Means for reusing chromatography

Abstract

To provide a method for purifying or regenerating a chromatography material which can be used for purifying or regenerating a chromatography material for reuse in large-scale production of a plurality of polypeptide products.SOLUTION: A method includes: a) a step of allowing 2 material volume or more of elution buffer to pass through a chromatography material; b) a step of statically holding the material in the elution buffer for a range of approximately 10 minutes to approximately 30 minutes; c) a step of allowing approximately 2 material volume or more of elution buffer to pass through the material; and d) a step of allowing approximately 2 material volume or more of regeneration buffer to pass through the material.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] Cross-reference to related applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 61 / 874,305, filed September 5, 2013, the entire contents of which are incorporated herein by reference.

[0002] The present invention provides a method for chromatography. [Background technology]

[0003] Recombinant monoclonal antibodies (mAbs) are used in medicine and diagnostics (Albrecht, H. et al., Drugs Today 2009, 45:199-211; Takimoto, CH; Principles of Oncologic Pharmacotherapy Calvo, E. In Cancer Management: A Multidisciplinary Approach Medical, Surgical & Radiation Oncolog, 11th ed.; Pazdur, R.; Wagman, LD; Camphausen, KA; Hoskins, WJ, eds. CMP Healthcare Media LLC: Lawrence, KS, USA, 2008). Industrially, recombinant mAbs are bioproduced in living cells, such as Chinese hamster ovary (CHO) cells (Fahrner, RL et al., Biotechnol. Genet. Eng. Rev. 2001, 18:301-327). After production, the desired mAb must be isolated from the cellular and media components used in its production. This purification process has two major steps: (a) a primary isolation process, followed by (b) a final purification process. This primary mAb isolation process begins after cells are harvested for the mAb of interest. After the mAb is harvested, the product pool contains the mAb of interest as well as cellular components (media components, proteins, DNA) and viruses that may be present during the mAb production process. In the first chromatography step of an exemplary isolation process, this product pool is run through a Protein A affinity column (Step 1). The purpose of the Protein A affinity step is to remove media components, cell debris, and putative viruses. After Step 1, the product pool containing the mAb is further purified on an ion exchange column (IEX, Step 2). Step 2 serves to remove additional contaminants such as aggregates and DNA. After IEX chromatography, the final step of the primary isolation process involves removing viruses using a virus reduction filter (Step 3).Typically, after step 3, final purification of the mAb is performed using a second ion exchange step (step 4) to remove any residual CHO protein (CHOP). After final purification, ultrafiltration / diafiltration (UF / DF, step 5) is performed to remove small molecules, concentrate the mAb, and exchange the buffer in order to formulate the purified mAb into its final formulation buffer. This is followed by a bulk filtration step (step 6) to ensure sterility of the mAb pool.

[0004] Protein A affinity chromatography is commonly used in industrial mAb purification because it is efficient, scalable, and reproducible (Affinity Chromatography Principles and Methods, Amersham Biosciences, Uppsala, Sweden, 2002, see the World Wide Web at gelifesciences.com / webapp / wcs / stores / servlet / productById / en / GELifeSciences-us / 18102229, accessed July 13, 2012; Fahrner, RL et al., Bioprocess Eng. 1999, 22:287). However, the cost of Protein A resin is significant and constitutes a significant portion of raw material costs in MAb production (Fahrner, RL et al., Biotechnol. Appl. Biochem. 1999, 30, 121-128; Kelley, B., Biotechnol. Prog. 2007, 23:995-1008). This expense is exacerbated by underuse of the resin, such as when a single packed Protein A column is used for only 10% of its potential life (in pilot plants and during clinical production). To reduce these costs, reuse of Protein A resin for multiple different mAb products is desirable. Reuse of Protein A resin for multiple products is not common practice because reuse can result in protein carryover not only from previous runs but also from previously purified products. Therefore, an efficient purification process enables reuse. Such a process not only saves money, space, and time, but is also environmentally friendly. Additionally, time savings result from avoiding repacking the column for every new mAb synthesized. Reusing Protein A resin also results in less environmental contamination, as less Protein A resin is wasted, stored, or shipped.Note that a typical MabSelect™ SuRe resin can be used for up to 250 cycles (Fahrner, RL Biotechnol. Appl. Biochem. 1999, 30, 121-128; Kelley, B., Biotechnol. Prog. 2007, 23:995-1008; MabSelect™ Sure resin; Application note 28-9872096 AA; Lifetime performance study of MabSelect™ Sure LX during repeated cleaning-in-place; GE Healthcare, Piscataway, NJ. February 2011. See worldwide web at gelifesciences.com / gehcls_images / GELS / Related%20Content / Files / 1314807262343 / litdoc28987296AA_20110831222625.pdf). However, in a typical pilot-plant scale clinical or toxicology run, a Protein A column is used for only 3–4 runs total (18–30 cycles), resulting in roughly 220–232 wasted cycles (Fahrner, RL et al., Biotechnol. Genet. Eng. Rev. 2001, 18:301–327). As described herein, the reuse of chromatography columns such as the MabSelect™ SuRe resin column for multiple CHO products at laboratory and pilot scale has been enabled and optimized. An improved Protein A resin cleanup procedure, used between mAb purification runs, was developed and validated to reduce the level of mAb carryover from the previous purification to an acceptable level.This was accomplished by addressing: (a) the amount of pre-cleanup protein carryover (if any) from a previous purification to a subsequent purification using the same Protein A resin, and (b) identifying a method to clean up the Protein A affinity resin before or after use so that multiple products can be purified on the same Protein A resin with little protein carryover and no safety concerns.

[0005] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety. Summary of the Invention

[0006] The present invention provides a method for purifying or regenerating chromatographic materials, such as chromatography resins, for reuse. The chromatographic materials can be purified and / or regenerated for use with the same product or a different product.

[0007] In some aspects, the present invention provides a method for purifying a chromatography material for reuse, the method comprising the steps of: a) passing at least two material volumes of an elution buffer through the material, wherein the elution buffer comprises about 0.15 M acetic acid and has a pH of about 2.9; b) statically holding the material in the elution buffer for a time ranging from about 10 minutes to about 30 minutes; c) passing at least two material volumes of the elution buffer through the material; and d) passing at least two material volumes of a regeneration buffer through the material, wherein the regeneration buffer comprises about 0.1 N NaOH and has a pH of about 13.

[0008] In some aspects, the present invention provides a method of purifying a chromatography material for reuse, the method comprising the steps of: a) passing about 2 material volumes of an elution buffer through the material, the elution buffer comprising about 0.15 M acetic acid and having a pH of about 2.9; b) statically holding the material in the elution buffer for about 30 minutes; c) passing about 2 material volumes of the elution buffer through the material; and d) passing about 4 material volumes of a regeneration buffer through the material, the regeneration buffer comprising about 0.1 N NaOH and having a pH of about 13.

[0009] In some aspects, the present invention provides a method of purifying a chromatography material for reuse, the method comprising the steps of: a) passing about 2 material volumes of an elution buffer through the material, the elution buffer comprising about 0.15 M acetic acid and having a pH of about 2.9; b) statically holding the material in the elution buffer for about 30 minutes; c) passing about 2 material volumes of the elution buffer through the material; and d) passing about 2.5 material volumes of a regeneration buffer through the material, the regeneration buffer comprising about 0.1 N NaOH and having a pH of about 13; e) statically holding the material in the regeneration buffer for about 30 minutes; and f) passing about 2.5 material volumes of the regeneration buffer through the material.

[0010] In some aspects, the present invention provides a method for purifying a chromatographic material for reuse, the method comprising the steps of: a) passing about 2 material volumes of an equilibration buffer through the material, the equilibration buffer comprising about 25 mM Tris and about 25 mM NaCl, and having a pH of about 7.1; b) statically holding the material in the equilibration buffer for about 30 minutes; c) passing about 2 material volumes of the equilibration buffer through the material; d) passing about 2 material volumes of an elution buffer through the material, wherein the elution buffer is eluted. e) statically holding the material in the elution buffer for about 30 minutes; f) passing about 2 material volumes of the elution buffer through the material; g) passing about 2 material volumes of the regeneration buffer through the material, the regeneration buffer comprising 0.1 N NaOH and having a pH of 13; h) statically holding the material in the regeneration buffer for about 30 minutes; i) passing about 2 material volumes of the regeneration buffer through the material.

