Process for concentration of antibodies and therapeutic products thereof

Description

Background of the invention

[0001] Methods for isolating, purifying and concentrating biological materials are known and include, for example, chromatography, ultrafiltration and lyophilization, see generally R. Hatti-Kaul et al., "Downstream Processing in Biotechnology," in Basis Biotechnology, Ch. 9, pages 187-211, 2nd edition, Cambridge University Press (2001). Methods for producing concentrated monoclonal antibody preparations for administration to humans are known, see, for example, US Patent No. 6,252,055, which uses ultrafiltration and recirculation of the resulting filtrate.

[0002] Some of the challenges associated with the available methods of concentrating antibodies include, for example, low flux density, long process times, large membrane areas, mechanical recovery efficiency and losses, high intensity of operator or handler intervention, slow mass transfer rate, shortages energy, and limits of hydraulic pressure against the concentration equipment. These and other challenges can contribute to a high overall manufacturing cost and consequently higher costs for consumers of the pharmaceutical preparations.

[0003] There is a need for improved methods for the preparation of highly concentrated protein formulations such as liquid antibody preparations and therapeutic products thereof.

Summary of the invention

[0004] In general, the disclosure generally relates to methods of concentrating proteins, such as methods of concentrating an antibody preparation, pharmaceutical formulations containing such preparation, and their use in human or animal therapy.

[0005] In embodiments, the disclosure provides methods for obtaining highly concentrated proteins such as the antibody preparations of the invention as defined in the claims; and therapeutic products obtained by the method, such as therapeutic antibody products. Accordingly, the disclosure provides a protein concentration method comprising, according to the invention: first ultrafiltering a first antibody preparation to provide a second antibody preparation; diafiltrating the second antibody preparation to provide a diafiltered antibody intermediate; and a second ultrafiltration of the diafiltered intermediate antibody preparation to provide a third antibody preparation, wherein one or more of the first ultrafiltration, second ultrafiltration, and diafiltration are performed at elevated temperatures, for example, from about 30 ° C to about 50 ° C, and such as defined in the claims.

[0006] The disclosure also provides, in embodiments of the invention, a method for concentrating a protein comprising: first ultrafiltering a first protein mixture to provide a second protein mixture; diafiltrating the second protein mixture to provide a diafiltered protein mixture; and second ultrafiltration of the diafiltered protein mixture,

To provide a third protein mixture, one or more of the first ultrafiltration, second ultrafiltration, and diafiltration being performed, for example, at a temperature of about 45 ° C as defined in the claims.

[0007] The disclosure also relates to highly concentrated antibody compositions obtained by the above methods.

Brief description of the drawings

[0008]

FIG. 1 illustrates an apparatus for carrying out a preparative method in embodiments of the disclosure.

FIG. 2 through 17 illustrate various observed or measured method values ​​for different phases or modes of the method in embodiments of the disclosure.

FIG. 18 and 19 illustrate the effect of elevated temperature on product quality in embodiments of the disclosure.

FIG. 20 and 21 illustrate the effect of elevated temperature in controlling bioburden, in embodiments of the disclosure.

FIG. 22 illustrates the effects of elevated temperature on the process stream and process time, in embodiments of the disclosure.

FIG. 23 to 25 illustrate different observed or measured values ​​of the method for different phases or modes of the method on an enlarged scale, in embodiments of the disclosure.

Detailed description of the invention

[0009] The various embodiments of the invention disclosed herein will be described in detail with reference to the drawings, if any. Reference to various embodiments is not intended to limit the scope of the invention, which is only limited by the scope of the claims appended hereto. In addition, any examples set forth in the specification are not intended to be limiting, and merely represent some of the many possible embodiments of the claimed invention.

[0010] The following applies unless otherwise specified:

"Ultrafiltration", "Ultrafiltration" "Ultrafiltered" "UF," and similar terms refer to, for example, the use of synthetic semi-permeable membranes, with appropriate physical and chemical properties, to distinguish between molecules in a mixture based primarily on the molecular size and shape , and separating different molecules or concentrating similar molecules.

"Diafiltration", "diafiltered", "diafiltered," "DF," and similar terms refer to, for example, the use of an ultrafiltration membrane to remove, replace or replace

-3 lowering the concentration of salts or solvents of solutions or mixtures containing proteins, peptides, nucleic acids or other biomolecules.

"Transmembrane pressure" or "TMP" refers to the mean applied pressure on the membrane - from the feed side to the filtrate side - calculated as TMP [bars] = [(P_F. + Pr) / 2] -Pf. with P_F. is the feed pressure, Pr is the retentate pressure, and Pf is the filtrate pressure.

"Tangential flow filtration", "cross flow filtration" "TFF" and similar terms refer to a filtration mode in which a solute containing solution passes tangentially through a UP membrane and lower molecular weight salts or solutes are transferred to the other side by applying pressure.

"Antibody" is used in the broadest sense and specifically includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies) made of at least two intact antibodies, and antibody fragments as long as they exhibit the desired biological activity. An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. Structurally described, the antibody is a Y-shaped protein consisting of four amino acid chains, two heavy and two light. In a simplified model sufficient for this reference, each antibody primarily has two regions: a variable region and a constant region. The variable region, located at the ends of the Y arms, binds to and interacts with the target antigen. This variable region includes a complementarity determining region (CDR) that recognizes and binds to a specific binding site on a particular antigen. The constant region, located on the "tail" of Y, is recognized by and interacts with the immune system (Janeway, C "Travers, P" Walport, M "Shlomchik (2001) Immuno Biology, 5th edition, Garland Publishing, New York). A target antigen generally has multiple binding sites, also called epitopes, which are recognized by CDRs on many antibodies. Each antibody that specifically binds to a different epitope has a different structure. Hence, one antigen may have more than one corresponding antibody.

[0011] The basic unit of a 4-chain antibody is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains (an IgM antibody consists of 5 basic heterotetrameric units, along with an additional polypeptide called the J chain, and consequently which contains 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent clusters, containing 2-5 basic 4-chain units, along with the J chain). In the case of IgG, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds, depending on the isotype of the H chain. Each H and L chain also has

- 4-spaced intra-chain disulfide bridges. Each H chain has at its N terminus a variable domain (Vh) followed by three constant domains (Ch) for each of the a and γ chains and four Ch domains for the μ and ε isotypes. Each L chain has, at its N terminus, a variable domain (V_L.) followed by a constant domain (Cl) at its other end. Vl is aligned with Vh and Cl is aligned with the first heavy chain constant domain (ChI). Certain amino acid residues are believed to form an interface between the heavy and light chain variable domains. Pairing Vh and Vl with each other creates a single antigen-binding site. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th Edition, D. Stites, A. Terr and T. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0012] The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (Ch), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with the heavy chains designated α, δ, γ, ε, and μ, respectively. The γ and a classes are further subdivided into subclasses based on relatively small differences in sequence and Ch function, e.g., the following subclasses are expressed in humans: IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2.

[0013] The term "variable" refers to the fact that certain segments of the variable domains differ significantly in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable domains. Instead, V regions are composed of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions", each of which is 9-12 amino acids long. Each of the native heavy and light chain variable domains comprises four FR regions largely in the β sheet configuration, connected by three hypervariable regions that form the connecting loops and in some cases forming part of the β sheet structure. The hypervariable regions in each chain are held in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antibody antigen-binding site (see Kabat et aL, Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not directly involved in binding an antibody to an antigen, but represent various effector functions, such as participation of the antibody in antibody dependent cellular toxicity (ADCC).

[0014] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally includes amino acid residues from the "complementarity determining region" or "CDR" (e.g., approximately Kabat residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in V1 and approximately residues according to Kabat - 31-35 (HI), 50-65 (H2) and 95-102 (H3) in Vh (see Kabat

-5 et al., Suprd) and / or those residues from the "hypervariable loop" (e.g., approximately Chothia residues - 26-32 (LI), 50-52 (L2) and 91-96 (L3) in V_L. and 26-32 (HI), 53-55 (H2), and 96-101 (H3) in V._H.(Chothia and Lesk, J. Mol. Biol, 196: 901-917 (1987)).

[0015] The term "monoclonal antibody" as used herein refers to an antibody from a population of substantially homogeneous antibodies, i.e. the individual antibodies making up the population are identical and / or bind the same epitope (s), except for possible variants that may arise during the preparation of the monoclonal antibody. such variants are generally present in minor amounts. Such a monoclonal antibody typically comprises an antibody comprising a polypeptide sequence that binds a target, wherein the target binding polypeptide sequence is obtained by a method that comprises selecting a single target binding polypeptide sequence from multiple polypeptide sequences. For example, a selection method may mean selecting a unique clone from among a plurality of clones, such as pooling hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that the selected target binding sequence may be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to formation of a multispecific antibody, etc., wherein the antibody comprising an altered target binding sequence is also a monoclonal antibody of the invention. Unlike polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in the monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically unpolluted by other immunoglobulins. The modifier "monoclonal" indicates the nature of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibodies by any particular method. For example, monoclonal antibodies for use in accordance with the invention can be obtained by a variety of techniques including, for example, a hybridoma method (e.g., Kohler et al "Nature, 256: 495 (1975); Harlow et al" Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd edition 1988); Hammerling et al in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), by recombinant DNA methods (see e.g. U.S. Patent No. 4,816,567), using phage display technology (see, e.g., Clackson et al "Nature, 352: 624-628 (1991); Marks et al" J. Mol. Biol., 222: 581-597 ( 1991); Sidhu et al "J. Mol. Biol. 338 (2): 299-310 (2004); Lee et al." J. Mol. Biol. 340 (5): 1073-1093 (2004); Fellouse, Proc Nat Acad Sci USA 101 (34): 12467-12472 (2004) and Lee et al. J. Immunol. Methods 284 (1-2): 119-132 (2004) and technologies for producing human or human-like antibodies in animals that have some or all of human immunoglobulin loci or gene sequences encoding human immunoglobulins (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits, et al. "Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits, et al., Nature, 362: 255-258 (1993); Bruggemann, et al., Year in Immuno., 7:33

-6 (1993); U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669 (all for GenPharm); US 5,545,807; WO 1997/17852, US Patent Nos. 5,545,807; US 5,545,806; US 5,569,825; US 5,625,126; US 5,633,425; and US 5,661,016, and Marks et al., Bio / Technology, 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature, 368: 812-813 (1994); Fishwild, et al., Nature Biotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology, 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol., 13: 65-93 (1995).

[0016] "Chimeric" antibodies (immunoglobulins) have a portion of the heavy and / or light chain identical to or homologous to corresponding sequences in antibodies obtained from a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain (s) is identical to or homologous to the corresponding sequences in antibodies derived from a different species or belonging to a different class or subclass of antibodies, fragments of such antibodies are also included, as long as they exhibit the desired biological activity (US Patent No. 4,816,567; and Morrison, et al., Proc. Natl. Acad. Sci. USA 81: 68516855 (1984)). A humanized antibody as used herein is a subset of chimeric antibodies.

[0017] "Humanized" forms of non-human (eg, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (recipient or acceptor antibody) in which the recipient hypervariable region residues are replaced with hypervariable region residues from a nonhuman species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity and fitness. In some instances, human immunoglobulin framework region (FR) residues are replaced with corresponding non-human residues. In addition, humanized antibodies may contain residues that are not found in the recipient antibody or donor antibody. These modifications are made to further refine antibody performance, as well as binding affinity. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence. although the FR regions may contain one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FR is typically no more than 6 in the H chain and no more than 3 in the L chain. The humanized antibody will optionally also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details see Jones, et aL, Nature 321: 522-525 (1986); Reichmann, et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593596 (1992).

