plating slide and the well was sealed with the inverted slide generating a pending drop experiment. The plates were stored at room temperature. Droplet microscopy was performed several times during the next five days. The conditions were classified as clear drops, drops containing random precipitation, drops containing crystals and drops containing mixtures of species and precipitated crystals.

RESULTS: In the 24 evaluated wells, the crystals were observed in a PEG 4,000 concentration of about 10% weight / volume, 14% weight / volume, 16% weight / volume, 20% weight / volume and 22% weight / volume, respectively. The crystals showed a morphology similar to the needle or bunch of needles.

Example 15 - Broad Classification of Conditions in Steam Diffusion Mode

A broad classification of the drop droplet vapor diffusion crystallization method was performed in ABT-874. ABT-874 was buffered in a 20 mM HEPES / 150 mM sodium chloride buffer at pH 7.4. The protein concentration was adjusted to 10 mg / ml. In another case, the protein concentration was adjusted to 5 mg / ml. In another case, the protein concentration was adjusted to 20 mg / ml. Employing the Hydra II crystallization robot, the 96-well Greiner plates were adjusted to room temperature, employing several commercially available crystallization filters. The protein solution and the crystallizing agent were mixed in a ratio of about 1: 1, preferably 1: 1. The following filters have been employed. Hampton Crystal Screen 1 &amp; 2. Hampton Index Screen, Hampton SaltRX Screen (all from Hampton Research), Nextal The Classics, The Classics Lite, The PEGs, The Anions, The pH clear and The Ammonium sulphate (all from Nextal Biotechnologies).

After adding the protein to the crystallizing agent (three drops per addition, containing the three different concentrations of protein described above), the plates were sealed with Clearseal film. Any plate was adjusted in quadruplicate and stored at room temperature, 4 ° C, 27 ° C and 37 ° C, respectively. Drops microscopy was performed after six days. The conditions were classified as clear drops, drops containing random precipitation, drops containing crystals and drops containing mixtures of species and precipitated crystals.

RESULTS: Among the 10,368 conditions tested, 4 yielded crystals. The conditions comprised the following concentration of proteins and crystallizing agents, as declared by the manufacturers:

- room temperature, ABT-874 at about 20 mg / mL, 0.2M ammonium sulfate, 30% weight / volume PEG 8,000 (Hampton Crystal Screen, C6)

- 4 ° C, ABT-874 at about 5 mg / mL, 0.1 M HEPES pH 7.5, 5% weight / volume PEG 8,000 (Nextal The Classics Lite, F4)

- 4 ° C, ABT-874 at about 10 mg / mL, 0.1 M HEPES pH 7.5, 5% weight / volume of PEG 6,000, 2.5% volume / volume MPD (Nextal The Classics Lite, H9 )

- 4 ° C, ABT-874 at about 20 mg / mL, 0.1 M HEPES, 5% weight / volume of PEG 6,000, pH 7.00 (Nextal clear pH, C4)

The crystals showed needle-like or curl-like morphologies.

Example 16 - Filtration by PEG 4000 / Sodium Acetate Grid in Drop Droplet Steam Diffusion Mode, Different Adjustment

A pendant drop vapor diffusion crystallization method was performed on ABT-874 employing PEG 400 with 4,000 / sodium acetate with a different setting. ABT874 was buffered in a 20 mM HEPES / 150 mM sodium chloride buffer at pH 7.4. The protein concentration was adjusted to 10 mg / ml. In another case, the protein concentration was adjusted to 5 mg / ml.

A shined VDX plate and circular siliconized glass cladding sheets were used. 500 μ \ - of a specific reservoir solution was prepared by mixing acetate buffer, 50% w / v volume of PEG 4,000 solution and Mil li Q water in each well. In this example, the molarity of the acetate buffer was kept constant at about 0.1 M and PEG 4,000 concentration was used at about 12% weight / volume, 18% weight / volume, 24% weight / volume and 30% weight / volume, respectively. The pH varied from about 3.6 to about 5.6 in 0.2 step, generating 48 different conditions. Any condition was adjusted with the two protein concentrations as described above. About 1 pL of the protein solution was mixed with about 1 pL of a specific reservoir solution on a siliconized glass coating slide and the well was sealed with the inverted slide generating a drop drop experiment. The plates were stored at room temperature. Drops microscopy was performed after six days. The conditions were classified as clear drops, drops containing random precipitation, drops containing crystals and drops containing mixtures of species and precipitated crystals.

RESULTS: In the 96 conditions tested, crystals in the form of needle curls were observed with 5 mg / mL of ABT-874 and about 24% PEG 4,000 at a pH of about 5.6.

Example 17 - PEG 4,000 / Sodium Citrate Grid Filtration in Drop Drop Steam Diffusion Mode, Different Adjustment

A pendant drop vapor diffusion crystallization method was performed on ABT-874 employing PEG 4,000 / Sodium Citrate with a different setting. ABT-874 was buffered in a 20 mM HEPES / 150 mM sodium chloride buffer at pH 7.4. The protein concentration was adjusted to 10 mg / ml. In another case, the protein concentration was adjusted to 5 mg / ml.

A shined VDX plate and circular siliconized glass cladding sheets were used. 500 // L of a specific reservoir solution was prepared by mixing citrate buffer, 50% w / v of PEG 4,000 solution and Milli Q water in each well. In this example, the molarity of the citrate buffer was kept constant at about 0.1 M, and the PEG 4,000 concentration was employed at about 12% weight / volume, 18% weight / volume, 24% weight / volume or 30% weight / volume. The pH varied from about 4.2 to about 6.4 in 0.2 step, generating 48 different conditions. Any condition was adjusted with the two protein concentrations as described above. About 1 // L of the protein solution was mixed with about 1 / vL of a specific reservoir solution in a siliconized glass coating slide and the well was sealed with the inverted slide generating a drop drop experiment. The plates were stored at room temperature. Drops microscopy was performed after six days. The conditions were classified as clear drops, drops containing random precipitation, drops containing crystals and drops containing mixtures of species and precipitated crystals.

RESULTS: In the 96 conditions tested, no crystals were observed.

D. Batch Crystallization Experiments

A batch crystallization method was performed on ABT-874. The concentration values ​​provided in the following examples are initial values ​​with reference to the antibody solution and crystallization solution before mixing the two solutions.

All pH values, if not described otherwise, refer to the pH of a master acetate buffer solution before being combined with other substances, such as the crystallizing agent.

All buffer molarities, if not otherwise described, refer to the concentrations of sodium acetate in a master solution prior to pH adjustment, typically performed using glacial acetic acid.

Example 18 - PEG 4,000 / Sodium Acetate Condition in mL Batch Volume

A crystallization method was performed on ABT-874 using PEG 4,000 / Sodium acetate in a 1 mL tapping volume. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.2. The protein concentration was adjusted to 10 mg / ml Batch crystallization was performed by mixing about 500 pL of the protein solution with an equal volume of the crystallization buffer in a 1.5 ml Eppendorff reaction tube. 500 pL of a specific reservoir solution was prepared by mixing acetate buffer, 50% weight / volume of PEG 4,000 solution and Milli Q water. In this example, the grind of the acetate buffer was 0.1 M, and the pH of the acetate buffer was about 6.7. PEG 4,000 was used at a concentration of about 14% weight / volume. The reaction tube was stored at room temperature. Microscopy of the 1 pL aliquot was performed after 16 days.

RESULTS: Crystals were not observed after 16 days.