[0011] In some aspects, the present invention provides a method for purifying a chromatography material for reuse, comprising: a) passing about 4 material volumes of an equilibration buffer through the material, wherein the re-equilibration buffer comprises about 25 mM Tris and about 25 mM NaCl and has a pH of 7.1; b) performing six cycles of the following steps: i) passing about 3 material volumes of an elution buffer through the material, wherein the elution buffer comprises about 0.15 M acetic acid and has a pH of about 2.8; ii) statically holding the material in the elution buffer for about 10 minutes; iii) passing about 1 material volume of the elution buffer through the material; iv) passing about 3 material volumes of a regeneration buffer through the material, wherein the regeneration buffer comprises about 0.1 N NaOH and has a pH of about 13; v) statically holding the material in the regeneration buffer for about 10 minutes; and vi) passing about 1 material volume of the regeneration buffer through the material.

[0012] In some aspects, the present invention provides a method of purifying a chromatographic material for reuse, the method comprising the steps of: a) passing about 3 material volumes of an elution buffer through the material, the elution buffer comprising about 0.15 M acetic acid and having a pH of about 2.8; b) statically holding the material in the elution buffer for about 15 minutes; c) passing about 1 material volume of the elution buffer through the material; d) passing about 3 material volumes of a regeneration buffer through the material, the regeneration buffer comprising about 0.1 N NaOH. and has a pH of about 13; e) statically holding the material in the refolding buffer for about 15 minutes; f) passing about 1 material volume of the refolding buffer through the material; g) passing about 3 material volumes of a storage buffer through the material, the storage buffer comprising about 100 mM sodium acetate, about 2% benzyl alcohol, and having a pH of about 5.0; e) statically holding the material in the storage buffer for about 15 minutes; f) passing about 1 material volume of the storage buffer through the material.

[0013] In some embodiments of the above aspects, the chromatographic material is present in a chromatographic column. In some embodiments, the chromatographic material is an affinity material. In further embodiments, the affinity material is a Protein A affinity material, such as, but not limited to, a MAbSelect material, a MAbSelect SuRe material, or a MAbSelect SuRe LX material. In some embodiments of the above aspects, the chromatographic material is used for large-scale production of polypeptides.

[0014] In some aspects, the invention provides a method of purifying a chromatography material for reuse, the method comprising the steps of: a) passing about 3 material volumes of an equilibration buffer through the material, the equilibration buffer comprising about 40 mM sodium acetate and about pH 5.5; b) passing about 2 material volumes of about 0.5 N NaOH through the material; c) statically holding the material in about 0.5 N NaOH for about 10 minutes; d) passing about 1 material volume of about 0.5 N NaOH through the material; and e) statically holding the material in about 0.5 N NaOH for about 10 minutes; f) passing about 1 material volume of about 0.5 N NaOH through the material.

[0015] In some embodiments of the above aspects, the chromatographic material is present in a chromatographic column. In some embodiments, the chromatographic material is an ion exchange material. In some embodiments, the ion exchange material is a cation exchange material, e.g., POROS HS50 material. In some embodiments, the chromatographic material is used for large-scale production of antibodies.

[0016] In some aspects, the invention provides a method of purifying a chromatography material for reuse, the method comprising the steps of: a) passing about 3 material volumes of an equilibration buffer through the material, the equilibration buffer comprising about 50 mM Tris, 85 mM sodium acetate, and having a pH of about 8.8 and about 8.6 mS / cm; b) passing about 2 material volumes of about 0.5 N NaOH through the material; c) statically holding the material in about 0.5 N NaOH for about 10 minutes; d) passing about 1 material volume of about 0.5 N NaOH through the material; and e) statically holding the material in about 0.5 N NaOH for about 10 minutes; f) passing about 1 material volume of about 0.5 N NaOH through the material.

[0017] In some embodiments of the above aspects, the chromatographic material is present in a chromatographic column. In some embodiments, the chromatographic material is an ion exchange material. In some embodiments, the ion exchange material is an anion exchange material, e.g., a QSFF material. In some embodiments, the chromatographic material is used for large-scale production of antibodies.

[0018] In some embodiments of any of the above aspects, the buffers are passed through the material at about 30 material volumes / hour, about 20 material volumes / hour, or about 15 material volumes / hour. In some embodiments, the buffers are passed through the material in a downflow or upflow direction. In some embodiments, purification of the chromatographic material is measured by performing a mock elution after purifying the chromatographic material. In some embodiments, the eluent of the mock elution contains one or more of <0.25 mg / mL total protein, <1 ppm IgG fragments, <1 ppm leached Protein A, <1 μg / mL CZE LIF, <1 ppm CHOP, and <1 pg / mL CHO DNA, which are indicators of effective purification of the material for multi-product use. In some embodiments, the chromatographic material is stable in alkali.

[0019] In some embodiments of any of the above aspects, the chromatography material is used to purify a polypeptide. In some embodiments, the chromatography material is purified after purification of a first polypeptide, and after purification, the chromatography material is used to purify a second polypeptide. In some embodiments, the polypeptide is an antibody or immunoadhesin. In some embodiments, the antibody is a monoclonal antibody. In further embodiments, the monoclonal antibody is a chimeric, humanized, or human antibody. In further embodiments, the monoclonal antibody is an IgG monoclonal antibody. In some embodiments, the antibody is an antigen-binding fragment. In some embodiments, the antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an scFv, a di-scFv, a bi-scFv, a tandem (di, tri)-scFv, an Fv, an sdAb, a trifunctional antibody, a BiTE, a diabody, or a triabody. In other embodiments, the polypeptide is an enzyme, hormone, fusion protein, Fc-containing protein, immunoconjugate, cytokine, or interleukin. In some embodiments, the first polypeptide is a first antibody or a first immunoadhesin and the second polypeptide is a second antibody or a second immunoadhesin. [Brief explanation of the drawings]