[0018] "Antibody fragments" contain a portion of an intact antibody, preferably the antigen-binding region or the variable region of an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab') 2, and Fv fragments; diabodies; linear antibodies (see US Patent No. 5,641,870, Example 2; Zapała et al., Protein Eng. 8 (10): 1051-1062 (1995)); single-chain antibody molecules; and multispecific antibodies made of antibody fragments.

[0019] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. The Fab fragment consists of the entire L chain along with the variable region domain of the H chain (Vh), and the first constant domain of one heavy chain (ChI). Each Fab fragment is monovalent with respect to antigen binding, ie it has a single antigen-binding site. Treatment of the antibody with pepsin yields a single large F (ab ') 2 fragment that corresponds roughly to two disulfide-linked Fab fragments with bivalent antigen-binding activity, still capable of cross-linking antigen. Fab 'fragments differ from Fab fragments by a few additional residues to the carboxy terminus of the ChI domain, including one or more cySteins from the antibody hinge region. Fab'-SH is the designation as described for Fab 'in which the cysteine ​​residues of the constant domains have a free halo group. F (ab ') 2 antibody fragments were originally produced as pairs of Fab' fragments that had hinge cysteines between them. Other chemical combinations of antibody fragments are also known.

[0020] The Fc fragment comprises portions of the carboxyl terminals of both H chains, held together by disulfides. Antibody effector functions are determined by sequences in the Fc region; this region is also the part recognized by Fc receptors (FcRs) found on certain types of cells.

[0021] "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and-binding site. This fragment consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. From the folding of these two domains, six hypervariable loops (3 loops from each of the H and L chains) emerge which provide amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind an antigen, albeit with a lower affinity than a complete binding site.

[0022] "Single-chain Fv", also abbreviated as "sFv" or "scFv", are antibody fragments that contain the VH and V1 antibody domains in which these domains are joined in a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the V H and V L domains that allows the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0023] "About" modifying, for example, the amount of an ingredient in the compositions, active ingredient concentration, buffer volumes, diafiltered volumes, pore size, apparent molecular weight, molecular weight cutoff; process temperature, process time, throughput, flow rate, pressures, bioburden, and the like, and ranges thereof, used in the methods of the invention refer to variability in numerical values ​​that may occur, for example, with typical measurement and handling procedures used in for obtaining concentrates or applying solutions; by inadvertent error in these procedures; by differences in the manufacture, source, or purity of the ingredients used to prepare the compositions or carry out the methods; and for similar reasons. The term "about" also includes amounts that vary with the aging of the composition at a particular starting concentration or mixture. The term "about" also includes amounts that vary due to the mixing or processing of a composition with a particular starting concentration or mixture. Regardless of the modification associated with "about", the claims include equivalent amounts.

[0024] "Consisting essentially of" refers to a method of preparing a concentrated protein or antibody composition that includes the steps and components recited in the claim, plus other steps and components that do not materially affect the basic and novel properties of the composition, such as a multiplicity of steps or buffering agents. Ingredients that materially affect the essential properties of the composition and method of the disclosure impart undesirable characteristics, including, for example, biological bias, such as undesirable toxicity or sensitivity associated with impurities.

[0025] Indefinite article [in English ] "a" or "an" and the corresponding article "the" as used herein are meant to mean at least one or one or more, unless otherwise stated.

[0026] The disclosure provides, in its embodiments, the above-mentioned methods of the invention as defined in the claims and their associated concentrated antibody products.

[0027] In embodiments of the present disclosure, the preparative methods and products thereof can be used in the preparation of highly concentrated preparations of antibodies and similar products, such as the purification and concentration of proteins or similar substances from natural or synthetic sources, which products may be useful in the treatment of conditions. pathological conditions such as asthma, cancer, psoriasis, inhibition of angiogenesis, and similar pathological conditions.

[0028] In the embodiments of the above-mentioned method for preparing the highly concentrated antibody compositions of the disclosure, the following further illustrates how to make and use the preparation methods and products of the disclosure.

[0029] In embodiments of the disclosure, there is provided a method of making highly concentrated antibody compositions, for example, by following the steps below in the sequence provided, including:

First ultrafiltering a first antibody preparation having a concentration in the order of, for example, about 0.1 to about 10 grams per liter (g / L), to provide a second antibody preparation as a retentate having a higher antibody concentration, for example, about 10 to about 50 grams per liter;

diafiltrating the resultant second antibody preparation to provide a diafiltered intermediate antibody preparation as a retentate having approximately the same concentration as the resultant retentate of the second antibody preparation, i.e., diafiltration to obtain a constant volume buffer exchange; and a second ultrafiltration of the diafiltered intermediate antibody preparation to provide a third antibody preparation as a retentate having a higher antibody concentration, for example, about 150 to about 200 grams per liter.

[0030] The preparative methods of the disclosure can further include, for example, an optional step or steps for recovering a product, and as disclosed and illustrated herein.

[0031] In embodiments of the above-mentioned method of the disclosure, one or more of the first ultrafiltration, diafiltration, and second ultrafiltration may be performed, for example, at a temperature from about 30 ° C to about 70 ° C. In embodiments, these steps may also be performed at, for example, from about 30 ° C to about 50 ° C. In embodiments, these steps can also be conducted at, for example, from about 35 ° C to about 50 ° C. In embodiments, these steps can also be performed, for example, at about 45 ° C, such as from about 45 ° C plus or minus 5 ° C. Depending on the nature of the antibody preparation, for methods conducted at temperatures above about 70 ° C, the preparation may show signs of deterioration such as denaturation, clumping, and the like. For processes operated at temperatures below from about 30 to about 35 ° C, flow rates are typically undesirably low and process times are undesirably long, making the process less attractive for efficient commercial production at lower temperatures.

[0032] The first antibody preparation may have an antibody concentration of, for example, from about 0.1 to about 100 grams per liter (g / L). Antibody concentration means, for example, the customary concentration typically available for other preliminary steps or methods for purifying a protein or antibodies, such as centrifugation, filtration, chromatography and similar procedures. The resulting second antibody preparation to be obtained from the first ultrafiltration may have an antibody concentration of, for example, from about 10 to about 50 grams per liter, and for example, from about 20 to about 40 grams per liter, such as 30 grams per liter. The range of antibody concentrations in an antibody intermediate preparation may depend on, for example, the balance of factors such as sample volume and sample stream achievable with the particular buffer containing the second antibody preparation. The intermediate antibody preparation may have an antibody concentration of, for example, from about 25 to about 35 grams per liter and the third antibody preparation may have an antibody concentration, for example, from about 170 to about 200 grams per liter. The third antibody preparation, in embodiments, can have an antibody concentration of, for example, from about 50 to about 250 grams per liter, such as from about 100 to about

-10 about 230 grams per liter, and from about 170 to about 200 grams per liter, such as 185 grams per liter.

[0033] It will be apparent to those skilled in the art, from the analysis of the disclosure, that the intermediate antibody preparation and the third antibody preparation contain the same ultra-filtered retentate except, for example, for differences in antibody concentration resulting from the first and second ultrafiltration concentration, and differences in suspending buffering agents, resulting from replacement of the diafiltration buffer. Hence, there is little, if any, change in the composition, such as distribution, of the target protein or antibody product in the embodiments of the disclosure.

[0034] Conventional concentration ultrafiltration methods may generally have longer times and less inefficiencies in throughput, since they have much longer process times, such as several days to several weeks, the process involves much smaller volumes, or both.

[0035] In embodiments, the protein concentration method of the disclosure may be conducted, for example, from about 1 to 10 hours, preferably from about 2 to 5 hours, and more preferably for about 3 hours. A higher throughput flux and smaller membrane regions are favored as preferred.

[0036] The first ultrafiltration can be accomplished, for example, in about 35 percent of the total process time. Hence, for example, in the concentration and purification method of the disclosure with about 3 hours of total process time, the first ultrafiltration may be performed for about 45 minutes. The second ultrafiltration can be accomplished, for example, in about 15 percent of the total process time. Thus, for example, in the process of the disclosure, after about 3 hours of total process time, the second ultrafiltration may be performed for about 15 minutes. Diafiltration can be accomplished, for example, in about 50 percent of the total process time. Thus, for example, in a method of the disclosure with about 3 hours of total process time, the diafiltration may be performed for about 90 to about 120 minutes.

[0037] In embodiments, the first ultrafiltration and the second ultrafiltration can be performed, for example, with an ultrafiltration membrane having a nominal pore size, or molecular weight cutoff, on the order of about 5 to about 50 kiloDalton. Another suitable nominal pore size is, for example, from about 10 to about 40 kiloDalton. Yet another suitable nominal pore size, or molecular weight cut-off, is about 30 kiloDalton.

[0038] In embodiments, the first antibody preparation may contain, for example, an antibody having an apparent molecular weight in the order of, for example, about 100 to about 200 kiloDalton. In other embodiments, the first antibody preparation may comprise an antibody with an apparent molecular weight in the order of, for example, about 150 kiloDalton, such as when the antibody preparation comprises anti-IgE or IgE antibodies, see, for example, US Patent No. 6,172,213 to Genentech, Inc.

[0039] Other antibodies suitable for use in the disclosure include antibodies for the treatment of cancer, see generally, for example: PCT / US02 / 19592; PCT / US01 / 20118; PCT / USO1 / 25464; PCT / USO1 / 26626; PCT / US02 / 28859; PCT / US02 / 41798;

PCT / US02 / 12206; PCT / US03 / 11148; PCT / US02 / 12619; and PCT / US02 / 33050. Still other antibodies suitable for use in the disclosure include the anti-CD20 antibody and similar antibodies, including human, non-human, murine, hybrid, and chimeric forms. See, for example, US Patent No. 6,582,959 (VEGF) and US Patent Application No. US 2002/0122797 Al (human VEGF).

[0040] In embodiments, the antibodies encompassed by the scope of this disclosure include hybrid and recombinant antibodies (eg, "humanized" and "human" antibodies), regardless of species of origin or immunoglobulin class or subclass determination, as well as antibody fragments (eg, Fab, F). (ab ') 2 and F_v). See U.S. Patent No. 4,816,567; Mage and Lamoyi, in Monoclonal Antibody Production Techniques and Applications, 79-97, Marcel Dekker, Inc., New York, (1987).

[0041] Monoclonal antibodies can also be used and isolated from antibody phage libraries, eg, using the techniques described in Clackson et al. (1991) Nature, 352: 624-628 and Marks, et al. (1991) J. Mol. Biol., 222: 581-597. Monoclonal antibodies include "chimeric" antibodies that have a portion of the heavy and / or light chain identical to or homologous to corresponding sequences in antibodies obtained from a particular species or belonging to a particular class or subclass of antibodies. while the remainder of the chain (s) is identical with or homologous to the corresponding sequences in antibodies derived from a different species or belonging to a different class or subclass of antibodies, fragments of such antibodies are also included, as long as they exhibit the desired biological activity (US Patent No. 4,816,567; and Morrison, et al. (1984) Proc. Natl. Acad. Sci. USA, 81: 6851-6855). Chimeric antibodies can include "primatized" antibodies comprising variable domain antigen binding sequences derived from a non-human primate (eg, tail apes, apes, etc.) and human constant region sequences.

[0042] Monoclonal antibodies are highly specific by targeting a single antigenic site. In addition, unlike polyclonal antibody preparations that contain different antibodies to different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized unpolluted by other antibodies. Hence, the modifier "monoclonal" indicates the nature of the antibody as being obtained from a substantially homogeneous population of antibodies, i.e. the individual antibodies making up the population are identical except for possible naturally occurring mutations which may be present in minor amounts and should not be construed as requiring the production of antibodies in any particular manner. For example, the monoclonal antibodies for use according to the disclosure can be

Obtained using the hybridoma method first described by Kohler and Milstein, Nature, 256: 495 (1975), or may be obtained by recombinant DNA methods. Other known methods of producing antibodies are described, for example, in Goding, Monoclonal Antibodies: Principles and Practice, 59-103, Academic Press (1986); Kozbor, J. Immunol., 133: 3001 (1984). Brodeur, et al., Monoclonal Antibody Production Techniques and Applications, 51-63, Marcel Dekker, Inc., New York (1987).