Example 19 - PEG 4000 / Sodium Acetate Grade Filtration in Batch Volume Mode of 300 z / L

A crystallization method was performed on ABT-874 using PEG

4,000 / Sodium acetate in a batch volume mode of 300 pL. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.5. The protein concentration was adjusted to 10 mg / mL

Batch crystallization was performed by mixing about 150 μΐ_ of the protein solution with an equal volume of the crystallization buffer in a well. The well plate was subsequently sealed with adhesive tape to prevent water evaporation. 150 / vL of a specific reservoir solution was prepared by mixing acetate buffer, 50% weight / volume of PEG 4,000 solution and Mil Li Q water in each well. In this example, the molarity of the acetate buffer was kept constant at about 0.1 M, and the pH of the acetate buffer was about 5.5 from start to finish. PEG 4,000 ranged from about 12% weight / volume to about 34% weight / volume in 2% steps. Any condition was assessed in triplicate. The plate was stored at room temperature. Droplet microscopy was performed for the next two days.

RESULTS: Crystals were observed in the 36 wells examined in the experiments, which were adjusted between 22% weight / volume and 26% weight / volume of PEG 4,000.

Example 20 - Condition of PEG 4,000 / Sodium Acetate in Batch Volume of 1 mL, Different Concentrations of PEG 4,000

A crystallization method was performed on ABT-874 using PEG 4,000 / Sodium Acetate in a batch volume of 1 mL using different concentrations of PEG 4,000. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.5. The protein concentration was adjusted to 10 mg / ml.

Batch crystallization was performed by mixing about 500 pL of the protein solution with an equal volume of the crystallization buffer in a 1.5 ml Eppendorff reaction tube. 500 pL of a specific reservoir solution was prepared by mixing acetate buffer, 50% weight / volume of PEG 4,000 solution and Milli Q water. In this example, the molarity of the acetate buffer was 0.1 M, and the The pH of the acetate buffer was about 5.5. PEG 4,000 was used at a concentration of about 22% weight / volume. The experiment was adjusted in quadruplicate. The reaction tubes were stored at room temperature. Microscopy of 1 pL aliquots was performed several times during the following 78 days. In addition, the crystal yield was as determined by OD 280. An aliquot of the suspension was centrifuged at 14,000 rpm, and the concentration of the protein in the supernatant was assessed.

RESULTS: Swordlike crystals appeared after seven days. No precipitated species were observed during the following months of storage. The crystal yield as determined by OD280 from the concentration of residual protein in the supernatant was between 50 and 70% after sixty days.

Example 21 - Condition of PEG 4,000 / Sodium Acetate in Batch Volume of 1 mL, Different Concentrations of PEG 4,000

A crystallization method was performed on ABT-874 using PEG 4,000 / Sodium Acetate in a batch volume of 1 mL using different concentrations of PEG 4,000. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.5. The protein concentration was adjusted to 10 mg / ml.

Batch crystallization was performed by mixing about 500 pL of the protein solution with an equal volume of the crystallization buffer in a 1.5 ml Eppendorff reaction tube. 500 pL of a specific reservoir solution was prepared by mixing acetate buffer, 50% weight / volume of PEG 4,000 solution and Milli Q water. In this example, the molarity of the acetate buffer was 0.1 M and the pH of the acetate buffer was about 5.5. PEG 4,000 was used at a concentration of about 26% weight / volume. The reaction tube was stored at room temperature. Microscopy of a 1 pL aliquot was performed several times during the following months.

RESULTS: After one day, precipitated species were observed. Swordlike crystals, in addition to the precipitate, were observed after five days.

Example 22 - Condition of PEG 4,000 / Sodium Acetate in Batch Volume of mL, Different Concentrations of PEG 4,000

A crystallization method was carried out on ABT-874 using PEG 4,000 / Sodium Acetate in a batch volume of 1 mL using different concentrations of PEG 4,000. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.5. The protein concentration was adjusted to 10 mg / ml.

Batch crystallization was performed by mixing about 500 pL of the protein solution with an equal volume of the crystallization buffer in a 1.5 ml Eppendorff reaction tube. 500 pL of a specific reservoir solution was prepared by mixing acetate buffer, 50% weight / volume of PEG 4,000 solution and Milli Q water. In this example, the molarity of the acetate buffer was 0.1 M, and the The pH of the acetate buffer was about 5.5. PEG 4,000 was used at a concentration of about 24% weight / volume. The reaction tube was stored at room temperature. Microscopy of the 1 pL aliquot was performed several times during the following months. In addition, the crystal yield was as determined by OD 280. An aliquot of the suspension was centrifuged at 14,000 rpm, and the concentration of the protein in the supernatant was assessed.

RESULTS: Needle-like crystals appeared after one day.

After five days, needle-like crystals and platelets were observed in addition to needle-like crystals. The crystal yield as determined by OD280 from the concentration of residual protein in the supernatant was between 60 and 70% after thirteen days.

Example 23 - PEG 4000 / Sodium Acetate Grade Filtration In 1 mL Batch Volume Mode, Different Protein Concentration

A crystallization method was performed on ABT-874 using PEG 4,000 / Sodium Acetate in a batch volume of 1 mL employing different concentrations of protein. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.5. The protein concentration was adjusted to 5 mg / ml.

Batch crystallization was performed by mixing about 500 µl of the protein solution with an equal volume of the crystallization buffer in a well. The well plate was subsequently sealed with adhesive tape to prevent water evaporation. 500 // L of a specific reservoir solution was prepared by mixing acetate buffer, 50% weight / volume of PEG 4,000 solution and Milli Q water in each well. In this example, the molarity of the acetate buffer was kept constant at about 0.1 M and the pH of the acetate buffer was about 5.5 from start to finish. PEG 4,000 ranged from about 12% weight / volume to about 34% weight / volume in 2% steps. Any condition was evaluated in duplicate. The plate was stored at room temperature. Microscopy of the drops was performed during the following month.

RESULTS: In the 24 wells examined, sword-like crystals were observed in the experiments, which were adjusted to approximately 24% weight / volume and 26% weight / volume of PEG 4,000.

Example 24 - PEG 4000 / Sodium Acetate Grade Filtration in 1 mL Batch Volume Mode, Different Adjustment

A crystallization method was performed on ABT-874 using PEG 4,000 / Sodium Acetate in a batch volume of 1 mL using a different setting. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.5. The protein concentration was adjusted to 10 mg / ml.

Batch crystallization was performed by mixing about 500 pL of the protein solution with an equal volume of the crystallization buffer in a well. The well plate was subsequently sealed with adhesive tape to prevent water evaporation. 500 pL of a specific reservoir solution was prepared by mixing acetate buffer, 50% weight / volume of PEG 4,000 solution and Milli Q water in each well. In this example, the molarity of the acetate buffer was kept constant at about 0.1 M and the pH of the acetate buffer was about 4.1, 4.6 and 5.1, respectively. PEG 4,000 ranged from about 20% weight / volume to about 28% weight / volume in 2% steps. The plate was stored at room temperature. Droplet microscopy was performed for the next four days.

RESULTS: In the 18 wells examined, sword-like crystals were observed in the experiments which was adjusted with 28% weight / volume of PEG 4,000 and sodium acetate buffer at pH 5.1.