[0020] [Figure 1] Figure 1 shows a plot of total protein carryover (intact IgG + Fc fragments) as a function of elution sample cycle from sequential lab-scale purification of mAbA, mAbB, and mAbC on a MabSelect™ SuRe column without further resin cleanup. Legend: mAbA carryover in mAbB elution (black and gray), mAbB carryover in mAbC elution (gray), and mAbA carryover in mAbC elution (black). [Figure 2]Figure 1 shows protein removal seen in the initial purification cycle as a function of elution buffer (0.15 M acetic acid) or regeneration buffer (0.1 N NaOH) CV wash. Arrows point to the 30 minute static hold. Note that there is a 5-fold increase in the amount of protein washed off the column after the 30 minute static hold, and that no protein was detected after the static hold with regeneration buffer. Legend: Method 4 (Table 2): black; Method 5 (Table 2): black and gray. [Figure 3] Figure 1 shows intact IgG protein detected after a "mock run" performed on a MabSelect™ SuRe column after cleanup using Method 6 (black bars, Table 2) and Method 7 (none detected, Table 2). Three "mock runs" were performed using Method 7 (Table 2) and the results were reproducible. [Figure 4] FIG. 1 shows that capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) analysis of the "mock elution" after cleanup of a MabSelect™ SuRe column following the Method 7 cleanup procedure (Table 2) for a 94 ng / mL mock elution sample revealed that the mAb was >90% fully intact. [Figure 5] Figure 1 shows an Akta chromatogram using the Method 7 cleanup procedure (Table 2). The "mock elution" chromatogram suggests efficient cleanup of the column, as evidenced by the spikes in UV intensity when the shift from elution buffer (0.15 M acetic acid) to regeneration buffer (0.1 N NaOH) is performed for each of six cycles. Blue line = UV 280 nm, red line = pH, magenta line = conductivity. [Figure 6]Figure 1 shows the results for lab-scale purification of mAbA using the optimized purification procedure (entry 7, Table 2) before a "mock run" to determine protein carryover. A. Protein carryover (ng / mg protein) as a function of elution buffer cycle wash; B. Protein carryover (ng / mg protein) as a function of regeneration buffer cycle wash; C. Protein carryover (ng / mg protein) as a function of step during the "mock run." Intact IgG is shown in black, Fc fragments are shown in gray. [Figure 7] Figure 1 shows a plot of total protein carryover as a function of purification cycles using storage buffer (100 mM sodium acetate and 2% benzyl alcohol, pH 5) according to the conditions outlined in Method 8, Table 2. Legend: Elution cycles (black and gray), regeneration cycles (gray), storage buffer cycles (black). [Figure 8] Figure 1 shows a plot of protein carryover as a function of sample from a 3.23 L pilot-scale purification of mAbZ on a MabSelect™ SuRe column followed by column washes with the optimized six-cycle cleanup procedure (entry 7, Table 2). Intact IgG is shown in black and Fc fragments are shown in gray. [Figure 9] Figure 1 shows a schematic overview of the optimized purification protocol using a 15 minute static hold time (A) and a "mock run" (B). The equilibration buffer is 25 mM Tris, 25 mM NaCl (pH 7.1). The elution buffer is 0.15 M sodium acetate (pH 2.9). The renaturation buffer is 0.1 N NaOH (pH 13). [Figure 10]Figure 9 shows the results for the lab-scale purification of mAbC using the cleanup procedure (Figure 9) before a "mock run" to determine protein carryover. A. Protein carryover (ng / mg protein) as a function of elution buffer cycle wash; B. Protein carryover (ng / mg protein) as a function of regeneration buffer cycle wash; C. Protein carryover (ng / mg protein) as a function of step during the "mock run". [Figure 11] Figure 9 shows 10% Tris-HCl gels taken at different stages of the optimized cleanup protocol for the MabSelect™ SuRe column (Figure 9). Samples were taken after purification of mAbC. The renatured samples were concentrated (25x); lanes 2, 4, 6, 8, 10, and 12 contain samples after a 15-minute static hold. [Figure 12] Figure 1 shows the results for a pilot-scale column (3 L) purification of mAbC using the optimized cleanup procedure (entry 7, Table 2) prior to a "mock run" to determine protein carryover. A. Protein carryover (ng / mg protein) as a function of elution buffer cycle washes; B. Protein carryover (ng / mg protein) as a function of regeneration buffer cycle washes. Intact IgG is shown in black, Fc fragments are shown in gray. [Figure 13] Figure 1 shows the results of intact IgG carryover detected after mock elution of a cation exchange column (POROS) and an anion exchange column (QSFF), where MAbA or MAbB had previously been loaded and eluted from the column. [Figure 14]Figure 14A shows the results of intact IgG carryover detected after mock elution of a cation exchange column (POROS) and an anion exchange column (QSFF) before and after a clean-in-place procedure. MAbA has been previously loaded and eluted from the column. Figure 14B shows the results of intact IgG carryover detected after mock elution of a cation exchange column (POROS) and an anion exchange column (QSFF) before and after a clean-in-place procedure. MAbB has been previously loaded and eluted from the column. [Figure 15] FIG. 1 shows the amount of intact IgG (MAbC) eluted from a POROS column or a QSFF column at the end of selected steps of the purification protocol. [Figure 16] FIG. 1 shows MAbD carryover at different steps of the pilot-scale column cleanup protocol. [Figure 17] FIG. 1 shows a plot of intact human IgG carryover as a function of different washing conditions. [Figure 18] Figure 1 shows a plot of protein carryover as a function of CV washes (resin cleanup attempts) with different buffer solutions on a ProSep® vA column. Legend: 6M guanidine HCl: magenta; 19% ethanol: red; 2M arginine HCl: brown; 20% hexene glycol: gray; 8M urea / 1M NaCl: orange; equilibration buffer: blue; 1% v / v phosphoric acid: yellow; 0.1M imidazole / 19% ethanol: black; 0.1M acetic acid: green; 2M potassium phosphate: turquoise. [Figure 19] Chromatogram showing five sequential mock runs of a 0.1 M acetic acid cleanup solution. [Figure 20] 1 is a chart showing carryover after sequential mock runs with various sanitizing solutions. [Figure 21] 1 is a chart showing carryover after sequential mock runs using sanitizing solution, 6 M guanidine hydrochloride, 2 M arginine hydrochloride, or 20% hexylene glycol. [Figure 22] Chromatogram of product elution without column cleanup (top panel). Carryover in each fraction is shown in the bottom panel. [Figure 23] FIG. 10 shows product elution throughout pulse purification. [Figure 24] 10 is a graph showing carryover across sequential mock runs with and without pulse purging. [Figure 25] 10 is a graph showing carryover across sequential mock runs using either downflow or upflow conditions. [Figure 26] Figure 1 shows chromatograms of product elution throughout pulse purification: black line indicates downflow conditions, grey line indicates upflow conditions, and light grey indicates pH. [Figure 27] FIG. 1 shows carryover across sequential mock runs at flow rates of 30 CV / hr and 15 CV / hr. [Figure 28] 1 shows carryover across sequential mock runs using a single static hold with equilibration buffer or regeneration buffer. Normal pulse indicates sample without static hold. [Figure 29] 1 shows carryover across sequential mock runs with multiple static holds with equilibration buffer. Normal pulse represents sample without static hold. [Figure 30] Figure 1 shows chromatograms of product elution throughout pulse purification: black line indicates 30 CV / hr conditions, grey line indicates 15 CV / hr conditions, light grey indicates pH. [Figure 31] Figure 1 shows chromatograms of product elution over a pulse purification using static hold: the black line indicates the normal pulse, the medium grey line indicates the equilibration buffer hold conditions, the dark grey line indicates the regeneration buffer hold conditions, and the light grey line indicates the pH. [Figure 32]Figure 1 shows chromatograms of product elution over a pulse purification using single or multiple static holds. The black line represents the normal pulse, the medium grey line represents 1x equilibration buffer hold conditions, the dark grey line represents 4x equilibration buffer hold conditions, and the light grey line represents pH. [Figure 33] FIG. 1 shows the effect of reduced cycle duration on carryover under pulsed purification conditions. [Figure 34] Figure 1 shows chromatograms of product elution over a pulse purification using reduced cycle duration: the black line represents IgG and the light grey line represents pH. DETAILED DESCRIPTION OF THE INVENTION

[0021] Provided herein are methods for purifying or regenerating chromatographic materials, such as chromatography resins, for reuse. Chromatographic reuse is a switchover procedure in which chromatographic materials are purified and / or regenerated for use with the same or different products. The methods of the present invention can be used for large-scale, e.g., manufacturing-scale, regeneration of chromatographic materials. Significant cost savings can be achieved when resins, e.g., Protein A resins, are reused for multiple products. In some embodiments, the purification procedure results in less than 1 ppm carryover of intact protein, e.g., IgG, into subsequent purified samples. In some embodiments, this low protein carryover is within a safety margin of 10 ppm of that set. 3 The resulting chromatographic material is present in a chromatography column, demonstrating that the same resin can be used to purify multiple products.

[0022] I. Definition The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also encompass amino acid polymers that are naturally modified or modified by intervention, e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art, are also included within this definition. The terms "polypeptide" and "protein," as used herein, specifically include antibodies.

[0023] A "purified" polypeptide (e.g., an antibody or immunoadhesin) means that the polypeptide has been increased in purity such that it exists in a purer form than when the polypeptide exists in its natural environment and / or when originally synthesized and / or amplified under laboratory conditions. Purity is a relative term and does not necessarily imply absolute purity.

[0024] A polypeptide that "binds" an antigen of interest, e.g., a tumor-associated polypeptide antigen target, is one that binds to the antigen with sufficient affinity so that it is useful as a diagnostic and / or therapeutic agent in targeting cells or tissues that express the antigen and does not significantly cross-react with other polypeptides. In such embodiments, the extent of binding of the polypeptide to a "non-target" polypeptide is less than about 10% of the binding of the polypeptide to its particular target polypeptide, as determined by fluorescence-activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA).

[0025] With respect to binding of a polypeptide to a target molecule, the term "specific binding," or "specifically binding to" or "specific for" a particular polypeptide or epitope on a particular polypeptide target, refers to binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which is generally a molecule of similar structure that has no binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target, e.g., an excess of unlabeled target. In this case, specific binding is indicated when binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target.

[0026] The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term "immunoglobulin" (Ig) is used interchangeably with antibody herein.

[0027] Antibodies are naturally occurring immunoglobulin molecules with variable structures all based on the immunoglobulin fold. For example, IgG antibodies have two "heavy" chains and two "light" chains disulfide-bonded to form a functional antibody. Each heavy and light chain itself contains a "constant" (C) region and a "variable" (V) region. The V region determines the antigen-binding specificity of the antibody, while the C region provides structural support and functions in non-antigen-specific interactions with immune effectors. The antigen-binding specificity of an antibody or an antigen-binding fragment of an antibody is the ability of the antibody to specifically bind to a particular antigen.

[0028] The antigen-binding specificity of antibodies is determined by structural features of the V regions. Variability is not evenly distributed across the 110-amino acid span of the variable domains. Instead, V regions consist of relatively invariant stretches of 15–30 amino acids called framework regions (FRs) separated by shorter regions of extreme variability called "hypervariable regions," each 9–12 amino acids long. Native heavy and light chain variable domains each contain four FRs, largely adopting a β-sheet configuration, connected by three hypervariable regions ("HVRs") that form loops connecting, and in some cases, part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by these FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (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 involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0029] Each V region typically contains three hypervariable regions, e.g., complementarity-determining regions ("CDRs"), each containing a "hypervariable loop," and four framework regions. Thus, an antibody combining site, the minimum structural unit required for binding to a particular desired antigen with substantial affinity, typically contains three CDRs and at least three, and preferably four, framework regions interspersed therebetween to hold and present the CDRs in the appropriate conformation. Classical four-chain antibodies contain a V H Domain and V L Certain antibodies, such as camel and shark antibodies, lack light chains and rely on binding sites formed exclusively by heavy chains. H and V LSingle domain engineered immunoglobulins can be prepared in which the binding site is formed by the heavy or light chain alone, in the absence of cooperation between the heavy and light chains.