[0043] Various methods have been used to produce monoclonal antibodies (MAbs). The hybridoma technique, which involves a cloned cell line that produces a single type of antibody, uses cells from a variety of species, including mice (mice), hamsters, rats, and humans. Another method of obtaining MAbs uses genetic engineering, including recombinant DNA techniques. Monoclonal antibodies produced by these techniques include, chimeric antibodies and humanized antibodies. A chimeric antibody joins DNA coding regions from more than one type of species. For example, a chimeric antibody can use a mouse-derived variable region and a human-derived constant region. A humanized antibody is primarily human, even when it contains non-human fragments. Like a chimeric antibody, a humanized antibody can contain an entirely human constant region. But unlike a chimeric antibody, the variable region can be partially human. The non-human, synthetic portions of a humanized antibody are often derived from CDRs in murine antibodies. In any event, these regions are crucial in enabling the antibody to recognize and bind to a specific antigen.

[0044] As noted, murine antibodies play an important role in antibody technology. While useful for diagnosis and short-term therapy, murine antibodies cannot be administered long-term to humans without the increasing risk of a deleterious immune response. This response, called human anti-mouse antibodies (HAMAs), occurs when the human immune system recognizes the murine antibody as foreign and attacks it. A HAMA response can cause toxic shock or even death. Chimeric and humanized antibodies reduce the likelihood of a HAMA response by minimizing the amount of non-human portions in the administered antibodies. In addition, chimeric and humanized antibodies have the added benefit of activating secondary human immune responses such as antibody-dependent cellular cytotoxicity.

[0045] An "intact" antibody is one that comprises an antigen binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CHI, CH2, and CH3. The constant domains may be native sequence constant domains (e.g., native human constant domain sequence) or an amino acid sequence variant thereof. An intact antibody may have one or more "effector functions" that relate to those biological activities attributed to the Fc region (native Fc region sequence or amino acid sequence variant of the Fc region) of the antibody. Examples of antibody effector functions include Clq binding; system dependent cytotoxicity

-13 complement; Fc receptor binding; antibody-dependent cellular cytotoxicity (ADCC); phagocytosis; lowering the expression of receptors on the cell surface (e.g. B cell receptor; BCR) etc.

[0046] Depending on the amino acid sequence of the constant domain of their heavy (Ch) chains, intact antibodies can be assigned to different "classes". There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of them can be further subdivided into "subclasses" (isotypes), eg, IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of various classes of immunoglobulins are well known.

[0047] In embodiments, the first ultrafiltration concentrates the first antibody preparation to provide a second antibody preparation with an antibody concentration of about 30 grams per liter, and the second ultrafiltration concentrates the intermediate antibody preparation (obtained after diafiltration) to provide a third antibody preparation with an antibody concentration, for example, about 170 to about 200 grams per liter. The first ultrafiltration and the second ultrafiltration can be performed with the same ultrafiltration membrane and, if desired, within the same vessel or process circuit, for example, to minimize handling, losses, leakage, and similar impacts on efficiency, effectiveness and cost effectiveness. . The first ultrafiltration and the second ultrafiltration can be performed with any suitable ultrafiltration apparatus or ultrafiltration membrane. Many suitable ultrafiltration apparatus and ultrafiltration membranes that are capable of carrying out tangential flow filtration (TFF) operations for ultrafiltration and diafiltration purposes are commercially available, such as from Millipore fum, Pall Corp., Sartorius, and similar suppliers. In embodiments, a suitable ultrafiltration membrane can be, for example, any regenerated cellulose composites, which composite has a relatively low protein adsorption profile compared to other available ultrafiltration membranes such as polyethersulfone.

[0048] The diafiltration operation exchanges the first buffer composition present in the first and second antibody preparations for a second buffer desired in the third antibody preparation. In embodiments, the first buffer may contain, for example, a mixture of aqueous sodium chloride and TRIS buffer, and the second buffer may contain, for example, a mixture of aqueous histidine chloride and arginine chloride. Diafiltration can exchange buffer at constant volume, constant concentration, or both. In embodiments, diafiltration performs a buffer exchange at a constant volume and constant concentration. Diafiltration can exchange the buffer, for example, from about 5 to about 15 times the volume (i.e., the volume of the diafiltration). Diafiltration can also exchange the buffer, for example, about 8 times the volume (8 diafiltration volumes), i.e., 8 times the volume of the sample containing the antibody preparation to be exchanged [buffer]. For example, a 10 liter antibody preparation can be diafiltered with 5 times (diafiltration volume) or 50 liters of exchange buffer volume. The exchanged volume and the preferences for the exchanged volume take into account a balance of factors, for example, process throughputs, product purity, government and client-patient.

Acceptance standards, and similar standards, and may depend on, for example, the concentration and type of buffer (e.g., first buffer) in the first antibody preparation, and similar considerations.

[0049] The first ultrafiltration, the second ultrafiltration, and the diafiltration are preferably achieved by tangential flow filtration (TFF mode) through an ultrafiltration membrane, and the ultrafiltration membrane is preferably the same membrane for each step. The product yield of the final pool (i.e., the third antibody preparation) can be, for example, greater than about 70 weight percent, such as from about 80 to about 100 weight percent based on the weight of the antibody in the first antibody preparation. The yield of the third antibody preparation may be, in embodiments, greater than about 90% by weight, in embodiments, greater than about 95% by weight, and in embodiments, even greater than about 98% by weight, based on the weight of the antibody in the first antibody preparation. .

[0050] The first ultrafiltration may have a recirculation rate of, for example, from about 50 to 1,000 ml / min, and preferably from about 100 to 1,000 ml / min. The recirculation rate can be scaled according to the available diaphragm area, for example, 5, 20, 200, 1,000 square feet [ft.²], and the like regions allow ever faster recirculation rates. Hence, a properly scaled recirculation rate, in exemplary embodiments, may be, for example, from about 0.5 l / min / ft.² to about 5 1 / min / ft². Ultrafiltration and diafiltration can be performed, for example, at transmembrane pressures on the order of about 5 to about 50 psi. Ultrafiltration and diafiltration may be performed, for example, at transmembrane pressures on the order of about 10 to about 50 psi. In embodiments of the disclosure, a method of preparing an antibody concentrate for a more dilute antibody formulation is provided, the antibody concentrate having a minimal biological burden, for example, less than or below the limit of detection, such as, less than about 100 CFU / ml.

[0051] The antibody compositions of the disclosure can be, for example, a concentrated monoclonal antibody preparation for human administration, such as at a concentration of greater than or equal to about 100 g / L (mg / mL), such as about 120 to about 170 g / L. .

[0052] The antibody compositions of the disclosure can be, for example, immunoglobulins such as from the group of IgA, IgD, IgE, IgG, and IgM; their subclasses; their recombinants; their fragments; and mixtures thereof of any of the above. A preferred antibody composition of the disclosure includes recombinant humanized anti-IgE antibodies. The antibody compositions of the disclosure can include a buffer. A preferred buffer can be, for example, a mixture of aqueous histidine chloride and arginine chloride.

[0053] The preparative methods of the disclosure are preferably performed in the same apparatus and with no operator intervention, or with minimal operator intervention, for example, as illustrated in FIG. 1.

[0054] The first antibody preparation may be provided or obtained by a variety of chemical, physical, mechanical or non-mechanical, or biochemical methods.

For methods such as trituration, ultrasound, homogenization, enzymatic digestion, solvent extraction, centrifugation, chromatography, and similar methods, and combinations thereof, see, for example, R. Hatti-Kaul et al., "Downstream Processing in Biotechnology" , ”In Basic Biotechnology, Chap. 9. The third antibody preparation may be further processed, if desired, using, for example, nanofiltration (to remove e.g. divalent ions), reverse osmosis (to remove, e.g., monovalent ions), and similar fluid purification methods. A third antibody preparation of the disclosure can be packaged, stored, or used directly. The third antibody preparation may be further processed as desired using, for example, additional concentration steps such as drying, lyophilization, lyophilization-reconstitution, and the like. The resulting concentrated third antibody product can be later reconstituted, if desired, with an appropriate fluid.

[0055] Referring to the figures, FIG. 1 illustrates an apparatus, in embodiments of the disclosure, for performing a preparative method, including an ultrafiltration-diafiltration system (100) having a TFF ultra-filtration-diafiltration (UF-DF) unit (110) having a UF-DF membrane (115) that it is in communication with the recirculation tank (120) which tank serves as the primary feed and retentate tank. In exemplary embodiments, the vessel (120) may have a temperature control system including, for example, an insulating jacket (125), a thermostatic element, or a temperature controlled heating element (126), such as a heating element with a resistance adjuster or a circulating heated fluid system that includes a heating element (not shown), a flow regulator (127) such as a recirculation pump, and a suitable heat transfer fluid such as water, glycols, or mixtures thereof. System components or a component contributing to a system flow or treatment, such as pipes, valves, pumps, tanks, and similar components, can be optionally insulated or optionally adapted for external heating to maintain tight control of temperature specifications and to avoid sudden spikes in temperature in the recirculation fluid loop within and between the filter chamber (110) and the recirculation tank (120). In exemplary embodiments, for example, when the system (100) performs a first ultrafiltration or a first ultrafiltration, such as in fed batch mode, the system may include an optional feed tank (128) that is in fluid communication with the feed recycling tank (120). and can be used, for example, to refill, refill, or add a lean liquid phase from a recirculation tank (120).

The pump (130) pumps the feed fluid from the reservoir (120) through the UF / DF unit (110) and then recirculates the resulting retentate (non-filtered or membrane-excluded portion of the feed fluid) to the recirculation tank (120). The second reservoir (140) holds and optionally pumps (not shown) the buffer to the main circuit (loop 110-120) for a constant diafiltration volume. For example, the addition rate and the volume of buffer introduced into the main circuit preferably occur at the same rate and volume at which the filtrate leaves the main circuit via the membrane (115). The buffer vessel (140) may optionally be insulated with a jacket (143) and may contain a counterpart

-16 of the above-mentioned heating element and a recirculation pump (not shown). An optional inert gas source (145) such as nitrogen or other compressed gas sources can be used, for example, for product recovery, for compressing the returning retentate, for oxygen exclusion, for flushing, for cleaning, for membrane integrity testing, and the like. The third vessel (160) is used to collect and recover the filtrate leaving the unit (110). Valves (150, 170) may be used to suit the direction and optionally the flow rate of the fluid in the system. All values ​​and pumps can be started manually, via coordinated computer control, or both. Optional fourth tank (190) and outflow stream can provide auxiliary waste washing, product recovery, or monitoring system, for example, when equipped with an optional monitoring device (180) such as optical density meter, optional filter (s) (185) such such as the limit filter, product filter, and similar optional subsystems. In exemplary embodiments, the main liquid circuit (loop 110-120) may optionally be equipped with an integrated monitoring system.

[0057] Concentrated antibody preparations obtained by the methods of the disclosure can be used for therapeutic administration to humans, including immunoglobulin products, for intramuscular (IMIG) or intravenous (IVIG) administration. The concentrated antibody preparations of the disclosure can include a stabilizer, for example, a buffered salt solution of amino acids, simple sugars, or similar stabilizers, suitable ion chelators, such as EDTA or citrate, and combinations thereof, see, for example, Wang, Y.-CJ et al. al., "Parenteral formulations of proteins and peptides: stability and stabilizers," J. Parenteral Sci. Technol., 42, Suppl. S3-S26 (1988). Summary JP01268646A (AN89-359879) of Derwent reports that the application describes the injection of a monoclonal IgGs preparation with a concentration of 0.1 micrograms / ml to 100 mg / ml. The subject matter disclosed in these publications is considered to be outside the scope of disclosure.