Example 25 - PEG 4,000 / Sodium Acetate Condition in 2 mL Batch Volume, Different Temperature

A crystallization method was performed on ABT-874 using PEG 4,000 / Sodium acetate in a batch volume of 2 ml using different temperatures. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.5. The protein concentration was adjusted to 10 mg / ml. Batch crystallization was performed by mixing about 1 ml of the protein solution with an equal volume of the crystallization buffer in a 2 ml Eppendorff reaction tube. 1 mL of a specific reservoir solution was prepared by mixing acetate buffer, 50% w / v of PEG 4,000 solution and Mil Li Q water. In this example, the molarity of the acetate buffer was 0.1 M, and the pH of the acetate buffer was about 5.5. PEG 4,000 was used at a concentration of about 22% weight / volume. The reaction tube was stored at 4-8 ° C. Microscopy of the 1 μ \ - aliquot was performed several times during the following month.

RESULTS: Precipitated species were observed after overnight storage.

Example 26 - Crystallization Condition of PEG 4,000 / 10 mL Sodium Acetate in Batch Volume, With Stirring

A crystallization method was performed on ABT-874 using PEG 4,000 / Sodium Acetate in a batch volume of 10 mL and with stirring. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.5. The protein concentration was adjusted to 10 mg / ml.

Batch crystallization was performed by mixing about 5 ml of a protein solution with an equal volume of the crystallization buffer in a 50 ml Falcon tube. 5 ml of the crystallization buffer was prepared by mixing acetate buffer, 50% weight / volume of PEG 4,000 solution and Milli Q water in the tube. In this example, the molarity of the acetate buffer was about 0.1 M and the pH of the acetate buffer was about 5.5. PEG 4,000 was used at a concentration of about 24% weight / volume. The tube was stored at room temperature, shaking the batch on a laboratory shaker. Microscopy of the 1 pL aliquot of the solution was performed several times during the following weeks.

RESULTS: Swordlike crystals appeared after six days, but were almost completely absorbed on the surface of the container. It could be concluded from microscopy that the batch was free of precipitated species. The crystallization liquor was almost completely clear.

Example 27 - Crystallization Condition of PEG 4,000 / Sodium Acetate in Batch Volume of 10 mL, Without Shaking

A crystallization method was performed on ABT-874 using PEG 4,000 / Sodium Acetate in a batch volume of 10 mL without stirring. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.5. The protein concentration was adjusted to 10 mg / ml.

Batch crystallization was performed by mixing about 5 ml of the protein solution with an equal volume of the crystallization buffer in a 50 ml Falcon tube. 5 ml of the crystallization buffer was prepared by mixing acetate buffer, 50% weight / volume of PEG 4,000 solution and Milli Q water in the tube. In this example, the molarity of the acetate buffer was about 0.1 M and the pH of the acetate buffer was about

5.5. PEG 4,000 was used at a concentration of about 24% weight / volume. The tube was stored at room temperature. Microscopy of the 1 pL aliquot of the solution was performed several times during the following weeks. In addition, the crystal yield was as determined by OD 280. An aliquot of the suspension was centrifuged at 14,000 rpm and the concentration of the protein in the supernatant was assessed.

RESULTS: Needle-like crystals appeared after one day. After four days, needle-like crystals were observed in addition to needle-like crystals. The crystal yield as determined by OD280 from the concentration of residual protein in the supernatant was between 30 and 40% after seven days.

Example 28 - Crystallization Condition of PEG 4,000 / Sodium Acetate in 10 mL Batch Volume, With Stirring, Different Container Material

A crystallization method was performed on ABT-874 using PEG 4,000 / Sodium Acetate in a batch volume of 10 ml using different agitation and container materials. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.5. The protein concentration was adjusted to 10 mg / ml.

Batch crystallization was performed by mixing about 5 ml of the protein solution with an equal volume of the crystallization buffer in a 50 ml class I glass vial. 5 ml of the crystallization buffer were prepared by mixing acetate, 50% weight / volume of PEG 4,000 solution and Milli Q water in a bottle. In this example, the molarity of the acetate buffer was about 0.1 M and the pH of the acetate buffer was about 5.5. PEG 4,000 was used at a concentration of about 24% weight / volume. The flask was stored at room temperature, shaking the batch on a laboratory shaker. Microscopy of the 1 // L aliquot of the solution was performed several times during the following weeks. In addition, the crystal yield was as determined by OD 280. An aliquot of the suspension was centrifuged at 14,000 rpm and the concentration of the protein in the supernatant was assessed.

RESULTS: Swordlike crystals were observed after eighteen days. The crystal yield as determined by OD280 from the concentration of residual protein in the supernatant was between 40 and 50% after eighteen days. A light microscopic photo of the needle clusters (photo width corresponding to a length of 450 // m) is shown in figure 7.

Example 29 - Crystallization Condition of PEG 4,000 / Sodium Acetate in Batch Volume of 10 mL, with Stirring, Different Container Material and Influence of Polysorbate 80

A crystallization method was performed on ABT-874 using PEG 4,000 / Sodium Acetate in a batch volume of 10 mL, with agitation, different container materials and influence of polysorbate 80. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.5. The protein concentration was adjusted to 10 mg / ml.

Batch crystallization was performed by mixing about 5 ml of the protein solution with an equal volume of the crystallization buffer in a 50 ml glass bottle of ciasis i. 5 ml of the crystallization buffer was prepared by mixing acetate buffer, 50% weight / volume of PEG 4,000 solution and Milli Q water in the flask. In this example, the molarity of the acetate buffer was about 0.1 M and the pH of the acetate buffer was about 5.5. PEG 4,000 was used at a concentration of about 24% weight / volume. In addition, polysorbate 80 at a concentration of 0.1% was added to the buffer. The flask was stored at room temperature, shaking the batch on a laboratory shaker. Microscopy of the 1 // L aliquot of the solution was performed several times during the following weeks. In addition, the crystal yield was as determined by OD 280. An aliquot of the suspension was centrifuged at 14,000 rpm and the protein concentration in the supernatant was assessed.

RESULTS: Swordlike crystals were observed after eighteen days. No difference was observed between the shape of the crystal in this example and in Example 28 (without adding polysorbate 80). The crystal yield as determined by OD280 from the concentration of residual protein in the supernatant was between 25 and 35% after eighteen days.

Example 30 - Different Crystallization Conditions of PEG 4,000 / 48 Acetate Sodium in 10 mL batch volume and Comparison of Stirred and Unstirred Batches

A crystallization method was performed on ABT-874 using PEG 4,000 / Sodium Acetate in a batch volume of 10 mL using a comparison of stirred and unstirred batches. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.5. The protein concentration was adjusted to 10 mg / ml.

Batch crystallization was performed by mixing about 5 ml of the protein solution with an equal volume of the crystallization buffer in a 50 ml class I glass vial. 5 ml of the crystallization buffer were prepared by mixing a acetate, 50% weight / volume of PEG 4,000 solution and Milli Q water in the bottle. In this example, the molarity of the acetate buffer was about 0.1 M and the pH of the acetate buffer was about 5.5. PEG 4,000 was used at a concentration of about 22% weight / volume and 24% weight / volume. The flasks were stored at room temperature, either without agitation or by shaking the batch by tipping. Microscopy of the 1 / zL aliquot of the solution was performed several times during the following weeks. In addition, the crystal yield of a batch was as determined by OD 280. An aliquot of the suspension was centrifuged at 14,000 rpm and the protein concentration in the supernatant was assessed.