[0030] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. In both the light-chain and heavy-chain variable domains, variability is concentrated in three segments called hypervariable regions. The more highly conserved portions of the variable domains are called framework regions (FRs). Native heavy and light chain variable domains each contain four FRs, mostly in a β-sheet configuration, connected by three hypervariable regions that form loops connecting, and in some cases, part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by these FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (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 involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0031] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. This hypervariable region is made up of amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., V L Approximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in Hamino acid residues from approximately 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the VH1 sequence (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or the "hypervariable loops" (e.g., V L Residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in V H Among these, the sequences may include 26-32 (H1), 52A-55 (H2), and 96-101 (H3) (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0032] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0033] "Antibody fragments" include portions of intact antibodies, preferably including their antigen-binding regions. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; tandem diabodies (taDbs); linear antibodies (e.g., U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); one-armed antibodies, single variable domain antibodies, minibodies, single-chain antibody molecules; multispecific antibodies formed from antibody fragments (e.g., Db-Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, di-scFv, bi-scFv, or tandem (di, tri)-scFv); and bispecific T cell engagers (BiTEs).

[0034] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0035] "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In this configuration, the three hypervariable regions of each variable domain interact to form the V H -V L The six hypervariable regions define an antigen-binding site on the surface of the dimer. Collectively, these six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0036] Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear at least one free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0037] The "light chains" of antibodies (immunoglobulins) from any vertebrate species are assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0038] Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., 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 different classes of immunoglobulins are well known.

[0039] "Single-chain Fv" or "scFv" antibody fragments are fragments of the V H Domain and V L In some embodiments, the Fv polypeptide comprises a V domain, which enables the scFv to form the desired structure for antigen binding. H Domains and V L For a review of scFvs, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York, pp. 269-315 (1994).

[0040] The term "diabody" refers to small antibody fragments with two antigen-binding sites, which contain a light chain variable domain (V L ) connected to the heavy chain variable domain (V H )(V H -V L(Diabodies generally comprise a diabody, typically a diabody with a complementary domain on another chain.) By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains on another chain to create two antigen-binding sites. Diabodies are more fully described in, for example, EP 404097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

[0041] The term "multispecific antibody" is used in the broadest sense and specifically covers antibodies with polyepitopic specificity. Such multispecific antibodies include V H V L The heavy chain variable domain (V H ) and the light chain variable domain (V L ) antibodies containing each V H V L Two or more V units bind to different epitopes L Domain and V H Examples of multispecific antibodies include, but are not limited to, antibodies having domains, antibodies having two or more single variable domains, where each single variable domain binds to a different epitope, full-length antibodies, antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies, triabodies, trifunctional antibodies, and covalently or non-covalently linked antibody fragments. "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different target(s). "Monospecificity" refers to the ability to bind to only one epitope. In one embodiment, the multispecific antibody is an IgG antibody that binds to each epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0.1 μM to 0.001 pM.

[0042] The expression "single domain antibody" (sdAb) or "single variable domain (SVD) antibody" generally refers to an antibody in which a single variable domain (VH or VL) is capable of conferring antigen binding. In other words, this single variable domain does not need to interact with another variable domain to recognize the target antigen. Examples of single domain antibodies include those derived from Camelidae (llamas and camels) and cartilaginous fish (e.g., nurse sharks), as well as those derived by recombinant methods from human and murine antibodies (Nature (1989) 341:544-546; Dev Comp Immunol (2006) 30:43-56; Trend Biochem Sci (2001) 26:230-235; Trends Biotechnol (2003):21:484-490; WO 2005 / 035572; WO 03 / 035694; Febs Lett (1994) 339:285-290; WO 00 / 29004; WO 02 / 051870).

[0043] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variations that may arise during the production of the monoclonal antibody, and such variations are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against 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 are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the methods provided herein may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in, e.g., Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0044] The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, so long as they exhibit the desired biological activity, as well as fragments of such antibodies (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies for purposes herein also include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., an Old World monkey, e.g., a baboon, a rhesus monkey, or a cynomolgus monkey) and human constant region sequences (U.S. Patent No. 5,693,780).

[0045] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in either the recipient or donor antibody. These modifications are made to further refine antibody performance. In general, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence, except for the FR substitution(s) described above. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0046] For purposes herein, an "intact antibody" is an antibody comprising heavy and light chain variable domains and an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Preferably, an intact antibody has one or more effector functions.

[0047] "Native antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, although the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain contains at one end a variable domain (V H ) followed by several constant domains. Each light chain has at one end a variable domain (V L ) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain.

[0048] A "naked antibody" is an antibody (as defined herein) that is not conjugated to a heterologous molecule, such as a cytotoxic moiety or radiolabel.

[0049] In some embodiments, antibody "effector functions" refer to the biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down-regulation of cell surface receptors.

[0050] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcR), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibody on target cells, subsequently causing lysis of the target cell. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on p464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998).

[0051] "Human effector cells" are leukocytes that express one or more FcRs and perform effector function. In some embodiments, these cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with PBMCs and NK cells being preferred.

[0052] The terms "Fc receptor" or "FcR" are used to describe a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native-sequence human FcR. Furthermore, preferred FcRs are those that bind IgG antibodies (gamma receptors), which include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. This term also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

[0053] The term "sequential," as used herein with respect to chromatography, refers to having a first chromatography followed by a second chromatography. Additional steps may be included between the first and second chromatography.

[0054] The term "continuous," as used herein with respect to chromatography, refers to having a first chromatographic material and a second chromatographic material connected directly or via some other mechanism that allows for continuous flow between the two chromatographic materials.

[0055] The term "isolated," as used herein, refers to a molecule that is separated from at least some of the components with which it is typically found or produced in nature. For example, a polypeptide is said to be "isolated" if it is separated from at least some of the components of the cell in which it is produced. If a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to "isolate" the polypeptide. Similarly, a polynucleotide is typically said to be "isolated" if it is not part of a larger polynucleotide in which it is found in nature (e.g., in the case of a DNA polynucleotide, such as genomic DNA or mitochondrial DNA), or, in the case of an RNA polynucleotide, if it is separated from at least some of the components of the cell in which it is produced. Thus, a DNA polynucleotide contained in a vector inside a host cell may also be said to be "isolated."

[0056] "Acceptable Daily Exposure (ADE)," as used herein, is a substance-specific dose that is unlikely to cause adverse health events or undesirable physiological effects when an individual is exposed to this dose or lower doses over their lifetime (Teschner, W. et al., Vox Sang. 2007, 92:42-55; Food and Drug Administration, HHS. Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers. Rockville, MD. July 2005, on the World Wide Web, accessed August 7, 2012, google.com / url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CE8QFjAA&url=http%3A%2F%2Fwww.fda.gov%2Fdownloads%2FDrugs%2F...%2FGuidances%2FUCM078932.pdf&ei=f4QhUJv4K9Ov6gGQ-4DgAg&usg=AFQjCNFbTE75U0nDbFpfdpxK85uWXT8frg;European Medicines Agency. Impurities: Residual Solvents, Note for Guidance on Impurities: Residual Solvents (CPMP / ICH / 283 / 95). London, UK, September 1997, on the World Wide Web at ema.europa.eu / ema / index.jsp?curl=pages / regulation / general / general_content_000431.jsp&mid=WC0b01ac0580029593, accessed August 7, 2012.) In addition to the ADE, an "Estimated Daily Intake (EDI)" for IgG is determined based on the amount of IgG administered per dose.

[0057] "Contaminants" refer to materials that differ from the desired polypeptide product. Contaminants include, but are not limited to, host cell materials such as CHOP; leached Protein A; nucleic acids; mutants, fragments, aggregates, or derivatives of the desired polypeptide; other polypeptides; endotoxins; viral contaminants; cell culture media components, etc. In some instances, the contaminants may be host cell proteins (HCPs) from cells such as, but not limited to, bacterial cells, e.g., E. coli cells, insect cells, prokaryotic cells, eukaryotic cells, yeast cells, mammalian cells, avian cells, fungal cells, etc.

[0058] Reference to "about" a value or parameter described herein includes (and describes) variations on that value or parameter itself. For example, a statement referring to "about X" includes a statement of "X."

[0059] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is understood that aspects and variations of the invention described herein include "consisting of" and / or "consisting essentially of" aspects and variations.