[0058] The formulations of the disclosure can be substantially free of aggregates. Acceptable levels of aggregate impurities would be less than, for example, about 5% by weight, and ideally less than 2% by weight. Levels as low as 0.2 wt% can be achieved, although aggregate impurities of about 1 wt% are more typical. The formulations in the embodiments may also advantageously be free of excipients conventionally used to stabilize polyclonal formulations, for example glycine and / or maltose.

[0059] The disclosure can provide a monoclonal antibody preparation for administration to humans, characterized in that the antibody in the preparation is a recombinant antibody and can be present at a concentration of 100 mg / ml or greater, preferably greater than 150 mg / ml. The preparation is preferably substantially free of any protein aggregates.

[0060] The pH of the pharmaceutical formulations of the disclosure will depend on the particular route of administration. However, to maximize the solubility of the antibody in the concentrated solution, the pH of the solution should be different from the pH of the isoelectric point (pi) of the antibody.

[0061] In embodiments of the disclosure, monoclonal formulations for use in human therapy may be envisaged. Various human disorders, such as cancer or infectious diseases, for example those mentioned above, and immune system dysfunctions such as T-cell regulated disorders, including severe vasculitis, rheumatoid arthritis, systemic lupus, also autoimmune disorders such as such as multiple sclerosis, graft versus host disease, psoriasis, juvenile diabetes mellitus, Sjógren's disease, thyroid disease, severe myasthenia gravis, transplant rejection, inflammatory bowel disease, asthma, IgE regulated disorders, and similar disorders or conditions, or combinations thereof.

[0062] The disclosure therefore provides in the embodiments the use of a concentrated preparation of monoclonal antibodies as set forth herein in the manufacture of a medicament for the treatment of any of the above-mentioned disorders, and similar disorders. There is also provided a method of treating humans suffering from such a disorder, comprising administering to the individual a therapeutically effective amount of a formulation of the disclosure. Dosages of such antibody preparations will vary with the conditions being treated and the recipient of treatment, but may, for example, be in the range of about 50 to about 2,000 mg for an adult patient, preferably about 100 to about 1,000 mg administered daily or weekly. for between 1 and 30 days, and repeated as needed. Doses can be administered in single or multiple doses.

Description of the method. The formulation step typically exchanges the purified greater amount of drug substance, for example, resulting from ion exchange chromatography, for a final excipient composition and concentrated. Typically no purification was achieved at this stage except for removal of small particles. Emphasis was placed on high yield, buffer exchange and strength of the formulation step. During formulation with TFF (tangential flow filtration), the protein-containing feed solution was pumped through the membrane system and back into the recycle vessel. The TFF membrane retained the protein (as part of the retentate) while the filtrate (or permeate) passed through the membrane under pressure. The pressure is called transmembrane pressure (TMP) and is typically controlled using a retentate pressure control valve. The method was typically carried out by a sequence of first ultrafiltration (concentration), diafiltration (constant buffer exchange volume), and second ultrafiltration (further concentration). The number of diafiltration volumes (volumetric equivalents) necessary to remove the buffer components of the process can be easily calculated or experimentally determined.

[0064] UF / DF method generally for anti-IgE. The pH of the anion exchange pool from the chromatography was adjusted to a pH of about 6 using 0.5 M aqueous phosphoric acid. The adjusted pH anion exchange pool was formulated by the ultrafiltration / diafiltration (UF / DF) method of the disclosure using a membrane with a nominal molecular weight cutoff of 10,000 to 30,000 Daltons. Before workup, the UF membrane was equilibrated with a diafiltration buffer (0.02 M histidine, 0.2 M arginine-HCl, pH 6).

[0065] The anion exchange product (anion exchange pool) was then loaded into the system and concentrated to an intermediate concentration by the first ultrafiltration. The pool was then diafiltered (8 X or diafiltration volumes) to its formulation (0.02 M histidine, 0.2 M arginine-HCl, pH 6). The pool was then concentrated by a second ultrafiltration to a final mass concentration of> 170 g / L and recovered by filtration through a 0.22 micron sterile filter. The entire UF / DF process was performed with a temperature setpoint of about 45 degrees C. This temperature control was achieved using the incoming pool temperature control after anion exchange, diafiltration buffer, and using a jacketed recirculation vessel in the UF / DF process as illustrated herein.

[0066] After UF / DF, the recovered pool was diluted (i.e., conditioned) to a mass concentration of about 150 g / L in 0.02 M histidine, 0.2 M arginine-HCl, 0.04% polysorbate-20, pH 6 (final formulation). During the conditioning steps, the temperature of the mass was allowed to return to ambient temperature. After conditioning, the mass formed was recovered by filtration through a 0.22 micron sterile filter.

[0067] The UF / DF system can be regenerated with 0.1 N sodium hydroxide and decontaminated with 1.4% Minncare.® When not in use, the system can be stored in 0.1 N aqueous sodium hydroxide. UF / DF membranes can be stored, for example, in 0.1% Roccal® / 20% glycerol-water between application cycles.

General procedures of the ultrafiltration / diafiltration process

[0068] Operation parameters: Feed flow rate at 0.5 l / min / ft². A constant pressure control of the retentate (e.g., 10 psig) was used for cleaning and equilibration prior to use, while C_waii, constant retentate pressure or constant TMP during work-up.

[0069] Equilibration Before Use: The following formulations were obtained for Pellicon-2 cleaned membrane cartridges prior to use to insure proper equilibration of the membranes.

<td>Volume (1 / ft²)</td><td>Solution (room temperature)</td><td>Mode</td>

<td> -</td><td> -</td><td>SPFO</td>

<td> 1,0</td><td>WFI</td><td>SPFO</td>

<td> 1,0</td><td>Buffer DF</td><td>SPFO</td>

<td> 0,5</td><td>Buffer DF</td><td>TRFO, 10 minutes</td>

<td> -</td><td> -</td><td>SPFO</td>

[0070] Application of the method: The following was performed on the resulting anion exchange pool (Q pool) obtained from a preceding separation step, for example, a Q-Sepharose chromatography step:

A first ultrafiltration or a first ultrafiltration (UF1) to a concentration of about 5 g / 1 to a concentration for diafiltration (Cdf);

diafiltration or diafiltration (DF1) with four (4) volumes of diafiltration (DV) with DF buffer;

continued diafiltration (DF2) with four (4) volumes of diafiltration (DV) of buffer DF;

second ultrafiltration or second ultrafiltration (UF2) to final concentration (CFinai); and optional product recovery.

[0071] The above steps were typically performed with a low dP Recycle (mix) of, for example, 15 min.

[0072] Cleanup After Use: The following tabulated sequence and conditions were used to clean Pellicon-2 cassette membranes immediately after use.

<td>Volume (1 / ft²)</td><td>Solution (temp, room)</td><td>Mode</td>

<td> 1,0</td><td>0.1NNaOH</td><td>SPFO</td>

<td> 0,5</td><td>0.1NNaOH</td><td>TRFO, 30 minutes</td>

<td> -</td><td> -</td><td>SPFO</td>

<td> 1,0</td><td>WFI</td><td>SPFO</td>

<td> 0,5</td><td>300 ppm Minncare®</td><td>TRFO, 30 minutes</td>

<td> -</td><td> -</td><td>SPFO</td>

<td> 1,0</td><td>WFI</td><td>SPFO</td>

<td> -</td><td> -</td><td>Integrity test @ 30 psig</td>

<td> 0,5</td><td>0.1 N NaOH</td><td>TRFO, 15 minutes storage</td>

Definitions of operating modes in TFF.

[0073] Single pass with filtrate in open form (SPFO). The retentate and filtrate are directed to the runoff. Filtrate valve open.

[0074] Full Recycle with Filtrate in Open Form (TRFO). The retentate and filtrate are directed to a recirculation vessel. Filtrate valve open.

[0075] Fed Batch Ultrafiltration (FB-UF). The retentate is directed to the recirculation tank, the filtrate is directed to the drain and the incoming pool is transferred to the recirculation tank.

Batch Ultrafiltration (B-UF). The retentate is directed to a recirculation tank and the filtrate is directed to the drain.

[0077] Diafiltration (DF). The retentate is directed to the recirculation tank, the filtrate is directed to the drain and the diafiltration buffer is transferred to the recirculation tank.

dP refers to the pressure difference.

[0078] Product Transfer. The ultrafiltration membrane unit and the recirculation tank are open to the pool tank. The nitrogen boost pressure is controlled. The pool is transferred first using a recirculation pump and then using a per steel hand pump.

[0079] Conveying the Feed. The incoming pool is pumped to the recirculation tank.

[0080] Complete Recycle with Filtrate Closed (TRFC). The retentate is directed to a recirculation vessel. Filtrate valve closed.

[0081] "Q pool" refers to a protein pool resulting from, for example, a preceding Q-Sepharose chromatography step that has been buffer conditioned, also referred to as "conditioned pool".

[0082] WFI refers to Water for Injection.

EXAMPLES

[0083] The following examples are provided to further describe how to apply the above disclosure as well as to give the best modes considered for practicing the various aspects of the disclosure. It is understood that these examples are in no way intended to limit the true scope of the disclosure, but are presented rather for illustrative purposes.

Example 1

[0084] Highly concentrated formulation of rhuMAb E25. A pilot scale UF system for concentration / formulation of rhuMAb E25 (recombinant human monoclonal antibody, targeting IgE, USP 6,172,213) was used. Millipore Pelicon Ultrafiltration / Diafiltration System, with 5.7-ft² 10,000 Daltons Regenerated Cellulose Composite Membrane. The system consisted of a diaphragm holder, a Waukeskaw Model 6 feed pump with a rotary lobe piston, U ”316L stainless steel recirculation tubing, and a recirculation vessel. Pressure indicators / transmitters (Anderson) were located at the inlet (RETENTATE), outlet (RETENTAT) and permeate (FILTRAT) of the diaphragm holder. Flux indicators (Yokogawa ADMAG) were located at the inlet (FEED MATERIAL) and the permeate (FILTRAT) of the membrane holder. A back pressure regulating valve (Mikroseal) was placed at the outlet of the diaphragm holder to control the retentate pressure and act on the transmembrane pressure (TMP). A 40 liter 316L stainless steel jacketed tank was used as a recirculation vessel. This tank was equipped with a level indicator, an overhead stirrer (Lightnin), an anti-vortex device, and a bottom valve (NovAseptic). Control

-21 temperatures were achieved by using the feed material - temperature-modulated glycol, supplied to the tank shell.

[0085] During this cycle, the feed flow rate was kept constant at a rate of 2.85 l / min (0.5 l / min / ft²). The retentate pressure control was adjusted to a constant level of 10 psig during all pre-application and post-application operations. The system used Control Scheme C during the ultrafiltration and diafiltration operations_waii for controlling the flux across the membrane, see, for example, R. van Reis, et al., Constant Cwall Ultrafiltration Process Control, J. of Membranę Science, 130 (1997), 123140.

[0086] Prior to the process, the system storage solution (0.1 N NaOH) was rinsed in a single pass, in purge mode, first 2 1 / ft² purified water (PW), then 1 1 / ft² diafiltration buffer (50 mM histidine / pH 6.0). After flushing, the system was equilibrated by recirculating 0.5 l / ft² diafiltration buffer for 10 min. The pH of the recirculated solution was checked to confirm that it was equilibrated. The tank level was then reduced to the minimum measurable value to minimize dilution of the incoming protein pool. The protein pool from the preceding QSepharose chromatography step was measured, which gave 3.2 g E25 / 1 and had a volume of 43.1 1. The protein was in a solution of 25 mM TRIS buffer and about 200 mM NaCl, and the pH was adjusted to 6. 2. To start the cycle, the protein pool was transferred to a recirculation vessel. In the vessel, the pool was mixed with an overhead stirrer, and the temperature was maintained at ambient temperature (20-25 ° C).