RESULTS: In both agitated batches, the precipitated species were observed after 26 days. The unstirred batch with the buffer of about 22% weight / volume of PEG 4,000 contained swordlike crystals after 26 days, however the crystal yield was judged low as the suspension was almost macroscopically clear. The non-stirred batch with the buffer of about 24% weight / volume of PEG 4,000 contained sword-like crystals after 26 days. The yield, as determined from the supernatant after 70 days, was between 65 and 75%.

Example 31 - Influence of sowing

The influence of sowing on the crystal yield of ABT-874 was examined. The batch not stirred with the crystallization buffer containing about 22% weight / volume of PEG 4,000 from Example 30 showed very low crystal yield after 26 days. Therefore, the batch was incubated with about 100 µl of the un stirred batch with the crystallization buffer containing about 24% weight / volume of PEG 4,000 of the same example.

RESULTS: No obvious yield extension resulted from incubation with seed crystals.

Example 32 - Crystallization Conditions of PEG 4,000 / Sodium Acetate in 10 mL Batch Volume, Different Protein Concentration, Comparison of Stirred and Unstirred Batch

A crystallization method was performed on ABT-874 using PEG

4,000 / Sodium acetate in a 10 ml batch volume employing different protein concentrations and a stirred and unstuffed batch comparison. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.5. The protein concentration was adjusted to 5 mg / ml.

Batch crystallization was carried out by mixing about 5 ml of the protein solution with an equal volume of the crystallization buffer in a 15 ml class I glass vial. 5 ml of the crystallization buffer were prepared by mixing a acetate, 50% weight / volume of PEG 4,000 solution and Milli Q water in the bottle. In this example, the molarity of the acetate buffer was about 0.1 M and the pH of the acetate buffer was about 5.5. PEG 4,000 was used at a concentration of about 22% weight / volume, 24% weight / volume and 26% weight / volume. The flasks were stored at room temperature, either without agitation or by shaking the batch on a laboratory shaker. Microscopy of the 1 μΥ aliquot. of the solution was carried out several times during the following weeks. In addition, the crystal yield of a batch was as determined by OD 280. An aliquot of the suspension was centrifuged at 14,000 rpm and the protein concentration in the supernatant was assessed.

RESULTS: Batches containing the buffer with about 22% weight / volume and about 24% weight / volume of PEG 4,000 were clear after 65 days. Although the agitated batch containing the crystallization buffer with about 26% weight / volume of PEG 4,000 contained precipitated species after 4 days, the un stirred batch of the same crystallization buffer contained sword-like crystals after 4 days. The crystal yield of this specific batch as determined from the supernatant after 26 days was between 40 and 50%. A microscopic photo in light of the crystals (width of the photo corresponding to a length of 225 // m) obtained without agitation is shown in figure 8.

Example 33 - Crystallization Condition of PEG 4,000 / Sodium Acetate in Batch Volume of 10 mL, Different Setting

A crystallization method was performed on ABT-874 using PEG 4,000 / Sodium Acetate in a batch volume of 10 mL employing a different setting. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.5. The protein concentration was adjusted to 10 mg / ml.

Batch crystallization was performed by mixing about 5 ml of the protein solution with an equal volume of the crystallization buffer in a 15 ml Falcon tube. 5 ml of the crystallization buffer was prepared by mixing acetate buffer, 50% weight / volume of PEG 4,000 solution and Milli Q water in the tube. In this example, the molarity of the acetate buffer was about 0.1 M and the pH of the acetate buffer was about

5.5. PEG 4,000 was used at a concentration of about 22% weight / volume. The tube was stored at room temperature. Microscopy of the 1 μΥ aliquot. The solution was carried out several times during the following weeks. In addition, the crystal yield of the batch was as determined by OD 280. An aliquot of the suspension was centrifuged at 14,000 rpm and the concentration of the protein in the supernatant was assessed.

RESULTS: Swordlike crystals were observed after 11 days. The crystal yield of this batch as determined from the supernatant after 26 days was between 40 and 50%. A microscopic photo in crystal light (width of the photo corresponding to a length of 450 μπι) obtained without shaking after 26 days is shown in figure 9.

Example 34a - Crystallization Condition of PEG 4,000 / Sodium Acetate in Batch Volume of 50 mL

A crystallization method was performed on ABT-874 using PEG 4,000 / Sodium Acetate in a batch volume of 50 mL. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.5. The protein concentration was adjusted to 10 mg / ml.

Batch crystallization was performed by mixing about 25 mL of the protein solution with an equal volume of the crystallization buffer in a 50 mL Falcon tube. 25 ml of the crystallization buffer was prepared by mixing acetate buffer, 50% weight / volume of PEG 4,000 solution and Midi Q water in the tube. In this example, the molarity of the acetate buffer was about 0.1 M and the pH of the acetate buffer was about

5.5. PEG 4,000 was used at a concentration of about 22% weight / volume. The tube was stored at room temperature. Microscopy of the 1 μΥ aliquot. of the solution was carried out several times during the following weeks. In addition, the crystal yield of the batch was as determined by OD 280. An aliquot of the suspension was centrifuged at 14,000 rpm and the concentration of the protein in the supernatant was assessed.

RESULTS: Swordlike crystals were observed after 3 days. The crystal yield of this batch as determined from the supernatant after 16 days was between 50 and 60%.

Example 34b - Crystallization Condition of PEG 4,000 / Sodium Acetate in Batch Volume of 700 mL

A crystallization method was performed on ABT-874 using PEG 4,000 / Sodium Acetate in a batch volume of 700 mL. ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.5. The protein concentration was adjusted to 10 mg / ml.

Batch crystallization was performed by mixing about 350 mL of the protein solution with an equal volume of the crystallization buffer in a 1 L polypropylene bottle. 350 mL of the crystallization buffer were prepared by mixing acetate buffer, PEG 4,000 and Milli Q water. In this example, the molarity of the acetate buffer was about 0.1 M, and the pH of the acetate buffer was about 5.5. PEG 4,000 was used at a concentration of about 22% weight / volume. The bottle was stored at room temperature. Microscopy of the 1 // L aliquot of the solution was performed after 40 days. Additionally, the batch crystal yield was as determined by OD

280. An aliquot of the suspension was centrifuged at 14,000 rpm and the protein concentration in the supernatant was assessed.

RESULTS: Swordlike crystals were observed after 40 days. The crystal yield of this batch as determined from the supernatant after 40 days was between 50 and 60%. A microscopic photo in crystal light (width of the photo corresponding to a length of 450 pm) obtained after 40 days without shaking is shown in figure 10.