[0060] II. Column Purification Method (A) Chromatography The present invention provides methods for purifying or regenerating chromatographic materials for reuse. In some embodiments, these chromatographic materials are used for large-scale, e.g., manufacturing-scale, production of polypeptide products.

[0061] In some embodiments, the method comprises the steps of: a) passing at least about 2 material volumes of an elution buffer through the material, wherein the elution buffer comprises about 0.15 M acetic acid at about pH 2.9; b) statically holding the material in the elution buffer for a time ranging from about 10 minutes to about 30 minutes; c) passing at least about 2 material volumes of the elution buffer through the material; and d) passing at least about 2 material volumes of a regeneration buffer through the material, wherein the regeneration buffer is about 0.1 N NaOH, pH 13. In some embodiments, the chromatography material is in a chromatography column. In some embodiments, the chromatography column is used for large-scale, e.g., manufacturing-scale, production of a polypeptide product, such as an antibody product. In some embodiments, the chromatography material is a Protein A chromatography material. In some embodiments, the chromatography material is used to purify multiple antibody products. In some embodiments, carryover after the purification method includes one or more of <0.25 mg / mL total protein, <1 ppm IgG fragments, <1 ppm leached Protein A, <1 μg / mL CZE LIF, <1 ppm CHOP, and <1 pg / mL CHO DNA.

[0062] In some embodiments, the method comprises the steps of: a) passing about 2 material volumes of elution buffer through the material, wherein the elution buffer comprises about 0.15 M acetic acid at about pH 2.9; b) statically holding the material in the elution buffer for about 30 minutes; c) passing about 2 material volumes of elution buffer through the material; and d) passing about 4 material volumes of renaturation buffer through the material, wherein the renaturation buffer is about 0.1 N NaOH at about pH 13. In some embodiments, the chromatographic material is in a chromatographic column. In some embodiments, the chromatographic column is used for large-scale, e.g., manufacturing-scale, production of a polypeptide product, such as an antibody product. In some embodiments, the chromatographic material is a Protein A chromatographic material. In some embodiments, the chromatographic material is used to purify multiple antibody products. In some embodiments, carryover after the purification method includes one or more of <0.25 mg / mL total protein, <1 ppm IgG fragments, <1 ppm leached Protein A, <1 μg / mL CZE LIF, <1 ppm CHOP, and <1 pg / mL CHO DNA.

[0063] In some embodiments, the method comprises the steps of: a) passing about 2 material volumes of elution buffer through the material, wherein the elution buffer comprises about 0.15 M acetic acid at about pH 2.9; b) statically holding the material in the elution buffer for about 30 minutes; c) passing about 2 material volumes of elution buffer through the material; d) passing about 2.5 material volumes of renaturation buffer through the material, wherein the renaturation buffer is about 0.1 N NaOH at about pH 13; e) statically holding the material in the renaturation buffer for about 30 minutes; and f) passing about 2.5 material volumes of renaturation buffer through the material. In some embodiments, the chromatography material is in a chromatography column. In some embodiments, the chromatography column is used for large-scale, e.g., manufacturing-scale, production of a polypeptide product, such as an antibody product. In some embodiments, the chromatography material is a Protein A chromatography material. In some embodiments, the chromatography material is used to purify multiple antibody products. In some embodiments, carryover after the purification method includes one or more of <0.25 mg / mL total protein, <1 ppm IgG fragments, <1 ppm leached Protein A, <1 μg / mL CZE LIF, <1 ppm CHOP, and <1 pg / mL CHO DNA.

[0064] In some embodiments, the method comprises the steps of: a) passing about 2 material volumes of an equilibration buffer through the material, the equilibration buffer comprising about 25 mM Tris, about 25 mM NaCl, at about pH 7.1; b) statically holding the material in the equilibration buffer for about 30 minutes; c) passing about 2 material volumes of the equilibration buffer through the material; and d) passing about 2 material volumes of an elution buffer through the material, the elution buffer comprising about pH 2.8. e) statically holding the material in the elution buffer for about 30 minutes; f) passing about 2 material volumes of the elution buffer through the material; g) passing about 2 material volumes of the renaturation buffer through the material, the renaturation buffer being about 0.1 N NaOH at pH 13; h) statically holding the material in the renaturation buffer for about 30 minutes; i) passing about 2 material volumes of the renaturation buffer through the material. In some embodiments, the chromatography material is in a chromatography column. In some embodiments, the chromatography column is used for large-scale, e.g., manufacturing-scale, production of a polypeptide product, such as an antibody product. In some embodiments, the chromatography material is Protein A chromatography material. In some embodiments, the chromatography material is used to purify multiple antibody products. In some embodiments, carryover after the purification method includes one or more of <0.25 mg / mL total protein, <1 ppm IgG fragments, <1 ppm leached Protein A, <1 μg / mL CZE LIF, <1 ppm CHOP, and <1 pg / mL CHO DNA.

[0065] In some embodiments, the method includes: a) passing about 4 material volumes of an equilibration buffer through the material, the equilibration buffer comprising about 25 mM Tris, about 25 mM NaCl, at about pH 7.1; b) performing six cycles of the following steps: i) passing about 3 material volumes of an elution buffer through the material, where the elution buffer is about 0.15 M acetic acid, pH 2.8; ii) statically holding the material in the elution buffer for about 10 minutes; iii) passing about 1 material volume of the elution buffer through the material; iv) passing about 3 material volumes of a regeneration buffer through the material, where the regeneration buffer is about 0.1 N NaOH at about pH 13; v) statically holding the material in the regeneration buffer for about 10 minutes; and vi) passing about 1 material volume of the regeneration buffer through the material. In some embodiments, the chromatography material is in a chromatography column. In some embodiments, the chromatography column is used for large-scale, e.g., manufacturing-scale, production of a polypeptide product, such as an antibody product. In some embodiments, the chromatography material is a Protein A chromatography material. In some embodiments, the chromatography material is used to purify multiple antibody products. In some embodiments, carryover after the purification method comprises one or more of <0.25 mg / mL total protein, <1 ppm IgG fragments, <1 ppm leached Protein A, <1 μg / mL CZE LIF, <1 ppm CHOP, and <1 pg / mL CHO DNA.

[0066] In some embodiments, the method comprises six cycles of: a) passing about 3 material volumes of elution buffer through the material, wherein the elution buffer is about 0.15 M acetic acid at about pH 2.8; b) statically holding the material in elution buffer for about 15 minutes; c) passing about 1 material volume of elution buffer through the material; and d) passing about 3 material volumes of regeneration buffer through the material, wherein the regeneration buffer is about 0.1 N NaOH at about pH 13. e) statically holding the material in a renaturing buffer for about 15 minutes; f) passing about 1 material volume of renaturing buffer through the material; and g) passing about 3 material volumes of a storage buffer through the material, the storage buffer being about 100 mM sodium acetate, about 2% benzyl alcohol at about pH 5.0. In some embodiments, the chromatographic material is in a chromatographic column. In some embodiments, the chromatographic column is used for large-scale, e.g., manufacturing-scale, production of a polypeptide product, such as an antibody product. In some embodiments, the chromatographic material is a Protein A chromatographic material. In some embodiments, the chromatographic material is used to purify multiple antibody products. In some embodiments, carryover after the purification method includes one or more of <0.25 mg / mL total protein, <1 ppm IgG fragments, <1 ppm leached Protein A, <1 μg / mL CZE LIF, <1 ppm CHOP, and <1 pg / mL CHO DNA.

[0067] In some aspects of the invention, the chromatography material is an affinity chromatography material. Examples of affinity chromatography materials include, but are not limited to, chromatography materials derivatized with Protein A or Protein G. Examples of affinity chromatography materials include, but are not limited to, Prosep-VA, Prosep-VA Ultra Plus, Protein A sepharose fast flow, Tyopearl Protein A, MAbSelect™, MAbSelect™ SuRe, and MAbSelect™ SuRe LX. In some embodiments of the above, the affinity chromatography material is an affinity chromatography material. In some embodiments of the above, the affinity chromatography material is an affinity chromatography membrane. In some embodiments, the affinity chromatography material is a Protein G chromatography material. In some embodiments, the chromatography column is used for large-scale, e.g., manufacturing-scale, production of a polypeptide product, such as an antibody product.