[0087] During the process, the pool was concentrated in UF1 mode to 50 g E25 / 1 (about 2.8 L). At the beginning of the diafiltration, the temperature set point of the recirculation vessel was increased to 40 ° C. The temperature rise and control was influenced by the flow of warm glycol through the outer jacket of the tank. The pool was then diafiltered with 8 diafiltration volumes of diafiltration buffer. Diafiltration was performed at a constant volume, which was achieved by matching the flow rate of buffered solution transferred to the recirculation tank to the flow rate of filtrate withdrawn from the system. After the diafiltration was completed, the pool was further concentrated in UF2 mode. This phase was also performed using an elevated temperature set point, of the order of 40 ° C. The target final concentration was 110 g / L. This was achieved without the need to reduce the feed flow rate. Subsequently, a low pressure drop mixing was performed with the feed pump controlled to maintain a pressure drop of 5-10 psig for the feed channel. A sample was taken from the recirculation tank and a final concentration by weight of approximately 120 g / L was measured. Table 1 summarizes the bandwidth and flux results for UF1, DF (DF1 + DF2) and UF2.

-22 Table 1.

<td>Way phase</td><td>Normalized throughput (g / ft²/ hour)</td><td>Normalized flux (LMH / psig)</td>

<td>UF1</td><td> 13,8</td><td> 4,97</td>

<td>DF</td><td> 13,8</td><td> 2,92</td>

<td>UF2</td><td> 181,4</td><td> 1,64</td>

[0088] FIG. 2 shows the observed or measured values ​​of the method over time for the parameters of the feed flow rate (210), tank temperature (220), feed dP (230), TMP (240) and filtrate flow rate (250) during the various phases or modes of the method in including UF1 (10), DF (20), UF2 (30).

[0089] FIG. 3 shows the observed or measured values ​​of the method over time for concentration E25 (310), stream (320) and TMP (240).

[0090] FIG. 4 shows the observed or measured values ​​of the method over time for pressure drop vs. protein concentration observed with UF1 (410) and UF2 (420) at 37 ° C.

[0091] The protein pool was recovered in a series of steps. The first pool in the recirculation tank was pumped from the tank through a Millipac 200, 0.22 micron sterilization-grade filter, using a rotary lobe feed pump. The protein solution was then displaced from the tubing and membrane unit with a 5 psig nitrogen gas purge applied to the highest point on the retentate line. The final step was to purge the tank and the feed line, also with 5 psig nitrogen gas.

[0092] Product recovery was considered to be improved over Example 1 when carried out at ambient temperature because elevated temperature used in one or more of the ultrafiltration, diafiltration or recovery steps reduced the viscous effects. For example, when the temperature control was turned off during product recovery, the system would slowly cool down during this operation, making recovery from the membrane unit difficult. Alternatively, recovery may be performed first from the membrane holder and then from the recirculation vessel.

[0093] To determine the mass of the loss in recovery, 1.74 L of DF buffer was added to the system and recirculated for approximately 5 minutes, and recovered using the same sequence as described above. This volume was then analyzed for protein concentration with other pools. Table 2 summarizes the results.

-23 Table 2.

<td></td><td>Volume (1)</td><td>Concentration (g / 1)</td><td>Weight (g)</td><td>Efficiency or {loss} (%)</td>

<td>Pool Q</td><td> 43,1</td><td> 3,2</td><td> 137,9</td><td> 100</td>

<td>Recovered pool</td><td> 0,99</td><td> 120</td><td> 118,8</td><td> 86,1</td>

<td>Flushing with buffer</td><td> 1,74</td><td> 9,8</td><td> 17,1</td><td> 12,4</td>

<td>Filtrate</td><td> 65,3</td><td> 0,04</td><td> 2,6</td><td> 1,9</td>

[0094] After treatment, the membrane was regenerated with 0.1 N NaOH, 1 L / ft, with a single-pass wash followed by a full recirculation of 0.5 L / ft.² for 30 min. Then a 1 L / ft wash was performed² PW (clean water). The 300 ppm Minncare® solution was then fully recirculated for 30 min. The system was flushed again with 1 L / ft² PW and finally 0.1 N NaOH was recirculated for 15 min and stored. The recovered pool was diluted to 80 g E25 / 1 and conditioned for a final formulation of 50 mM histidine / 150 mM trehalose / 0.02% polysorbate 20 / pH 6.0. Product quality was assessed by size exclusion chromatography (SEC) for both the incoming Q pool and the final recovered weight. These data are presented in Table 3.

Table 3.

<td>Pool</td><td>SEC results (% monomer)</td>

<td>Pool Q</td><td> 99,8</td>

<td>Final mass</td><td> 99,8</td>

Comparative example 2

Highly concentrated formulation of rhuMAb E25 at ambient temperature

[0095] Example 1 was performed with the following exceptions. Prior to the process, the system storage solution (0.1 N NaOH) was rinsed in a single pass, in purge mode, first 2 1 / ft² purified water (PW), then 1 1 / ft² diafiltration buffer (20 mM histidine / pH 6.0). After rinsing, the system was equilibrated by recirculating 0.5 l / ft² diafiltration buffer for 10 min. The pH of the recirculated solution was checked to confirm that it was equilibrated. The tank level was then reduced to the minimum measurable value to minimize dilution of the incoming protein pool.

[0096] The protein pool from the preceding Q-Sepharose chromatography step was measured to give 3.3 g E25 / 1 and had a volume of 33.3 L. The protein was in a solution of 25 mM TRIS buffer and about 200 mM NaCl, and The pH was adjusted to 6.2. To start the cycle, the protein pool was transferred to a recirculation vessel. In the vessel, the pool was stirred with an overhead stirrer, and the temperature was kept at the temperature

-24 ambient (20-25 ° C). During the process, the pool was concentrated in UF1 mode to 50 g E25 / 1 (about 2.2 L). The pool was then diafiltered with 8 diafiltration volumes of diafiltration buffer. Diafiltration was performed at a constant volume, a volume achieved by adjusting the flow rate of the buffered solution transferred to the recirculation tank to the flow rate of the filtrate withdrawn from the system. Diafiltration was also performed at ambient temperature. After completion of diafiltration, the pool was further concentrated in UF2 mode. The target final concentration was 110 g / L. However, due to the high pressure drop in the feed channel, this concentration was not achieved. In an attempt to achieve this concentration, the feed flow rate was reduced to 1.4 l / min at a mass concentration of about 80 g E25 / L as the pressure drop in the feed channel reached 50 psig. UF2 was continued until the high pressure drop of 50 psig was reached again and the process was stopped. Next, a low pressure drop mixing was attempted whereby a feed pump was used to maintain a 5 psig pressure drop across the feed channel. Again, the viscous nature of the protein solution made this difficult to achieve as the rotary lobe pump was under excessive pressures. A sample was taken from the recirculation tank and a final concentration by weight of approximately 104 g / L was measured. Table 4 summarizes the results for throughput and flux measured during the UF1, DF (DF1 + DF2) and UF2 phases.

Table 4.

<td>Way phase</td><td>Normalized throughput (g / ft²/ hour)</td><td>Normalized flux (LMH / psig)</td>

<td>UF1</td><td> 14,5</td><td> 5,31</td>

<td>DF</td><td> 9,5</td><td> 1,47</td>

<td>UF2</td><td> 144,6</td><td> 0,78</td>

[0097] FIG. 5 shows the observed or measured values ​​of the process over time for the parameters of the feed flow rate (210), tank temperature (220), feed dP (230), TMP (240) and filtrate flow rate (250) during the various phases or modes of the process in including UF1 (10), DF (20), UF2 (30).

[0098] FIG. 6 shows the observed or measured values ​​of the method over time for the concentration of E25 (310), stream (320) and TMP (240).

[0099] FIG. 7 shows the observed or measured values ​​of the method over time for pressure drop vs. protein concentration observed with UF1 (410) and UF2 (420) at 24 ° C.

[0100] The protein pool was recovered in steps. The first pool in the recirculation tank was pumped from the tank through a Millipac 200, 0.22 micron sterilization-grade filter, using a rotary lobe feed pump. The protein solution was then displaced from the tubing and membrane unit with a 5 psig purge

-25 nitrogen gas applied to the highest point in the retentate line. Product recovery at this stage was very poor due to the sticky nature of the solution. The final step was to purge the tank and feed line, also using 5 psig of nitrogen gas.

[0101] To determine the weight of recovery loss, 1.85 L of DF buffer was added to the system, recirculated for approximately 5 minutes, and recovered using the same sequence as in Example 1. This volume was then analyzed for protein concentration with other pools. Table 5 summarizes the results.

Table 5.

<td></td><td>Volume (1)</td><td>Concentration (g / 1)</td><td>Weight (g)</td><td>Efficiency or {loss} (%)</td>

<td>Pool Q</td><td> 33,3</td><td> 3,3</td><td> 109,9</td><td> 100</td>

<td>Recovered pool</td><td> 0,77</td><td> 104,4</td><td> 80,4</td><td> 73,1</td>

<td>Flushing with buffer</td><td> 1,85</td><td> 14,7</td><td> 27,2</td><td> 24,7</td>

<td>Filtrate</td><td> 52,2</td><td> 0,03</td><td> 1,6</td><td> 1,5</td>

[0102] After the process, the membrane was regenerated with 0.1 N NaOH, 1 L / ft, with a single-pass wash, followed by a full recirculation of 0.5 L / ft.² for 30 min. Then a 1 L / ft wash was performed² PW. The 300 ppm Minncare® solution was then fully recirculated for 30 min. The system was flushed again with 1 L / ft² PW and finally 0.1 N NaOH was recirculated for 15 min and stored. The recovered pool was diluted to 80 g E25 / 1 and conditioned for a final formulation of 20 mM histidine / 250 mM sucrose / 0.02% polysorbate 20 / pH 6.0. Product quality was assessed by size exclusion chromatography (SEC) for both the incoming Q pool and the final recovered weight. These data are presented in Table 6.

Table 6.

<td>Pool</td><td>SEC results (% monomer)</td>

<td>Pool Q</td><td> 99,8</td>

<td>Final mass</td><td> 99,8</td>

Example 3

Highly concentrated rhuMAb E26 formulation with initial fed batch mode

[0103] Example 1 was repeated with the following exceptions. The concentrate / preparation was rhuMAb E26 (recombinant human monoclonal antibody, targeting IgE). The products of this example were used in the toxicological evaluation. Millipore system was assembled

-26Pelicon for ultrafiltration / diafiltration, with 11.4-ft² 30,000 daltons regenerated cellulose composite membrane. The feed flow rate was set to a fixed rate of 5.0 l / min (0.44 l / min / ft²). The retentate pressure was maintained between about 6-8 psig during the ultrafiltration and diafiltration operations. The protein pool from the preceding Q-Sepharose chromatography step was measured, giving 6.7 g E26 / 1 and having a volume of 59.3 L.

[0104] Since the incoming pool was larger than the recirculation vessel, the UF1 method was started in a fed batch mode. In this mode, pool Q was added to the recirculation vessel at approximately the same rate as filtrate passes through the TFF membrane to the drain. After the remaining Q pool was transferred to the recirculation vessel, the UF1 process was continued in batch mode. During UF1, the pool was concentrated to 50 g E25 / 1 (about 7.9 L). At the beginning of the diafiltration, the temperature set point of the recirculation vessel was increased to 40 ° C. The temperature rise and control was influenced by the flow of warm glycol through the outer jacket of the tank. The pool was then diafiltered with 8 diafiltration volumes of diafiltration buffer. Diafiltration was performed at a constant volume, which was achieved by matching the flow rate of buffered solution transferred to the recirculation tank to the flow rate of filtrate withdrawn from the system. After completion of the diafiltration, the pool was further concentrated in UF2 mode to a final concentration of 109 g E26 / 1 (3.6 L). This phase was also performed using an elevated temperature set point of 40 ° C. Subsequently, a low pressure drop mixing was performed with the feed pump controlled to maintain a pressure drop of 5-10 psig for the feed channel. Table 7 summarizes the throughput and flux results for UF1, DF (DF1 + DF2), and UF2.