The experimental conditions of the batch experiments above are summarized in Table 1 below:

Table 1: Batch experiments

<td>Ex in peep</td><td>Vol. ba- screen- gives, mL</td><td>Crystallization buffer (initial conc.</td><td>Agi OK- Here O</td><td>Crystals (yielded ment, %)</td><td>PH OK mp to</td><td>pH Fi- nal</td><td>Conc. Final protect ina, mg / m L</td><td>Tem- ratura</td><td>Day of con trolley visual</td>

<td> 18</td><td> 1</td><td> 14%¹ PEG 4,000, 0.1 M NaAc</td><td></td><td></td><td> 6,7</td><td></td><td> 5</td><td>ambience ente</td><td>16th day</td>

<td> 25</td><td> 2</td><td>22% PEG 4,000, 0.1M NaAc</td><td></td><td>Precip</td><td> 5,5</td><td> 5,6</td><td> 5</td><td>4-8 ° C</td><td>1st day</td>

<td> 19</td><td> 0,3</td><td>22% -26% PEG 4,000, 0.1M NaAc</td><td></td><td>+ (na)</td><td> 5,5</td><td></td><td> 5</td><td>ambience ente</td><td>2nd day</td>

<td> 20</td><td> 1</td><td>22% PEG 4,000, 0.1 M NaAc</td><td></td><td> +(50- 70)</td><td> 5,5</td><td> 5,6</td><td> 5</td><td>ambience ente</td><td>7th day</td>

<td> 21</td><td> 1</td><td>26% PEG 4,000, 0.1 M NaAc</td><td></td><td>+ (na)</td><td> 5,5</td><td> 5,6</td><td> 5</td><td>ambience ente</td><td>5th day</td>

<td> 22</td><td> 1</td><td>24% PEG 4,000, 0.1M NaAc</td><td></td><td>+ (60- 70) (13d)</td><td> 5,5</td><td> 5,6</td><td> 5</td><td>ambience ente</td><td>1st day</td>

<td> 23</td><td> 1</td><td>24% -26% PEG 4,000, 0.1 M NaAc</td><td></td><td>+ (na)</td><td> 5,5</td><td> 5,6</td><td> 2,5</td><td>ambience ente</td><td>2nd day</td>

<td> 24</td><td> 1</td><td>28% PEG 4,000, 0.1 M NaAc</td><td></td><td>+ (na)</td><td> 5,1</td><td> 5,2</td><td></td><td>ambience ente</td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td> 5,3</td><td></td><td></td><td></td>

<td> 26</td><td> 10</td><td>24% PEG 4,000, 0.1M Na Ac</td><td> +</td><td>+ (na)</td><td> 5,5</td><td> 5,6</td><td> 5</td><td>ambience ente</td><td>4th day</td>

<td> 27</td><td> 10</td><td>24% PEG 4,000, 0.1 M Na Ac</td><td></td><td> +(30- 40)</td><td> 5,5</td><td> 5,6</td><td> 5</td><td>ambience ente</td><td>6th day</td>

<td> 28</td><td> 10</td><td>24% PEG 4,000, 0.1 M Na Ac</td><td> +</td><td> +(40- 50)</td><td> 5,5</td><td> 5,6</td><td> 5</td><td>ambience ente</td><td>1st day</td>

<td> 29</td><td> 10</td><td>24% PEG 4,000, 0.1M Na Ac 0.1% polysorbate 80</td><td> +</td><td> +(25- 35)</td><td> 5,5</td><td> 5,6</td><td> 5</td><td>ambience ente</td><td>18th day</td>

<td rowspan="4"> 30</td><td rowspan="4"> 10</td><td>22% PEG 4,000, 0.1M Na Ac</td><td></td><td>+ (na)</td><td rowspan="4"> 5,5</td><td rowspan="4"> 5,6</td><td rowspan="4"> 5</td><td rowspan="4">ambience ente</td><td>26th day</td>

<td>22% PEG 4,000, 0.1M Na Ac</td><td> +</td><td>precip</td><td></td>

<td>24% PEG 4,000, 0.1M Na Ac</td><td></td><td>+ (65- 75) (70d )</td><td>26th day</td>

<td>24% PEG 4,000, 0.1M NaAc</td><td> +</td><td>precip</td><td></td>

<td> 32</td><td> 10</td><td>22% PEG 4,000, 0.1M NaAc</td><td></td><td>huh- no</td><td rowspan="4"> 5,5</td><td rowspan="4"> 5,6</td><td rowspan="4"> 2,5</td><td rowspan="4">ambience ente</td><td>64 ° day</td>

<td></td><td></td><td>24% PEG 4,000, 0.1M NaAc</td><td></td><td>huh- no</td><td>64 ° day</td>

<td></td><td></td><td>26% PEG 4,000, 0.1M NaAc</td><td></td><td> +(40- 50)</td><td>4th day</td>

<td></td><td></td><td>26% PEG 4,000, 0.1M NaAc</td><td> +</td><td>precip</td><td>4th day</td>

<td> 33</td><td> 10</td><td>22% PEG 4,000, 0.1 M NaAc</td><td></td><td>+ (40- 50) (26d )</td><td> 5,5</td><td> 5,6</td><td> 5</td><td>ambience ente</td><td>11th day</td>

<td>34 The</td><td> 50</td><td>22% PEG 4,000, 0.1M NaAc</td><td></td><td>+ (50- 60) (16d )</td><td> 5,5</td><td> 5,6</td><td> 5</td><td>ambience ente</td><td>3rd day</td>

<td>34 B</td><td> 700</td><td>22% PEG 4,000, 0.1M NaAc</td><td></td><td>+ (50- 60) (40d )</td><td> 5,5</td><td> 5,6</td><td> 5</td><td>ambience ente</td><td>40 ° day</td>

¹ % (weight / volume).

E. Methods for Processing and Analyzing the Crystal

Example 35 - Washing Crystals

After the crystals have formed, a washing step without redissolving the crystals may be favorable. After the crystallization process was finished, the crystal paste was transferred to a centrifuge tube and centrifuged at 500 x 1,000 xg for twenty minutes. Centrifugation was carried out at 4 ° C or at room temperature. After centrifugation, the supernatant was decanted and the crystal microspheres were easily resuspended in a buffer containing about 24% weight / volume of PEG 4,000 in about 0.1 M sodium acetate at a pH of about 5.5. No measured solubility of the ABT-874 crystals was run in such a wash buffer, as analyzed by OD280. The centrifugation / resuspension steps were subsequently repeated one to three times and after this washing procedure, the microspheres were suspended and stored in such a buffer.

Example 36 - Analysis of crystals by SDS PAGE

In order to confirm the protein character of the crystals, they were washed with a wash buffer as described in Example 32. After certifying by using OD280 that there was no more protein dissolved in the liquor, the crystals were centrifuged, the supernatant was decanted and the crystals were subsequently dissolved in distilled water. The OD280 measurement of this solution revealed that the protein was now present as the absorbance of the sample was now significantly higher as in the residual wash buffer. SDS PAGE analysis of this redissolved crystal solution, when compared to an original ABT-874 sample, showed the same pattern.

Example 37 - Analysis of crystals by SE-HPLC

In order to evaluate the content of aggregated species of the ABT-874 crystals, an aliquot of the washed crystals was centrifuged and redissolved in the SE-HPLC operating buffer (92 mM disodium hydrogen phosphate / 211 mM disodium sulfate, pH 7.0 ). Immediately after the end of the crystallization process, in this example, 16 days at room temperature, the aggregate content typically increased slightly from about 0.9% to about 1.6-1.7%. It is not yet clear whether such aggregates are contained in the crystals or on their surfaces and have not been properly removed by the washing process.

F. Miscellaneous Examples

The concentration values ​​provided in the following examples are initial values ​​with reference to the antibody solution and crystallization solution before mixing the two solutions.

All pH values, if not described otherwise, refer to the pH of a master acetate buffer solution before it is combined with other substances, such as the crystallizing agent.

All buffer molarities, if not otherwise described, refer to the concentrations of sodium acetate in a master solution prior to pH adjustment, typically performed using glacial acetic acid.

Example 38 - Solid Crystallization Agent

ABT-874 was buffered in a buffer containing about 0.1 M sodium acetate at a pH of about 5.5. The protein concentration was adjusted to 10 mg / ml.