[0068] In some aspects, the present invention provides a method for purifying an ion exchange chromatography material for reuse. In some embodiments, the method comprises the steps of: a) passing about 3 material volumes of an equilibration buffer through the material, the equilibration buffer comprising about 25 mM Tris, about 25 mM NaCl, at about pH 7.1; b) passing about 2 material volumes of about 0.5 N NaOH through the material; c) statically holding the material in about 0.5 N NaOH for about 10 minutes; d) passing about 1 material volume of about 0.5 N NaOH through the material; e) statically holding the material in about 0.5 N NaOH for about 10 minutes; and f) passing about 1 material volume of about 0.5 N NaOH through the material. In some embodiments, the ion exchange material is present in a chromatography column. In some embodiments, the chromatography column is used for large-scale, e.g., manufacturing-scale, production of a polypeptide product, such as an antibody product. In some embodiments, the chromatography material is used to purify multiple antibody products. In some embodiments, carryover after the purification method comprises one or more of <0.25 mg / mL total protein, <1 ppm IgG fragments, <1 ppm leached Protein A, <1 μg / mL CZE LIF, <1 ppm CHOP, and <1 pg / mL CHO DNA.

[0069] In some embodiments of any of the methods described herein, the chromatography material is an ion exchange chromatography material, such as an anion exchange chromatography material or a cation exchange chromatography material. In some embodiments of any of the methods described herein, the chromatography material is an anion exchange material. In some embodiments, the anion exchange material is present in a chromatography column. In some embodiments, the anion exchange chromatography material is a positively charged solid phase having free anions for exchange with anions in an aqueous solution passed over or through the solid phase. In some embodiments of any of the methods described herein, the anion exchange material may be a membrane, monolith, or resin. In one embodiment, the anion exchange material may be a resin. In some embodiments, the anion exchange material may comprise primary amine, secondary amine, tertiary amine, or quaternary ammonium ion functional groups, polyamine functional groups, or diethylaminoethyl functional groups. In some embodiments of the above, the anion exchange chromatography material is an anion exchange chromatography material. In some embodiments of the above, the anion exchange chromatography material is an anion exchange chromatography membrane. In some embodiments, the anion exchange chromatography material is used for large-scale, eg, manufacturing-scale, production of a polypeptide product, such as an antibody product.

[0070] In some embodiments of any of the methods described herein, the chromatographic material is a cation exchange material. In some embodiments, the cation exchange material is present in a chromatographic column. In some embodiments, the cation exchange material is a negatively charged solid phase having free cations for exchange with cations in an aqueous solution passed across or through the solid phase. In some embodiments of any of the methods described herein, the cation exchange material may be a membrane, monolith, or resin. In some embodiments, the cation exchange material may be a resin. The cation exchange material may include carboxylic acid or sulfonic acid functional groups, for example, but not limited to, sulfonate, carboxylic acid, carboxymethylsulfonic acid, sulfoisobutyl, sulfoethyl, carboxyl, sulfopropyl, sulfonyl, sulfoxyethyl, or orthophosphate. In some embodiments of the above, the cation exchange chromatographic material is a cation exchange chromatographic material. In some embodiments of the above, the cation exchange chromatographic material is a cation exchange chromatographic membrane. In some embodiments of the present invention, the chromatographic material is not a cation exchange chromatographic material. In some embodiments, the cation exchange chromatography material is used for large-scale, eg, manufacturing-scale, production of a polypeptide product, such as an antibody product.

[0071] In some embodiments of any of the methods described herein, the ion exchange material may utilize a conventional chromatography material or a convection chromatography material. These conventional chromatography materials include, for example, perfusable materials (e.g., poly(styrene-divinylbenzene) resin) and diffusive materials (e.g., cross-linked agarose resin). In some embodiments, the poly(styrene-divinylbenzene) resin may be a Poros resin. In some embodiments, the cross-linked agarose resin may be a sulfopropyl-Sepharose Fast Flow ("SPSFF") resin. The convection chromatography material may be a membrane (e.g., polyethersulfone) or a monolith material (e.g., a cross-linked polymer). The polyethersulfone membrane may be Mustang. The cross-linked polymer monolith material may be cross-linked poly(glycidyl methacrylate-co-ethylene dimethacrylate).

[0072] Examples of anion exchange materials are known in the art and include, but are not limited to, Poros HQ 50, Poros PI 50, Poros D, Mustang Q, Q Sepharose FF, and DEAE Sepharose.

[0073] Examples of cation exchange materials are known in the art and include, but are not limited to, Mustang S, Sartobind S, SO3 Monolith, S Ceramic HyperD, Poros XS, Poros HS50, Poros HS20, SPSFF, SP-Sepharose XL (SPXL), CM Sepharose Fast Flow, Capto S, Fractogel Se HiCap, Fractogel SO3, or Fractogel COO. In some embodiments of any of the methods described herein, the cation exchange material is Poros HS50. In some embodiments, the Poros HS resin can be Poros HS 50 μm particles or Poros HS 20 μm particles.

[0074] Mixed Mode In some embodiments of any of the methods described herein, the chromatography material is a mixed-mode material containing functional groups capable of one or more of the following functionalities: anion exchange, cation exchange, hydrogen bonding, and hydrophobic interaction. In some embodiments, the mixed-mode material contains functional groups capable of anion exchange and hydrophobic interaction. The mixed-mode material may contain N-benzyl-N-methylethanolamine, 4-mercapto-ethyl-pyridine, hexylamine, or phenylpropylamine as a ligand, or may contain cross-linked polyallylamine. Examples of mixed-mode materials include Capto Adhere resin, QMA resin, Capto MMC resin, MEP HyperCel resin, HEA HyperCel resin, PPA HyperCel resin, or ChromaSorb membrane or Sartobind STIC. In some embodiments, the mixed-mode material is Capto Adhere resin. In some embodiments of the above, the mixed-mode material is a mixed-mode chromatography material. In some embodiments of the above, the mixed-mode material is a mixed-mode chromatography column. In some embodiments of the above, the mixed mode material is a mixed mode membrane. In some embodiments, the mixed mode chromatography column is a large scale, e.g., manufacturing scale, chromatography column.

[0075] In some aspects of the present invention, the chromatography material is a hydrophobic interaction chromatography material. Hydrophobic interaction chromatography (HIC) is a liquid chromatography technique that separates biomolecules according to their hydrophobicity. Examples of HIC chromatography materials include, but are not limited to, Toyopearl hexyl 650, Toyopearl butyl 650, Toyopearl phenyl 650, Toyopearl ether 650, Source, Resource, Sepharose Hi-Trap, Octyl sepharose, and Phenyl sepharose. In some embodiments of the above, the HIC chromatography material is an HIC chromatography column. In some embodiments of the above, the HIC chromatography material is an HIC chromatography membrane. In some embodiments, the HIC chromatography column is a large-scale, e.g., production-scale, chromatography column.

[0076] In some aspects of the invention, the chromatography material is a hydroxyapatite (HAP) chromatography material. Examples of hydroxyapatite chromatography materials include, but are not limited to, HA Ultrogel and CHT hydroxyapatite. In some of the above embodiments, the HAP chromatography material is a HAP chromatography column. In some of the above embodiments, the HAP chromatography material is a HAP chromatography membrane. In some embodiments, the HAP chromatography column is a large-scale, e.g., manufacturing-scale, chromatography column.

[0077] In some embodiments, the present invention provides methods for cleaning or regenerating alkaline-stable chromatography materials, such as alkaline-stable chromatography columns.

[0078] The present invention provides buffers for use in the methods of the present invention. Elution buffers are generally used to remove materials, such as desired materials or undesired materials such as contaminants, from chromatographic materials. Examples of elution buffers include, but are not limited to, about 0.15 M acetic acid at about pH 2.8-2.9. Regeneration buffers are generally used to recharge the column after a chromatographic procedure. For example, a regeneration buffer for anion chromatography may be about 0.1 N NaOH at about pH 13. Equilibration buffers can be used to place the chromatographic material under the same conditions (salt concentration, pH, etc.) as the sample. A non-limiting example of an equilibration buffer is about 25 mM Tris and about 25 mM NaCl at about pH 7.1. Storage buffers are generally used to maintain chromatographic materials when not in use, e.g., with microcodes to prevent contamination. A non-limiting example of a storage buffer is about 100 mM sodium acetate, about 2% benzyl alcohol, and about pH 5.0.

[0079] In some embodiments of any of the methods described herein, the flow rate is less than about 50, 40, or 30 material volumes per hour. The flow rate can be between about 5 and 50 material volumes per hour, between about 10 and 40 material volumes per hour, or between about 18 and 36 material volumes per hour. In some embodiments, the flow rate is about 9, 18, 25, 30, 36, or 40 material volumes per hour.

[0080] In some embodiments, the chromatographic material is present in a chromatographic column. In some embodiments of any of the methods described herein, the flow rate is less than about 50 column volumes (CV) / hour, about 40 CV / hour, or about 30 CV / hour. The flow rate can be between about 5 CV / hour and about 50 CV / hour, between about 10 CV / hour and about 40 CV / hour, or between about 18 CV / hour and about 36 CV / hour. In some embodiments, the flow rate is about 9 CV / hour, about 18 CV / hour, about 25 CV / hour, about 30 CV / hour, about 36 CV / hour, or about 40 CV / hour. In some embodiments of any of the methods described herein, the flow rate is less than about 100 cm / hour, about 75 cm / hour, or about 50 cm / hour. The flow rate can be between about 25 cm / hour and about 150 cm / hour, between about 25 cm / hour and about 100 cm / hour, between about 50 cm / hour and about 100 cm / hour, or between about 65 cm / hour and about 85 cm / hour.