Table 7.

<td>Way phase</td><td>Normalized throughput (g / ft²/ hour)</td><td>Normalized flux (LMH / psig)</td>

<td>UF1</td><td> 26,1</td><td> 3,71</td>

<td>DF</td><td> 19,2</td><td> 2,34</td>

<td>UF2</td><td> 174,2</td><td> 1,80</td>

[0105] FIG. 8 shows the observed or measured process values ​​over time for the feed rate (210), the temperature of the vessel (220), the feed dP (230), the TMP (240) and the filtrate flow rate (250).

[0106] FIG. 9 shows the observed or measured values ​​of the method over time for concentration E26 (910), flux (920) and TMP (940).

[0107] FIG. 10 shows the observed or measured values ​​of the method over time for pressure drop vs. protein concentration observed for UF1 (1010) and UF2 (1020).

[0108] Just prior to product recovery, a 10 ml sample was analyzed for detection and bioburden titer, typical rejection limit is 1,000 colony forming units (CFU) per ml. The results of this test were 1.8 CFU / mL, a suitable value at this stage and well below the rejection limit. To determine the mass loss in recovery, 908.1 L of DF buffer was added to the system and recirculated for approximately 5 minutes, and recovered using the same sequence as described above. This volume was then analyzed for protein concentration with other pools. Table 8 summarizes the results.

Table 8.

<td></td><td>Volume (1)</td><td>Concentration (g / 1)</td><td>Weight (g)</td><td>Efficiency or {loss} (%)</td>

<td>Pool Q</td><td> 59,3</td><td> 6,7</td><td> 397,3</td><td> 100</td>

<td>Recovered pool</td><td> 3,41</td><td> 109,1</td><td> 372,0</td><td> 93,6</td>

<td>Flushing with buffer</td><td> 0,908</td><td> 20,4</td><td> 18,5</td><td> 4,7</td>

<td>Filtrate</td><td> 120</td><td>n / a</td><td>n / a</td><td>n / a</td>

[0109] The recovered pool was diluted to 80 g E26 / 1 and conditioned for a final formulation of 50 mM histidine / 150 mM trehalose / 0.02% polysorbate 20 / pH 6.0. Product quality was assessed by size exclusion chromatography (SEC) for incoming pool Q, post UF1 retentate pool, post DF retentate pool and final recovered weight. These data are summarized in Table 9.

Table 9.

<td>Pool</td><td>SEC results (% monomer)</td>

<td>Pool Q</td><td> 99,8</td>

<td>End of UF1</td><td> 99,8</td>

<td>End of DF</td><td> 99,8</td>

<td>Final mass</td><td> 99,8</td>

Example 4

[0110] High Concentrated formulation of rhuMAb E26 for Toxicological Assessment-Comparison of 10kD and 30kD. Example 3 was repeated with the following exceptions. Two pilot scale UF systems were used for the concentration / formulation of rhuMAb E26. Two Millipore Pelicon ultrafiltration / diafiltration systems were assembled, with 11.4-ft² regenerated composite cellulose membranes, one with a pore size of 10,000 Daltons and the other with a pore size of 30,000 Daltons. Retentate pressures were maintained at about 6-9 psig.

-28 10 kD method

[0111] The protein pool from the preceding QSepharose chromatography step was measured, giving 5.85 g E26 / 1 and having a volume of 62.4 1. During UF1 the pool was concentrated to 50 g E26 / 1 (approximately 7.3 L) . After the diafiltration was completed, the pool was further concentrated in UF2 mode to a final concentration of 107.5 g E26 / 1 (3.4 L). Table 10 summarizes the results for throughput and flux for UF 1, DF and UF2.

Table 10.

<td>Way phase</td><td>Normalized throughput (g / ft²/ hour)</td><td>Normalized flux (LMH / psig)</td>

<td>UF1</td><td> 21,8</td><td> 3,6</td>

<td>DF</td><td> 15,9</td><td> 2,6</td>

<td>UF2</td><td> 137,4</td><td> 1,93</td>

[0112] To determine the mass of recovery loss, 987 mL of DF buffer was added to the system, recirculated for approximately 5 minutes, and recovered using the same sequence as described above. This volume was then analyzed for protein concentration with other pools. Table 11 summarizes the results.

Table 11.

<td></td><td>Volume (1)</td><td>Concentration (g / 1)</td><td>Weight (g)</td><td>Efficiency or {loss} (%)</td>

<td>Pool Q</td><td> 62,4</td><td> 5,85</td><td> 365,4</td><td> 100</td>

<td>Recovered pool</td><td> 3,38</td><td> 107,5</td><td> 361,7</td><td> 98,9</td>

<td>Flushing with buffer</td><td> 0,987</td><td> 19,9</td><td> 19,6</td><td> 5,4</td>

<td>Filtrate</td><td> 125</td><td>n / a</td><td>n / a</td><td>n / a</td>

[0113] FIG. 11 shows the observed or measured values ​​of the method over time for the feed flow rate (210), vessel temperature (220), feed dP (230), TMP (240) and filtrate flow rate (250) during various process phases or modes including UF1 (10), DF (20), UF2 (30), and low dP (40), for the 10 kD method.

[0114] FIG. 12 shows the observed or measured values ​​of the method over time for the concentration of E26 (1210), flux (1220), and TMP (1240) during various phases or modes of the method, including UF1 (10), DF (20), UF2 (30), and low dP (40), for the 10 kD method.

[0115] FIG. 13 shows the observed or measured values ​​of the method over time for pressure drop vs. protein concentration observed with UF1 (1310) and UF2 (1320) for the 10 kD method.

-29 Way 30 kD

[0116] The protein pool from the preceding QSepharose chromatography step was measured, giving 5.85 g E26 / 1 and having a volume of 64.5L. During UF1, the initial pool was concentrated to 50 g E26 / 1 (about 7.5L). ). After the diafiltration was completed, the pool was further concentrated in UF2 mode to a final concentration of 117.5 g E26 / 1 (3.2 L). Table 12 summarizes the throughput and flux results for UF1, DF, and UF2.

Table 12.

<td>Way phase</td><td>Normalized throughput (g / ft²/ hour)</td><td>Normalized flux (LMH / psig)</td>

<td>UF1</td><td> 25,5</td><td> 4,01</td>

<td>DF</td><td> 17,6</td><td> 2,39</td>

<td>UF2</td><td> 180,5</td><td> 1,57</td>

[0117] To determine the mass of recovery loss, 918 mL of DF buffer was added to the system, recirculated for approximately 5 minutes, and recovered using the same sequence as described above. The recovered pool was diluted to 80 g E26 / L and conditioned to a 50 mM histidine / 150 mM trehalose / 0.02% polysorbate 20 / pH 6.0 formulation. Table 13 summarizes the results.

Table 13.

<td></td><td>Volume (1)</td><td>Concentration (g / 1)</td><td>Weight (g)</td><td>Efficiency or {loss} (%)</td>

<td>Pool Q</td><td> 64,5</td><td> 5,85</td><td> 377,3</td><td> 100</td>

<td>Recovered pool</td><td> 3,20</td><td> 117,5</td><td> 376,0</td><td> 99,6</td>

<td>Flushing with buffer</td><td> 0,918</td><td> 22,7</td><td> 20,8</td><td> 5,5</td>

<td>Filtrate</td><td> 125</td><td>n / a</td><td>n / a</td><td>n / a</td>

[0118] FIG. 14 shows the observed or measured values ​​of the method over time for the feed flow rate (210), vessel temperature (220), feed dP (230), TMP (240) and filtrate flow rate (250) during various process phases or modes including UF1 (10), DF (20), UF2 (30), and low dP (40), for the 30 kD method.

[0119] FIG. 15 shows the observed or measured values ​​of the method over time for the concentration of E26 (1510), flux (1520) and TMP (1540) during various phases or modes of the method including UF1 (10), DF (20), UF2 (30), and low dP (40), for the 30 kD method.

[0120] FIG. 16 shows the observed or measured values ​​of the method over time for the pressure drop vs. protein concentration observed with UF1 (1610) and UF2 (1620) for the 30 kD method.

Example 5

Scaling up for liquid rhuMAb E25. Example 1 was repeated with the following exceptions.

[0121] A production scale UF system was used for the concentration / formulation of liquid rhuMAb E25 (recombinant human monoclonal antibody, targeting IgE). The product can be used in therapeutic applications and in human bioequivalence studies. Millipore Pelicon ultrafiltration / diafiltration systems were assembled, with 226-ft² a regenerated composite cellulose membrane with a pore size of 30,000 Daltons. Each system consisted of a diaphragm holder, a Viking S3S feed pump with a rotary lobe piston, 1% U ”316L stainless steel recirculation tubing, and a 250L recirculation vessel.

[0122] One 250 liter 316L stainless steel jacketed vessel was used as a recirculation vessel. The temperature control of this tank was achieved by using the feed - temperature modulated glycol, supplied to the tank shell. The temperature of the glycol fed to the tank jacket was raised or lowered using a steam fed heat exchanger or suitably cold glycol supply.

[0123] During this cycle, the feed flow rate was kept constant at a rate of 114 l / min (0.5 l / min / hl). Diafiltration buffer (20 mM histidine / 200 mM arginine chloride / pH 6.0) was obtained in a separate tank. The temperature of this buffer was set to 45 ° C prior to the process. This ensured precise temperature control during the process.

[0124] Prior to processing, the system storage solution (0.1 N NaOH) was rinsed in a single pass, in purge mode, first 1 1 / ft.² water for injection (WFI) followed by 1 l / ft² diafiltration buffer. After flushing, the system was equilibrated by recirculating 0.5 l / ft² diafiltration buffer for 10 min. The pH of the recirculated solution was checked to confirm that it was equilibrated.

[0125] The protein pool from the preceding Q-Sepharose chromatography step was measured to give 5.2562 g E25 / 1 and had a volume of 1.141L. The protein was in a solution of 25 mM TRIS buffer and about 200 mM NaCl, and the pH was adjusted at 6.2. Just before the cycle started, the temperature setpoint for this pool was set at 45 ° C. To start the cycle, the protein pool was transferred to a recirculation vessel through a sterilization grade 0.22 micron filter to approximately 200L in the tank. In the vessel, the pool was mixed with an overhead stirrer, and the temperature was maintained at about 40-50 ° C. Since the incoming pool was larger than the recirculation vessel, the UF1 process was started in a fed batch mode. In this mode, pool Q was added to the recirculation vessel at approximately the same rate as filtrate passes through the TFF membrane to the drain. After moving the remaining Q pool to

From the recirculation vessel, the UF2 process was continued in a batch mode. During UF1 mode, the pool was concentrated to about 30 g E25 / 1 (about 200 L). The pool was then diafiltered with approximately 8 diafiltration volumes of diafiltration buffer. The temperature was kept between 40 ° and 50 ° C during the diafiltration. Diafiltration was performed at a constant volume, which was achieved by matching the flow rate of the buffer transferred to the recirculation tank to the flow rate of the filtrate removed from the system. After completion of the diafiltration, the pool was further concentrated in UF2 mode to a preset final concentration value of&gt; 170 g E25 / 1 (35 L). This UF2 mode was also performed using an elevated temperature setpoint of 45 ° C +/- 5 ° C. Subsequently, a low pressure drop mixing was performed with the feed pump controlled to maintain a pressure drop of 5-10 psig for the feed channel. A sample was taken and specialized scans were performed to confirm the concentration prior to recovery. The concentration of this sample was 219 g E25 / 1. Table 14 summarizes the results for throughput and flux measured during the UF1, DF (DF1 + DF2) and UF2 phases.