Batch crystallization was performed by mixing about 500 // L of the protein solution with about 380 μ \ - acetate buffer (0.1 M, pH 5.5) in a 2 mL Eppendorf reaction tube. Subsequently, solid polyethylene glycol was added to a final concentration of 12% w / v (120 mg / ml). The tube was subsequently closed and stirred until the crystallizing agent completely dissolved. The tube was stored at room temperature without stirring. Microscopy of the aliquots of the crystallization mixture was performed several times during the following weeks.

RESULTS: Swordlike crystals were observed after seven days.

Example 39 - Different Buffer Preparation and Crystals Preparation Protocol

In this example, acetate buffers were prepared as described below: 60 g of glacial acetic acid were diluted with about 840 ml of purified water. The pH was adjusted with sodium hydroxide solution and the volume adjusted to 1,000 mL. In this case, the amount of total acetate was fixed at 1 M (100 mM in the protein solution, in the crystallization buffer and in the crystallization mixture).

Crystallization was carried out according to Example 34a; sword-like crystals were observed after three days.

Example 40 - Preparation of Encapsulated Crystals

The crystals as obtained in Example 34 were positively charged as determined by measuring zeta potential using Malvern Instruments Zetasizer nano. The crystals were washed and suspended in a buffer containing excipients that preserved the crystallization and that had a pH that kept the crystals charged. Subsequently, an appropriate encapsulating agent was added to the crystal suspension. In this context, an appropriate encapsulating agent is a (polymeric) substance with low toxicity, biodegradability and contrionic character. Due to its contrionic character, the substance is attracted by the crystals and allows coating. With this technique, the dissolution of the crystals is preferably prolonged in media that do not contain any other excipients while maintaining crystallinity.

Example 41 - Preparation of Encapsulated / Embedded Crystals

The crystals are obtained as described in Example 34. The crystals are washed and suspended in a buffer containing excipients that retain crystallinity.

The crystals can then be embedded by drying them and combining these dry crystals with a vehicle, for example, by compression, dispersion by melting, etc.

- encapsulated / embedded by combining crystal suspension with a vehicle solution that is not miscible with water. The vehicle precipitates after removing the solvent from the vehicle. Subsequently, the material is dried.

- encapsulated / embedded by combining a crystal suspension with a water miscible vehicle solution. The vehicle precipitates as its solubility limit is exceeded in the mixture.

- embedded by combining the dry crystals or a crystal suspension with a water miscible vehicle solution.

- embedded by combining the dry crystals with a carrier solution that is not miscible in water.

Example 42 - Investigation of the precipitated ABT-874

a) Precipitation

Acetate buffer was prepared by dissolving 1 mol of sodium acetate in water and adjusting the pH to 5.5 with acetic acid (100%). The master solution was diluted 1:10 with water to change the buffer. The PEG 4,000 solution was prepared by dissolving 20 g of PEG 4,000 in 5 ml of 1 M sodium acetate, buffer pH 5.5 and water. After dissolution, the volume was adjusted to 50 ml with water. 5 mL of the 10 mg / mL of ABT874 (in 0.1 M sodium acetate buffer, pH 5.5) (original buffer exchanged for diafiltration) were mixed with 5 mL of 40% PEG 4,000 in 0.1 M acetate buffer sodium, pH 5.5.

The precipitated batch was kept at room temperature all night without stirring. There was no formation of birefringent particles in the magnitude of approximately 1-10 pm.

b) Washing of the precipitate mL of the precipitate paste was placed in a centrifuge and centrifuged at 500 xg for 20 minutes. The supernatants were discarded and the microspheres were resuspended in 2 ml of 40% PEG 4,000 in 0.1 M sodium acetate buffer, pH 5.5 (prepared according to the procedure above). The protein concentration of the final suspension was determined by OD280 to be 3.9 mg / ml.

G. Characterization of the Crystal

The following section summarizes the experiments that were carried out to determine whether the crystalline monoclonal antibody ABT-874 maintains the bioactivity characteristic of ABT-874 never crystallized by redissolving the crystalline material.

G1. Bioactivity Test by Determination of IFN-v Production of NK92 Cells

a) General Method

The biological activity of the dissolved ABT-874 crystals was measured by a cell-based assay that monitors the IFN-γ production of NK-92 cells in response to stimulation by IL-12. Before analysis, samples were first diluted to 30 µg / ml in cell culture medium (σ-ΜΕΜ medium with 20% FCS and 200 mM L-glutamine). Subsequently the samples were further diluted in 11 steps from 3 µg / ml to 0.1 ng / ml. The IL-12 solution was diluted to 10 ng / ml in the cell culture medium and added to the ABT-874 samples. The mixtures were then incubated at 37 ° C and CO₂ at 5% for 1 hour.

A suspension of NK-92 cells (2.0 x 10⁶ cells / mL) was pipetted into a 96-well microplate, ABT-874 / IL-12 mixtures were added to the cells and the microplates were then incubated at 37 ° C and CO₂ at 5% for about 20 hours. After incubation, the microplates were centrifuged at 1,000 rpm and 5 ° C for 10 minutes and 50 // L of the supernatant from each well was used to measure the amount of IFN-γ produced by the cells by an ELISA (ELISA Kit Human Interferon-γ, Pierce, Cat. No. EHIFNG).

The biotinylated anti-IFN-γ antibody solution was pipetted into the 96-well pre-coated microplate and cell culture supernatants were added (4 rows for each of both samples). After incubating the microplate for 2 hours at room temperature, it was washed. After that, the Streptavidin-HRP solution was added and the microplate was incubated for another 30 minutes and then washed. After the TMB substrate was added, the microplate was incubated at room temperature for about 20 minutes in the dark and the reaction was then stopped by adding the stop solution.

Finally, absorption was measured within the next 5 minutes on a microplate reader at 450 nm (correction wavelength 550 nm) and the results shown in a graph versus the ABT-874 concentration. The CI values₅₀ were then evaluated using a 4-parameter non-linear curve fit and the sample's reactive biological activity was calculated by dividing the IC value₅₀ of the reference standard by the IC value₅₀ of the sample and multiplying by 100%.

b) ABT-874 Crystals Related Activity

The test was performed as a comparison of the biological activity of the sample to that of a reference standard. The amounts of IFN-γ produced by the cells were measured by a commercially available ELISA kit and were reported as absorption units at 450 nm wave length. These values, plotted against the ABT-874 concentration and evaluated by a non-linear regression of 4 parameters, revealed the CI values₅₀ for inhibiting the effect of IL-12 by ABT-874. Since both samples were operated in four repetitions on a microplate, this results in four IC values₅₀ for ABT-874 reference standard and the sample, respectively57. Subsequently, the mean of the CI values₅₀ of the reference standard was calculated and the relative activity of each repetition of the sample was evaluated by dividing the IC value₅₀average of the reference standard by the CI value₅₀ sample and multiplied by 100%.

The sample test (crystal suspension 2.9 mg / mL) revealed a 98% relative biological activity. Thus, the sample can be considered as complete and biologically active.