[0081] The bed height is the height of the chromatography material used. In some embodiments of any of the methods described herein, the bed height is greater than about 3 cm, about 10 cm, or about 15 cm. The bed height can be between about 3 cm and about 35 cm, between about 5 cm and about 15 cm, between about 3 cm and about 10 cm, or between about 5 cm and about 8 cm. In some embodiments, the bed height is about 3 cm, about 5 cm, about 10 cm, about 15 cm, about 20 cm, about 25 cm, or about 30 cm. In some embodiments, the bed height is determined based on the amount of polypeptide or contaminants in the load. In some embodiments, the chromatography material is present in a column used for large-scale, e.g., manufacturing-scale production of a polypeptide. In some embodiments, the manufacturing-scale chromatography material has a bed height of about 10 cm, about 15 cm, about 20 cm, about 25 cm, or about 30 cm.

[0082] The bed diameter is the diameter of the chromatographic material used. In some embodiments of any of the methods described herein, the bed diameter is greater than about 80 cm, about 100 cm, or about 120 cm. In some embodiments, the bed diameter is about 50 cm, about 60 cm, about 70 cm, about 80 cm, about 90 cm, about 100 cm, about 110 cm, about 120 cm, about 130 cm, about 140 cm, about 150 cm, about 160 cm, about 170 cm, about 180 cm, about 190 cm, or about 200 cm. In some embodiments, the bed diameter is determined based on the amount of polypeptide or contaminants in the load. In some embodiments, the chromatographic material is present in a column used for large-scale, e.g., manufacturing-scale, production of a polypeptide. In some embodiments, the manufacturing scale chromatography material has a bed diameter of any of about 50 cm, about 60 cm, about 70 cm, about 80 cm, about 90 cm, about 100 cm, about 110 cm, about 120 cm, about 130 cm, about 140 cm, about 150 cm, about 160 cm, about 170 cm, about 180 cm, about 190, or about 200 cm.

[0083] In some embodiments, the chromatography is performed at a concentration of about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 15 mL, about 20 mL, about 25 mL, about 30 mL, about 40 mL, about 50 mL, about 75 mL, about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL The material is present in a container having a volume greater than about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L, about 25 L, about 50 L, about 100 L, about 200 L, about 300 L, about 400 L, about 500 L, about 600 L, about 700 L, about 800 L, about 900 L, or about 1000 L. In some embodiments, the container has a 14 cm bed height and 80 cm bed volume, such as a large-scale Protein A column. In some embodiments, the container has a 19 cm bed height and 100 cm bed volume, such as a large-scale anion exchange column. In some embodiments, the container has a 30 cm bed height and 120 cm bed volume, such as a large-scale cation exchange column.

[0084] As used herein, a load refers to a composition loaded onto a chromatographic material. In some embodiments, the load is a polypeptide loaded onto a chromatographic material that has previously been used to isolate a different polypeptide. A loading buffer is a buffer used to load a composition containing a product of interest onto the chromatographic material. The chromatographic material may be equilibrated with an equilibration buffer before loading the composition to be purified. In some instances, a wash buffer is used after loading the composition onto the chromatographic material and before eluting the polypeptide of interest from the solid phase. However, a portion of the product of interest, such as a polypeptide, may be removed from the chromatographic material by the wash buffer (e.g., similar to a flow-through mode).

[0085] Elution, as used herein, is the removal of a product, e.g., a polypeptide, from a chromatographic material. In some embodiments of the invention, the elution is a "mock elution," in which an elution procedure is applied to a chromatographic material on which no protein has been loaded following a final purification procedure. In some embodiments of the invention, the mock elution procedure is applied to the chromatographic material after any one of the purification procedures described herein. In some embodiments, the mock elution mimics the elution used to elute the protein, which is applied to the material in an attempt to determine whether carryover material (e.g., contaminants) may be present during an actual production run. Mock elution can be used as a means to evaluate the effectiveness of a purification procedure.

[0086] An elution buffer is a buffer used to elute a polypeptide or other product of interest from a chromatographic material. Often, the elution buffer has different physical properties than the load buffer. For example, the elution buffer may have a different conductivity or a different pH than the load buffer. In some embodiments, the elution buffer has a lower conductivity than the load buffer. In some embodiments, the elution buffer has a higher conductivity than the load buffer. In some embodiments, the elution buffer has a lower pH than the load buffer. In some embodiments, the elution buffer has a higher pH than the load buffer. In some embodiments, the elution buffer has a different conductivity and a different pH than the load buffer. The elution buffer may have any combination of a higher or lower conductivity or a higher or lower pH.

[0087] Conductivity refers to the ability of an aqueous solution to conduct electric current between two electrodes. In a solution, electric current flows by ion transport. Therefore, with increasing amounts of ions present in an aqueous solution, the solution has a higher conductivity. The basic unit of measure for conductivity is the siemens (or mho), mho (mS / cm), and can be measured using various models of conductivity meters, such as Orion conductivity meters. Since electrolytic conductivity is the ability of ions in a solution to conduct electric current, the conductivity of a solution can be altered by changing the concentration of ions therein. For example, the concentration of a buffering agent and / or the concentration of salt (e.g., sodium chloride, sodium acetate, or potassium chloride) in the solution can be changed to achieve a desired conductivity. Preferably, the salt concentrations of various buffers are modified to achieve a desired conductivity.

[0088] (B) Impurities The present invention provides a method for the reuse of chromatographic materials for large-scale use, such as manufacturing scale. This method provides for the multiple use of chromatographic materials for multiple polypeptide products. For example, using the methods of the present invention, a first antibody can be purified on a chromatographic material on an industrial scale, followed by a method for purifying / regenerating the chromatographic material described herein, which can then be followed by industrial-scale purification of a second antibody product. In some embodiments, the methods of the present invention are used to reduce "carryover" of a previous product purified using the chromatographic material. In some embodiments, this carryover contaminant includes, but is not limited to, whole antibodies, IgG fragments, Fc, and Fc fragments.

[0089] In some embodiments of any of the methods described herein, the at least one contaminant is any one or more of host cell material, such as CHOP; leached protein A; nucleic acids; variants, fragments, aggregates, or derivatives of the desired polypeptide; another polypeptide; endotoxin; viral contaminants; cell culture media components, carboxypeptidase B, gentamicin, etc. In some examples, the contaminant can be host cell proteins (HCPs) from cells such as, but not limited to, bacterial cells, e.g., E. coli cells, insect cells, prokaryotic cells, eukaryotic cells, yeast cells, mammalian cells, avian cells, fungal cells, etc.

[0090] Leached protein A is protein A that has been desorbed or washed from the solid phase to which it is bound. For example, leached protein A can be leached from a protein A chromatography material. The amount of protein A can be measured, for example, by ELISA. In some embodiments of any of the methods described herein, the amount of leached protein A is reduced by more than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. The amount of leached protein A can be reduced by any of between about 10% and about 99%, between about 30% and about 95%, between about 30% and about 99%, between about 50% and about 95%, between about 50% and about 99%, between about 75% and about 99%, or between about 85% and about 99%. In some embodiments, the amount of leached Protein A is reduced by any of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%, In some embodiments, this reduction is determined by comparing the amount of leached Protein A in the composition recovered from the purification step(s) to the amount of leached Protein A in the composition prior to the purification step(s).

[0091] A host cell protein (HCP) is a protein derived from the cell in which the polypeptide is produced. For example, CHOP is a host cell-derived protein, i.e., a Chinese hamster ovary protein. The amount of CHOP can be measured by enzyme-linked immunosorbent assay ("ELISA") or Meso Scale Discovery ("MSO"). In some embodiments of any of the methods described herein, the amount of HCP (e.g., CHOP) in the eluate is minimal in the mock elution. In some embodiments, the levels of host cell protein in the eluate from the mock elution are compared with and without a clarification method, or before and after a clarification method.

[0092] Methods for measuring DNA, such as host cell DNA, are known in the art and are described in the Examples section. In some embodiments of any of the methods described herein, the amount of DNA is reduced by more than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. The amount of DNA may be reduced by between about 10% and about 99%, between about 30% and about 95%, between about 30% and about 99%, between about 50% and about 95%, between about 50% and about 99%, between about 75% and about 99%, or between about 85% and about 99%. The amount of DNA may be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99%. In some embodiments, this reduction is determined by comparing the amount of DNA in a composition recovered from a purification step(s) to the amount of DNA in the composition before the purification step(s).