Table 14.

<td>Way phase</td><td>Normalized throughput (g / ft²/ hour)</td><td>Normalized flux (LMH / psig)</td>

<td>UF1</td><td> 43,8</td><td> 3,34</td>

<td>DF</td><td> 25,9</td><td> 2,46</td>

<td>UF2</td><td> 78,9</td><td> 0,66</td>

[0126] Just before product recovery, a 30 ml sample was taken and analyzed for detection and bioburden titer. The result was &lt;0.13 CFU / ml. The protein pool was recovered in a series of steps. First, the product was displaced from the membrane in single pass mode with 5 L of DF buffer added to the retentate line. The product was filtered into a recovery tank through a 7.4 ft restriction filter², 0.22 micron sterilization grade, followed by a 2 ft post filter², 0.22 microns sterilization grade. The pool in the recirculation tank was then pumped from the tank using a rotary lobe feed pump. The residual protein solution was then displaced from the tank and feed line with a 5 psig nitrogen gas purge. The final phase was a purge of the membrane unit which now mainly contained DF buffer from the initial product displacement. This phase also used 5 psig of nitrogen gas, at the highest point in the retentate line. The recovered pool was first diluted to approximately 153 g E25 / 1 with DF buffer. Finally, the pool was conditioned to a final formulation of 20 mM histidine / 200 mM arginine-HCl / 0.04% polysorbate 20 / pH 6.0. Each volume of the recovered pool, the diluted pool, and the conditioned pool (Q pool) were analyzed for protein concentration. Table 15 summarizes the results.

-32 Table 15.

<td></td><td>Volume (1)</td><td>Concentration (g / 1)</td><td>Weight (g)</td><td>Efficiency or {loss} (%)</td>

<td>Pool Q</td><td> 1,141</td><td> 5,2562</td><td> 5,997,3</td><td> 100</td>

<td>Recovered pool</td><td> 35,0</td><td> 170,0</td><td> 5,950,0</td><td> 99,2</td>

<td>Diluted pool</td><td> 39,0</td><td> 147,0</td><td> 5,726</td><td> 95,5</td>

[0127] FIG. 17 shows the parameters of the feedstock flow (210), the tank temperature (220), the feed temperature dP (230), TMP (240), and the filtrate flow rate (250) during various phases or modes of the process, including UF1 (10), DF1 ( 20), DF2 (25), UF2 (30), and low dP (50).

Example 6

[0128] Liquid formulation of rhuMAb E25. Example 5 was repeated with the following exceptions. A production scale UF system was used to concentrate / formulate liquid rhuMAb E25 (E25, recombinant human monoclonal antibody, targeting IgE). Millipore Pelicon ultrafiltration / diafiltration systems were assembled, with 226-ft² a regenerated composite cellulose membrane with a pore size of 30,000 Daltons. Each system consisted of a diaphragm holder, a Viking S3S feed pump with a rotating lobe piston, 1U ”316L stainless steel recirculation tubing, and a 230L recirculation vessel. One 250 liter 316L stainless steel jacketed tank was used as recirculation vessel. The feed flow rate was kept constant at a rate of 114 lpm (0.5 lpm / ft²). The retentate pressure control was adjusted to a constant level of 10 psig during all pre- and post-application operations. The system used Control Scheme C during the ultrafiltration and diafiltration operations_INaii for controlling the flow of the stream through the membrane. Diafiltration buffer (20 mM histidine / 200 mM arginine chloride / pH 6.0) was obtained in a separate tank. The temperature of this buffer was set to 45 ° C prior to the process. This ensured precise temperature control throughout the process. The protein pool from the preceding Q-Sepharose chromatography step was measured to give 5.5438 g E25 / 1 and had a volume of 1.082 L. The protein was in a solution of 25 mM TRIS buffer and about 200 mM NaCl, and the pH was adjusted to 6.2. Just before the cycle started, the temperature setpoint for this pool was set at 45 ° C. To start the cycle, the protein pool was transferred to a recirculation vessel through a sterilization grade 0.22 micron filter to approximately 200L in the tank. In the vessel, the pool was mixed with an overhead stirrer, and the temperature was maintained at ambient temperature (40-50 ° C). Since the incoming pool was larger than the recirculation vessel, the UF1 process was started in a fed batch mode. In this mode, pool Q was added to the recirculation vessel at approximately the same rate as filtrate passes through the TFF membrane to the drain. After the remaining Q pool was transferred to the recirculation vessel, it continued

Implementing the UF1 method in the batch mode. During UF1, the pool was concentrated to about 30 g E25 / L (about 200 L). The pool was then diafiltered with 8 diafiltration volumes of diafiltration buffer. The temperature was kept between 40 ° and 50 ° C during the diafiltration. Diafiltration was performed at a constant volume, which was achieved by matching the flow rate of the buffer transferred to the recirculation tank to the flow rate of the filtrate removed from the system. After the diafiltration was completed, the pool was further concentrated in the UF2 mode to a final concentration preset value greater than 170 g E25 / 1 (35 L). This phase was also performed using an elevated temperature setpoint of 45 ° C +/- 5 ° C. Subsequently, a low pressure drop mixing was performed with the feed pump controlled to maintain a pressure drop of 5-10 psig for the feed channel. A sample was taken and specialized scans were performed to confirm the concentration prior to recovery. The concentration of this sample was 191 g E25 / 1 and the pool volume was 31.9L. The plot of method parameters over time was comparable to those observed and summarized for the above FIG. 17.

Table 14.

<td>Way phase</td><td>Normalized throughput (g / ft²/ hour)</td><td>Normalized flux (LMH / psig)</td>

<td>UF1</td><td> 45,1</td><td> 3,21</td>

<td>DF</td><td> 25,9</td><td> 2,51</td>

<td>UF2</td><td> 121,4</td><td> 0,79</td>

[0129] Just prior to product recovery, a 30 ml sample was taken and analyzed for bioburden titer. The results of this test were below the limit of detection (&lt;0.13 CFU / ml).

The protein pool was recovered in a series of steps. First, the product was displaced from the membrane in single pass mode with 5 L of DF buffer added to the retentate line. The product was filtered into a recovery tank through a 7.4 ft restriction filter², 0.22 micron sterilization grade, followed by a 2 ft post filter², 0.22 microns sterilization grade. The pool in the recirculation tank was then pumped from the tank using a rotary lobe feed pump. The residual protein solution was then displaced from the tank and the feed line with a 5 psig nitrogen gas purge. The final phase was a purge of the membrane unit which contained mainly DF buffer from the initial product displacement. This phase also used 5 psig of nitrogen gas, at the highest point in the retentate line. The recovered pool was first diluted to approximately 153 g E25 / 1 with DF buffer. Finally, the pool was conditioned to a final formulation of 20 mM histidine / 200 mM arginine-HCl / 0.04% polysorbate 20 / pH 6.0. The volumes of the recovered pool, the diluted pool, and the conditioned pool were then analyzed for concentration

-34proteins. Table 15 summarizes the results. Upon completion of the process, the membrane was regenerated as described above.

Table 15.

<td></td><td>Volume (1)</td><td>Concentration (g / 1)</td><td>Weight (g)</td><td>Efficiency or {loss} (%)</td>

<td>Pool Q</td><td> 1,082</td><td> 5,5438</td><td> 5,998,4</td><td> 100</td>

<td>Recovered pool</td><td> 34,95</td><td> 167,08</td><td> 5,839,8</td><td> 97,4</td>

<td>Diluted pool</td><td> 38,2</td><td> 152,14</td><td> 5,810,3</td><td> 96,7</td>

Example 7

[0130] Effect of elevated temperature on product quality. The 30 g / L and 150 g / L E25 samples in histidine and Q buffers were kept at different temperatures for 24 hours. Samples were taken for turbidity measurements and SEC analyzes. Turbidity vs. the temperature for E25 at 30 g / L in buffer Q is shown in FIG. 18. FIG. 19 shows the amount of soluble E25 aggregate (150 g / L in 50 mM histidine buffer, pH 6.0) observed over time and at 23 ° C, 40 ° C, 50 ° C, 60 ° C and 70 ° C. The four time intervals (0 hours, 4 hours, 7.5 hours, and 24 hours) for each of these temperatures are shown as a cluster of four bars from left to right as 1810 and 1910, in FIG. 18 and 19. The turbidity of the solution remained essentially unchanged for 24 hours at 60 ° C. No significant soluble E25 aggregate was observed below the temp. 70 ° C, suggesting that the product samples were essentially temperature stable up to at least 60 ° C and for at least 24 hours.

Example 8

[0131] Effect of Elevated Temperature on Biological Load. Samples of E25 at 30 g / L in both arginine and histidine buffers were inoculated with 10³ colony forming units per ml for two challenge organisms: a Gram-positive strain (Staphylococcus aureus) and one Gram-negative strain (Pseudomonas chlororaphis). Samples were taken after 1.5 hours and 6 hours. The results shown in the bar graphs in FIG. 20 and 21 show that with increasing temperature, the numbers of both these provoking organisms decreased. Three temperature ranges (temp. 25 ° C, 40 ° C and 50 ° C; hours) for each of the observed time intervals is shown as a cluster of three bars from left to right as 2010 and 2110, in FIG. 20 and 21. The inoculations shown were carried out in arginine buffer at protein concentrations of 30 g / l. Example 9

[0132] Effect of elevated temperature on process stream. 10 g / L E25 samples in 0.2M arginine, 25 mM histidine, pH 6.0 buffer were evaluated for their effect on flux vs. transmembrane pressure (TMP). FIG. 22 shows that increasing

The system temperature also increased the process flux during the UF / DF operation. Sudden flux jumps were carried out for various mass concentrations and three different temperatures of 23 ° C (2210), 40 ° C (2220) and 46 ° C (2230). The mass transfer coefficient and filtrate flux increased by about 2 to about 3-fold, providing significantly reduced process times.

Example 10

[0133] Highly concentrated anti-CD20 rhuMAb formulation ("2H7"). A pilot scale UF system was used for concentration and formulation of anti-CD20 rhuMAb (2H7; recombinant human monoclonal antibody). Example 1 was repeated with the following exceptions. Millipore Pelicon ultrafiltration / diafiltration systems were assembled, with 17.5-ft² a regenerated composite cellulose membrane with a pore size of 30,000 Daltons. The system consisted of a diaphragm holder, a Viking SI L feed pump with rotating lobe piston, 1U ”316L stainless steel recirculation tubing, and a 40L recirculation vessel. Back pressure control valves were from HD Baumann, Inc. The temperature of the glycol fed to the vessel jacket was raised or lowered as needed using an electric heat exchanger to supply cold glycol, or both.

[0134] During this cycle, the feed flow rate was kept constant at 8.5 l / min (approximately 0.5 l / min / ft).²). FIG. 23 shows the value trends over time for the feed rate (210) scaled 0 to 20, pH (212) scaled 2 to 12, filtrate flow rate (250) scaled 0 to 5, recirculation tank level (2320) scaled from 0 to 45, and dP (2350) retentate scaled from 0 to 100 during various phases or modes of the method including UF1 (10), DF1 (20) and UF2 (30).

[0135] During the ultrafiltration and diafiltration operations, the system used a constant retentate pressure followed by a constant feed / delta retentate pressure control scheme to control the flow of the stream through the membrane. Diafiltration buffer (30 mM sodium acetate / pH 4.9) was obtained in a separate tank. The temperature of this buffer was set to 45 ° C upstream of the process for precise temperature control throughout the process. Prior to processing, the system storage solution (0.1 N NaOH) was rinsed in a single pass, in purge mode, first 1 l / ft² water for injection (WFI) followed by 1 l / ft² diafiltration buffer. After flushing, the system was equilibrated by recirculating 0.5 l / ft² diafiltration buffer for 10 min. The pH of the recirculated solution was checked to confirm that it was equilibrated.