G2. Microscopic Characterization

The microscopic characterization data for ABT-874 crystals will be presented below:

a) Optical analysis of mAb crystal batch samples

After homogenization, aliquots of 1 to 10 pL of sample volume were pipetted into an object holding plate and covered with a glass coating slide. The crystal preparations were evaluated using a Zeiss Axiovert 25 inverted light microscope equipped with 10x E-PI eyepieces and 10x, 20x and 40x objectives, respectively. The photos were taken using a digital camera (Sony Cybershot DSC S75).

b) Characterization of Scanning Electron Microscope (SEM) of ABT-874 Crystals

In order to image protein crystals with an electron microscope, they must be dry, electrically conductive and stable enough to tolerate high vacuum and the energy of an electron beam. This protocoio separates the crystals from its buffer by filtration, stabilizes the crystals by chemical fixation of them with a glutaraldehyde based fixative, dehydrates them through a graded series of ethanol, dries them by a critical point method and coats the plasma with gold to make them electrically conductive.

b1) Materials

- 0.2 M Sorensen's phosphate buffer (SPB) - 0.15 M disodium phosphate, 0.05 M monobasic potassium phosphate, pH 7.3

- Karnovsky fixative - 2.5% glutaraldehyde, 1.5% paraphoric aldehyde, 0.1 M

SPB

- 50%, 75%, 95% and 100% ethanol

- crystal sample of ABT-874 in crystallization buffer (from Example 34, stored in washing buffer from Example 35)

- ABT-874 crystallization buffer (Example 35 wash buffer)

- Millipore stainless steel filter mount for attachment to 13 mm filter membranes for syringes

- 0.4 pm polycarbonate filter membranes (Nucleopore, Cat # 110407) b2) Equipment

- Critical Point Dryer (CPD) - Baltec Model CPD030, Asset

LC978501

- Scanning Electron Microscope (SEM) - XL30 Phillips field emission scanning electron microscope

- Coating spray - Denton Desk II coating spray,

Asset LC827847 b3) Procedure

Steps 3-12 were performed by washing the solution through the filter assembly and keeping the syringe in the filter assembly for a predetermined waiting time.

1. Load the filter holder of the syringe with polycarbonate filter;

2. Mix 0.1 mL of the crystal sample with 0.4 mL of the crystal buffer in the 1.0 mL syringe;

3. Dispense the diluted crystal solution through the filter assembly;

4. Dispense 1 mL of the crystal buffer and wait 2 minutes;

5. Dispense 1 mL of 50% fixation buffer, 50% crystal buffer and wait 2 minutes;

6. Dispense 1 mL of the fixative at 100% and wait 2 minutes;

7. Dispense 1 mL of SPB and wait 2 minutes;

8. Dispense 1 mL of SPB and wait 2 minutes, again;

9. Dispense 1 mL of 50% ethanol and wait 2 minutes;

10. Dispense 1 mL of 75% ethanol and wait 2 minutes;

11. Dispense 1 mL of 95% ethanol and wait 2 minutes;

12. Dispense 1 mL of 100% ethanol and wait 2 minutes, repeat step 3 times;

13. Transfer the filter membrane with the attached crystals to CPD filled with 100% ethanol;

14. Process the filter through the CPD as follows:

The. five CO exchanges₂ liquid at 10 ° C, mixing for 5 minutes per change;

B. heat to 40 ° C, pressure 8,000 kPa; and

ç. slowly pour into the atmosphere for 20 minutes;

15. Mount the filter membrane on the SEM support;

16. Spray the coating with gold for 60 seconds;

17. Examination with SEM.

c) Results

The attached figures 1 to 5 illustrate the representative photos of the ABT-874 crystals.

Figure 1 shows a light micrograph of the crystals of ABT-74 in the crystallization buffer (from Example 34, stored in the washing buffer from Example 35) obtained according to Example 34. The crystal habit is similar to the fixed dry crystals shown in figures 2 to 5. The crystals exhibited birefringence.

Figures 2 to 5 show SEMs at different magnifications than the ABT-874 crystals obtained according to Example 34.

G3. Birefringence

The crystals as generated from all batch experiments showed birefringence.

G4. Syringe Usage Capacity

A suspension of ABT-874 crystal of 150 mg / ml of protein incorporated into the crystals and formulated in a washing buffer of Example 35 can be used in syringe through a 27G needle.

H. Capillary Isoelectric Focusing experiments (clEF) with ABT-874

a) Equipment

The ÍCE280 analyzer (Convergent Bioscience) was used for the analysis. System ID 1054 (IS number 2785).

b) Material

The capillary used was 50 mm long, ID column of 100 // m, coated (Convergent, Catalog number 101700. The electrolytes used were - Anolith (80 mM H3PO4) and Catholite (100 mM NaOH). (Convergent, Catalog number 101800 The vehicle ampholyte is Pharmalyte 4% (8-10.5), (GE Healthcare, Catalog number 17-0455-01. The additive was methyl cellulose (0.35%), (Convergent, Catalog number 101876) . The internal pl markers were a mixture of BioRad pl markers (8.4, 8.5, 10.1 and 10.4 - BioRad, Catalog number 148-2100, Lot number 482-511).

<td></td><td>Volume (// L)</td>

<td>Pl Marker 8.4</td><td> 2,5</td>

<td>Pl marker 8.5</td><td> 2,5</td>

<td>Pl marker 10.1</td><td> 2,5</td>

<td>Pl Marker 10.4</td><td> 2,5</td>

<td>Water</td><td> 40</td>

<td>Total</td><td> 50</td>

c) Methods

The focusing time was 2 minutes at 1,500V and 20 minutes at 3,000V. Sample preparation procedure - Mab crystals, Mab precipitate and reference standard were all diluted to about 1 mg / mL in Milli-Q water. Sample preparation procedure (with urea).

<td></td><td>Volume (// L)</td>

<td>Milli-Q Water</td><td> 92</td>

<td>Methyl cellulose 1%</td><td> 70</td>

<td>Vehicle - Pharmalyte</td><td> 8</td>

<td>Sample (1 mg / mL)</td><td> 30</td>

<td>Pl marker mix (Table 1)</td><td> 16</td>

<td>Total</td><td> 216</td>

The samples were mixed in 1.5 ml microcentrifuge tubes, as shown in the table above. The urea was then added (20 mg) to provide a final concentration of about 1.6 M. The centrifuge tubes were then vortexed, centrifuged for 10 minutes and then carefully transferred to the flasks for analysis.

d) Results

The following samples were analyzed:

ABT-874 crystal buffer (Example 35 wash buffer)

ABT-874 crystals (obtained according to Example 33, in the washing buffer of Example 35)

Reference Standard (Net sample of ABT-874)

The results are shown in the attached figures 6A to C.

Example 43: Retention of Native Secondary Structure Through Crystallization / Redissolution of Crystals

Infrared spectra were recorded with a Confocheck system on a Bruker Optics 27 Tensor according to the manufacturer's instructions. Liquid samples were analyzed using a MicroBiolytics AquaSpec cell. Measurements of protein suspensions were performed with a Harrick BioATRIl ™ cell. Each sample was evaluated by performing at least two measurements from 120 to 500 scans at 25 ° C. Crude buffer spectra were subtracted from the protein spectra, respectively. Spectra of the second protein derivative were generated by Fourier transformation and the vector normalized from 1,580-1,720 cm '¹ for relative comparison.

The redissolution of the crystals was carried out as follows. The crystal suspensions were centrifuged, the supernatant discarded and the crystal microsphere was dissolved in 0.1 M sodium acetate buffer, pH 5.5 at a protein concentration of 10 mg / mL.

Figure 11 illustrates spectra of the second FT-IV derivative of ABT-874 crystalline suspensions which were crystallized following the procedure as described in Example 34b, washed following the procedure introduced in Example 35 and redissolved. The spectra demonstrate that no significant alteration of the secondary structure was observed, either in the crystalline solid state or after redissolution.