[0136] The protein pool resulting from the preceding Q-Sepharose chromatography step was measured to give 2.31 g of 2H7 / 1 and had a volume of 356 1. The protein was in a solution of 6 mM HEPES free acid / 19 mM HEPES sodium salt and 25 mM sodium acetate, the pH of which was adjusted to 5.3 with 0.5 M acetic acid. Just before the cycle, the temperature setpoint for this pool was set to 45 ° C. To start the cycle, the protein pool was transferred to a recirculation vessel through a sterilization grade 0.22 micron filter, to a level of approximately 40 L in the tank. In the vessel, the pool was mixed with an overhead stirrer, and the temperature was maintained at 40-50 ° C.

[0137] Since the incoming pool was larger than the recirculation vessel, the UF1 method was started in the fed batch mode (see FIG. 23). In this mode, pool Q was added to the recirculation vessel at approximately the same rate as filtrate passes through the TFF membrane to the drain. After the remaining Q pool was transferred to the recirculation vessel, the UF1 process was continued in batch mode. During UF1, the pool was concentrated to about 50 g of 2H7 / 1 (about 16 L). The pool was then diafiltered with 10 diafiltration volumes of diafiltration buffer. The temperature was kept between 40 ° and 50 ° C during the diafiltration. Diafiltration was performed at a constant volume, which was achieved by matching the flow rate of the buffer transferred to the recirculation tank to the flow rate of the filtrate removed from the system. After completion of the diafiltration, the pool was further concentrated in UF2 mode to a predetermined target concentration value of 190 g 2H7 / L (4.3 L). See FIG. 23 inclusion of the constant control dP for 50 psig after completion of this phase. This phase was also performed using an elevated temperature setpoint of 45 ° C +/- 5 ° C. Subsequently, a low pressure drop mixing was performed with the feed pump controlled to maintain a pressure drop of 20 psig for the feed channel. A sample was taken and a density measurement performed to confirm the concentration before recovery. The concentration of this sample was 189 g 2H7 / 1. Table 16 summarizes the results for throughput and flux.

Table 16.

<td>Way phase</td><td>Normalized throughput (g / ft²/ hour)</td><td>Normalized flux (LMH / psig)</td>

<td>UF1</td><td> 32</td><td> 4,8</td>

<td>DF</td><td> 56</td><td> 2,4</td>

<td>UF2</td><td> 267</td><td> 1,6</td>

[0138] The protein pool was recovered in a series of steps. First, the product was displaced from the membrane in single pass mode with 0.2 L of DF buffer added to the retentate line. The product was filtered into a recovery tank through a sterilization-grade 0.22 micron post-filter. The pool in the recirculation tank was then pumped from the tank using a rotary lobe feed pump. The residual protein solution was then displaced from the tank and feed line with a 5 psig nitrogen gas purge. The final phase was a purge of the membrane unit which now mainly contained DF buffer from the initial product displacement. This phase also used 5 psig of nitrogen gas, at the highest point in the retentate line.

[0139] If necessary, the recovered pool was first diluted to about 175 g of 2H7 / 1 with dilution buffer (30 mM sodium acetate, pH 5.3). Finally, the pool was diluted to a target concentration of 150 g 2H7 / 1 and conditioned for a final formulation of 30 mM acetate

-37sodium, 7% trehalose, 0.03% polysorbate 20, pH 5, with 7X conditioning buffer (30 mM sodium acetate, 49% trehalose, 0.21% polysorbate 20, pH 5.3).

The volumes of the recovered pool, the diluted pool, and the conditioned pool were then analyzed for protein concentration. Table 17 presents the results.

Table 17.

<td></td><td>Volume (1)</td><td>Concentration (g / 1)</td><td>Weight (g)</td><td>Efficiency or {loss} (%)</td>

<td>Pool Q</td><td> 355,81</td><td> 2,31</td><td> 821,92</td><td> 100,0</td>

<td>Recovered pool</td><td> 4,64</td><td> 180,02</td><td> 835,3</td><td> 101,6</td>

<td>Final pot</td><td> 4,871</td><td> 149,40</td><td> 727,7</td><td> 88,5</td>

<td colspan="5">Note: The yields include losses due to sampling. The recovered pool volume and concentration include the addition of displaced buffer.</td>

[0140] After processing, the membrane was regenerated with 0.1 N NaOH, 1 L / ft², with a single-pass rinse followed by a full recirculation of 0.5 l / ft² for 30 min. Then a 1 L / ft wash was performed² PW. A complete recirculation of 0.5 L / ft was then performed² 1.4% Minncare® solution for 30 min. The system was flushed again with 1 L / ft² PW and finally 0.1 N NaOH was recirculated for 15 min and stored.

Example 11

[0141] Highly concentrated anti-CD20 rhuMAb formulation. A pilot scale UF system for concentration and formulation of anti-CD20 (2H7) rhuMAb was used for use in a Phase I human clinical trial in a manufacturing facility using good manufacturing practice (GMP). Example 10 was repeated with the following exceptions.

[0142] The protein pool from the preceding Q-Sepharose chromatography step was measured, giving 3.729 g of 2H7 / 1 and having a volume of 262 1. The protein was in a solution of 6 mM HEPES free acid / 19 mM HEPES sodium salt and 25 mM sodium acetate, the pH of which was adjusted to 5.3 with 0.5 M acetic acid. Just before the cycle, the temperature setpoint for this pool was set to 45 ° C. To start the cycle, the protein pool was transferred to a recirculation vessel through a sterilization grade 0.22 micron filter, to a level of approximately 40 L in the tank. In the vessel, the pool was mixed with an overhead stirrer, and the temperature was maintained at 40-50 ° C.

[0143] During UF1, the pool was concentrated to about 50 g 2H7 / 1 (about 20 L). FIG. 24 shows the time value trends for the recirculation tank level (210) scaled from 0.713963 to 295.989, dP retentate (2420) scaled from -0.237899 to 98.6629, feed rate (250) scaled from -0.356981 to 147.994, and the filtrate flow rate (2450) scaled from -0.118994 to 49.3315 during the process. The pool was then diafiltered with 10 diafiltration volumes of diafiltration buffer. During

The diafiltration temperature was kept between 40 ° and 50 ° C. Diafiltration was performed at a constant volume, which was achieved by matching the flow rate of the buffer transferred to the recirculation tank to the flow rate of the filtrate withdrawn from the system. Upon completion of diafiltration, the pool was further concentrated in UF2 mode to a predetermined target concentration value of 190 g 2H7 / L (5.25 L). Notice in FIG. 24 incorporation of the constant control dP for 40 psig after completion of this phase. This phase was also performed using an elevated temperature setpoint of 45 ° C +/- 5 ° C. Subsequently, a low pressure drop mixing was performed with the feed pump controlled to maintain a pressure drop of 20 psig for the feed channel. A sample was taken and a density measurement performed to confirm the concentration before recovery. The concentration of this sample was 194 g 2H7 / 1. Table 18 summarizes the results for throughput and flux.

Table 18.

<td>Way phase</td><td>Normalized throughput (g / ft²/ hour)</td><td>Normalized flux (LMH / psig)</td>

<td>UF1</td><td> 51</td><td> 3,8</td>

<td>DF</td><td> 46</td><td> 2,2</td>

<td>UF2</td><td> 286</td><td> 1,6</td>

[0144] Just before product recovery, a 30 ml sample was taken and analyzed for detection and bioburden titer. The results were negative (i.e. &lt;0.13 CFU / ml). The protein pool was recovered through the series of steps in Example 10. The volumes of the recovered pool, dilution pool, and conditioned pool were then analyzed for protein concentration. Table 19 presents the results. The membrane was regenerated as in Example 10.

Table 19.

<td></td><td>Volume (1)</td><td>Concentration (g / 1)</td><td>Weight (g)</td><td>Efficiency or {loss} (%)</td>

<td>Pool Q</td><td> 262</td><td> 3,72</td><td> 977</td><td> 100</td>

<td>Recovered pool</td><td> 5,0</td><td> 174,0</td><td> 863,0</td><td> 88,3</td>

<td>Diluted pool</td><td> 5,421</td><td> 149,6</td><td> 811,0</td><td> 83,0</td>

Example 12

[0145] Highly concentrated anti-CD20 GMP rhuMAb formulation. Example 11 was repeated with the following exceptions. The protein pool from the preceding QSepharose chromatography step was measured, which gave 5.106 mg of 2H7 / 1 and had a volume of 196L. The protein was in a solution of 6 mM HEPES - free acid / 19 mM HEPES sodium salt and 25 mM sodium acetate,

-39, the pH of which was adjusted to 5.3 with 0.5 M acetic acid. Just before the cycle, the temperature setpoint for this pool was set to 45 ° C. To start the cycle, the protein pool was transferred to a recirculation vessel through a sterilization grade 0.22 micron filter, to a level of approximately 40 L in the tank. In the vessel, the pool was mixed with an overhead stirrer, and the temperature was maintained at 40-50 ° C.

[0146] During UF1 the pool was concentrated to about 50 g of 2H7 / 1 (about 20 L). FIG. 25 shows the time value trends for the recirculation tank level (210) scaled 0 to 300, dP retentate (2520) scaled 0 to 100, feed flow rate (250) scaled 0 to 150, and filtrate flow rate (2550) scaled from 0 to 50 during the method. The pool was diafiltered with 10 diafiltration volumes (10X) of diafiltration buffer. The temperature was kept between 40 ° and 50 ° C during the diafiltration. Diafiltration was performed at a constant volume, which was achieved by matching the flow rate of the buffer transferred to the recirculation tank to the flow rate of the filtrate removed from the system. Upon completion of diafiltration, the pool was further concentrated in UF2 mode to a predetermined final concentration target of 190 g 2H7 / 1 (5.26 L), again using the dP constant control at the very end of this phase (see FIG. 25). This phase was also performed using an elevated temperature setpoint of 45 ° C +/- 5 ° C. Subsequently, a low pressure drop mixing was performed with the feed pump controlled to maintain a pressure drop of 20 psig for the feed channel. A sample was taken and a density measurement performed to confirm the concentration before recovery. The concentration of this sample was 191 g 2H7 / 1. Table 20 summarizes the results for throughput and flux.

Table 20.

<td>Way phase</td><td>Normalized throughput (g / ft²/ hour)</td><td>Normalized flux (LMH / psig)</td>

<td>UF1</td><td> 67</td><td> 3,6</td>

<td>DF</td><td> 47</td><td> 2,1</td>

<td>UF2</td><td> 292</td><td> 1,8</td>

[0147] Just before product recovery, a 30 ml sample was taken and analyzed for detection and bioburden titer. The results were negative (i.e. &lt;0.13 CFU / ml). The protein pool was recovered by a series of steps as in Example 11. The volumes of the recovered pool, dilution pool, and conditioned pool were then analyzed for protein concentration. Table 21 presents the results. The membrane was regenerated as in Example 11.

Table 21.

<td></td><td>Volume (1)</td><td>Concentration (g / 1)</td><td>Weight (g)</td><td>Efficiency or {loss} (%)</td>

<td>Pool Q</td><td> 196</td><td> 5,106</td><td> 1000</td><td> 100</td>

<td></td><td>Volume (1)</td><td>Concentration (g / 1)</td><td>Weight (g)</td><td>Efficiency or {loss} (%)</td>

<td>Recovered pool</td><td> 4,9</td><td> 187,1</td><td> 918,0</td><td> 91,8</td>

<td>Diluted pool</td><td> 6,075</td><td> 150,9</td><td> 916,9</td><td> 91,7</td>

[0148] The disclosure has been described with reference to various specific and preferred embodiments and techniques.

Mirosława Ważyńska

Patent Attorney