Example 44: Stability data (SE HPLC morphology, FT-IV)

ABT-874 was crystallized using the crystallization procedure described in

Example 34b. The crystals were washed as described in Example 35, with a dispersion buffer containing 22% PEG 4,000 and 0.1 M sodium acetate and the pH was adjusted to 5.5 with glacial acetic acid. Subsequently, the crystals were concentrated to 5 mg / ml and 50 mg / ml of protein by centrifugation, respectively and stored at 2-8 ° C.

The stability data of 5 mg / mL and 50 mg / mL of crystalline ABT-874 in 3 months of storage at 2-8 ° C indicated retention above 90% of the monomer.

(a) SE-HPLC

Table 2 - Stability data of 5 mg / mL of crystalline ABT-874 after redissolution

<td>Time Point</td><td>Aggregates (%)</td><td>Monomer (%)</td><td>Fragments (%)</td>

<td>T0</td><td> 3,9</td><td> 95,8</td><td> 0,3</td>

<td>1 month</td><td> 5,7</td><td> 94,0</td><td> 0,3</td>

<td>3 months</td><td> 8,7</td><td> 91,0</td><td> 0,3</td>

Table 3 - Stability data of 50 mg / mL of crystalline ABT-874 after redissolution

<td>Time Point</td><td>Aggregates (%)</td><td>Monomer (%)</td><td>Fragments (%)</td>

<td>T0</td><td> 3,8</td><td> 96,0</td><td> 0,2</td>

<td>1 month</td><td> 4,6</td><td> 95,0</td><td> 0,4</td>

<td>3 months</td><td> 6,3</td><td> 93,4</td><td> 0,3</td>

A Dionex HPLC system (P680 pump, ASI 100 autosampler, UVD170U) was used to measure the stability of the ABT-874 antibody. ABT-874 samples were separated on a GE Superdex® 200 column, applying a flow rate of 0.75 15 mL / minute. Detection was performed at a wavelength of 214 nm. The operating buffer consisted of 0.2 M disodium sulfate in 0.09 M sodium phosphate buffer, pH 7.0.

(b) FT-IV

Infrared spectra were recorded with a Confocheck system on a 20 Tensor Bruker Optics 27. Liquid samples were analyzed using a MicroBiolytics AquaSpec cell. Measurements of protein suspensions were performed with a Harrick BioATRIl ™ cell. Each sample was evaluated by performing at least two measurements from 120 to 500 scans at 25 ° C. Crude buffer spectra were subtracted from the protein spectra, respectively. Spectra of the second protein derivative were generated by Fourier transformation and the vector normalized from 1,580-1,720 cm '¹ for relative comparison.

The redissolution of the crystals was carried out as follows. The crystal suspensions were centrifuged, the supernatant discarded and the crystal microsphere was dissolved in 0.1 M sodium acetate buffer, pH 5.5 at a protein concentration of 10 mg / mL.

Figure 2 illustrates spectra of the second FT-IV derivative of the crystalline ABT874 suspensions (50 mg / mL shelf stability samples, prepared as described above and stored for 3 months at 25 ° C) and after redissolving such pretreated crystals . The spectra demonstrate that no significant alteration of the secondary structure was observed when stored at 25 ° C for three months, both in the crystalline solid state and after redissolution.

(c) Morphology

After 3 months of storage at 2-8 ° C, no significant morphological changes were observed in the light microscopic analysis of the crystals. Aliquots of 1 to 10 pL of the sample volume were pipetted into an object holding plate, diluted with formulation buffer (22% PEG) and covered with a glass coating slide. The preparations were evaluated using a Zeiss Axiovert 25 inverted light microscope equipped with 10x E-PI eyepieces and 10x, 20x and 40x objectives, respectively.

Example 45 - Extending the Yield of the Crystallization Process

The end point of a crystallization process can be defined as the time point when OD measurements₂The aliquots of the crystallization paste supernatant are constant, for example, for three subsequent days. An extension of the yield is possible by adding a certain additional amount of PEG 4,000 (50% w / v solution of about 0.1 M sodium acetate buffer at a pH of about 5.5) to the supernatant of the crystallization paste . Crystals that are similar to the first harvest form during the days that follow. With the application of this procedure, the total yield easily rises beyond 90%, without the introduction of precipitation.

For example, the concentration of PEG 4,000 is increased from about 11% weight / volume to about 22% weight / volume, about 20% weight / volume, about 18% weight / volume, about 16% weight / volume or about 14% w / v in aliquots of the supernatant of Example 34b. After storage for several days at room temperature (for example, between about 20 and about 25 ° C), precipitated species are observed in certain concentrations of PEG 4,000, for example, about 22% weight / volume, about 20% weight / volume or about 18% weight / volume of PEG 4,000. Crystals without concomitant precipitation are found in lower PEG 4,000 concentrations, for example, about 16% weight / volume and about 14% weight / volume PEG 4,000. By adding PEG 4,000 to a total concentration of, for example, about 14% weight / volume to the residual crystallization paste supernatant, the total crystal yield is increased from 60% to about 70% and in some 90% days.

Example 46: Yield Extension by Applying a Continuous Process

In this example, additional precipitant and / or protein are "titrated" in a batch of crystallization (optionally containing a certain amount of crystallizing agent) at a predetermined rate. Continuous crystallization over time is induced, finally resulting in a 90% crystal yield.

Example 47 - Sowing of Batches of Crystallization of ABT-874

Spontaneous nucleation is statistical in nature. The seeds, which may consist of the same protein (homogeneous seeding) or another substance (heterogeneous seeding) in relation to the one being crystallized, provide a template in which additional molecules can be assembled. Thus, seeding can thus accelerate crystallization.

A batch of crystallization of ABT-874 was prepared as described in Example 34b. After mixing the protein solution with the crystallization buffer, the mixture was seeded by homogeneous seeding with ABT-874 crystals. For example, a rate of a ridge suspension! prepared as described in Example 34b, exhibiting about 50 to 60% crystal yield, was added, for example, at a rate of 1/20 (volume / volume) to the batch of crystallization. By applying this strategy, the crystal yields and the total process duration periods have been further optimized in the direction of higher yields in shorter process time periods.

In summary, a crystallization mixture of ABT-874 (5 mg / ml of protein and 4000% 11% PEG in 0.1 M acetate buffer, pH 5.5) was prepared and divided into two 40 ml aliquots. The first batch was stored at room temperature without additional procedures and the second batch was seeded by adding 2 mL of a crystallization mixture of the same composition that already exhibited 65% crystal yield (6.5 mg of seeds, calculated on the basis of in crystallized protein, compared to 200 mg of ABT-874 in batch). The graphs shown in figure 13 illustrate that when applying this sowing approach, the total yield was extended by about 15% over 80 days, considering that the progression of the parallel curve suggested that the process times to reach the maximum yield were not significantly reduced. Figure 13 suggests that although the seedless batch reached a maximum yield after about 80 days, the theoretically possible yield can be as high as for the seeded batch, meaning that sowing reduced the duration of the crystallization process instead of extending the yield.

Incorporation as a Reference

The content of all references cited (including literature references, patents, patent applications and web sites) throughout this application is incorporated into this document expressly as a reference in its entirety and constitutes the references cited. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of crystallization and formulation which are well known in the art.

Equivalents

The invention can be realized in other specific ways, without escaping from its spirit or essential characteristics. The preceding modalities, therefore, must be considered in all points as not limiting the invention described in the present document. The scope of the invention is thus indicated by the appended claims and not by the preceding description and all changes that fall within the meaning and equivalence range of the claims are therefore covered by this document.