[0093] The fragment polypeptide can be a low molecular weight (LMW) protein. In some embodiments, the fragmented polypeptide is a fragment of a polypeptide of interest. Examples of LMW proteins include, but are not limited to, a Fab (fragment antigen binding), an Fc (fragment, crystallizable) region, or a combination of both, or any randomly fragmented portion of an antibody of interest. Methods for measuring fragmented proteins (e.g., LMW proteins) are known in the art and are described in the Examples section. In some embodiments of any of the methods described herein, the amount of LMW protein is reduced by more than any of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%. The amount of LMW protein may be reduced by between about 10% and about 99%, between about 30% and about 95%, between about 30% and about 99%, between about 50% and about 95%, between about 50% and about 99%, between about 75% and about 99%, or between about 85% and about 99%. The amount of LMW protein may be reduced by any of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%. In some embodiments, this reduction is determined by comparing the amount of fragmented protein (e.g., LMW protein) in a composition recovered from a purification step(s) to the amount of fragmented protein (e.g., LMW protein) in the composition prior to the purification step(s).

[0094] The aggregated polypeptide can be a high molecular weight (HMW) protein. In some embodiments, the aggregated polypeptide is a multimer of the polypeptide of interest. The HMW protein can be a dimer, up to 8x the monomer, or more of the polypeptide of interest. Methods for measuring aggregated proteins (e.g., HMW proteins) are known in the art. In some embodiments, the level of HMW in the mock elution is minimal, e.g., less than about 5 ppm, less than about 4 ppm, less than about 3 ppm, less than about 2 ppm, or less than about 1 ppm. In some embodiments of any of the methods described herein, the amount of aggregated protein is reduced by more than any of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%. The amount of aggregated protein may be reduced by any of about 10% to about 99%, about 30% to about 95%, about 30% to about 99%, about 50% to about 95%, about 50% to about 99%, about 75% to about 99%, or about 85% to about 99%. The amount of aggregated protein may be reduced by any of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%. In some embodiments, this reduction is determined by comparing the amount of aggregated protein (e.g., HMW protein) in a composition recovered from a purification step(s) to the amount of aggregated protein (e.g., HMW protein) in the composition prior to the purification step(s).

[0095] Cell culture medium components refer to components present in the cell culture medium. The cell culture medium can be the cell culture medium at the time of cell harvesting. In some embodiments, the cell culture medium component is gentamicin. The amount of gentamicin can be measured by ELISA. In some embodiments of any of the methods described herein, the amount of the cell culture medium component is reduced by more than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. The amount of the cell culture medium component can be reduced by any of between about 10% and about 99%, between about 30% and about 95%, between about 30% and about 99%, between about 50% and about 95%, between about 50% and about 99%, between about 75% and about 99%, or between about 85% and about 99%. In some embodiments, the amount of the cell culture media component is reduced by any of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 98%. In some embodiments, this reduction is determined by comparing the amount of the cell culture media component in the composition recovered from the purification step(s) to the amount of the cell culture media component in the composition prior to the purification step(s).

[0096] (C) Methods for detecting contaminants The present invention provides methods for evaluating the efficiency of purification of reusable chromatography materials. For example, a chromatography material previously loaded with a polypeptide and eluted at least once is purified by one of the methods described above. A mock elution is then performed on the material without any additional polypeptides loaded onto it after the purification procedure. This mock elution can follow the elution procedure used for the polypeptide previously loaded onto the material, or the elution procedure can follow the elution procedure for the polypeptide to be purified after the purification procedure. In some embodiments, a mock loading is performed on the material prior to the mock elution. The mock loading uses the same procedure for loading a polypeptide onto the material, except that the polypeptide is not included in the loading. In some embodiments, the eluent from the mock elution is collected in one or more fractions. In some embodiments, the eluent from the mock elution is collected in a single fraction. In some embodiments, the eluent, or a sample of the eluent, is analyzed for contaminants, including carryover polypeptides from a previous loading of the chromatography material, IgG fragments, leached Protein A, CHOP, and CHO DNA.

[0097] Polypeptide quantification The concentration of polypeptides, such as antibodies, can be determined via absorbance at 280 nm and 320 nm using a UV-visible spectrophotometer (8453 Model G1103A; Agilent Technologies; Santa Clara, CA, USA) or a NanoDrop 1000 Model ND-1000 (Thermo Fisher Scientific; Waltham, MA, USA). Species other than polypeptides (i.e., impurities) previously loaded onto reusable chromatography materials or onto materials purified by the methods of the present invention may be at concentrations too low to have a discernible effect on UV absorbance. If necessary, samples can be diluted with an appropriate non-interfering diluent in the range of 0.1–1.0 absorbance units. Sample preparation and UV measurements are performed in duplicate, and average values ​​are recorded. MAb absorption coefficients can range from 1.42 / mg·ml·cm to 1.645 / mg·ml·cm.

[0098] Total protein can be determined by capillary zone electrophoresis / laser-induced fluorescence detection assay.

[0099] IgG detection Intact human IgG and human IgG fragments can be detected using an intact human IgG-specific ELISA or an IgG fragment-specific ELISA. Human Fc can be detected using a human Fc-specific ELISA.

[0100] Quantification of CHO host cell protein (CHOP) ELISA can be used to quantify the levels of a host cell protein called CHOP. Anti-CHOP antibodies are immobilized on microtiter plate wells. Dilutions of CHOP-containing samples, standards, and controls are incubated in the wells, followed by incubation with anti-CHOP antibodies conjugated to horseradish peroxidase (HRP). HRP enzyme activity can be detected using o-phenylenediamine, and CHOP is quantified by reading absorbance at 490 nm in a microtiter plate reader. Based on the sandwich ELISA principle, the concentration of peroxidase corresponds to the CHOP concentration. The assay range for ELISA is typically 5-320 ng / ml with an intra-assay variability of <10%. CHOP values ​​can be reported in units of ng / ml. Alternatively, CHOP values ​​can be divided by the MAb concentration, and results can be reported in PPM (parts per million; e.g., ng of CHOP / mg of MAb). The CHOP ELISA can be used to quantify total CHOP levels in a sample, but does not quantify the concentration of individual proteins.

[0101] CHO DNA quantification CHO DNA in product samples can be quantified using real-time PCR (TaqMan PCR). DNA from samples and controls is first extracted using Qiagen's Virus Biorobot kit. The extracted sample, control, and standard DNA is subjected to TaqMan real-time polymerase chain reaction (PCR) using PCR primers and probes in a 96-well plate with ABI's sequence detection system. These primers are defined by a 110-base-pair segment of repetitive DNA sequence in the Chinese hamster (Cricetulus griseus) genome. The probe is labeled with a fluorescent reporter dye at the 5' end and a quencher dye at the 3' end. When the probe is intact, the reporter emission spectrum is suppressed by the quencher. The 5' nuclease activity of the polymerase hydrolyzes the probe, releasing the reporter, which results in an increase in fluorescence emission. The sequence detector quantifies the amplified product, which is directly proportional to the increase in fluorescence emission, which is measured continuously during DNA amplification. The cycle number at which the DNA has amplified above the threshold (CT) is calculated for a standard curve. A standard curve ranging from 1 pg / mL to 10,000 pg / mL can be generated and used to quantify the DNA in the sample.

[0102] Quantification of leached protein A The level of leached protein A in the protein A pool can be determined by sandwich protein A ELISA. Chicken anti-staphylococcal protein A antibodies are immobilized on microtiter plate wells. Sample processing procedures can include sample dilution and dissociation of protein A / IgG complexes using microwave-assisted heating as a pretreatment step before running the samples on the sandwich ELISA. Protein A, if present in the sample, can bind to the coated antibody. Bound protein A is detected using a horseradish peroxidase-conjugated anti-protein antibody. The enzymatic activity of horseradish peroxidase is quantified using a two-component TMB substrate solution, which generates a colorimetric signal.

[0103] III. Polypeptides The methods of the invention can be used to purify chromatography materials used in the purification of multiple polypeptides. In some embodiments, the chromatography material is used in large-scale, e.g., manufacturing-scale, production of polypeptides such as antibodies or fragments thereof. In some embodiments, the chromatography material is used in the purification of a first polypeptide, such as a first antibody, and then the material is purified by the methods of the invention, and then the chromatography material can be used to purify a second polypeptide, such as a second antibody. In some embodiments, the purification is effective, such that a preparation comprising the second purified polypeptide is essentially free of the first polypeptide. In some embodiments, a preparation comprising the second purified polypeptide (e.g., the second antibody) contains less than 1 ppm of the first polypeptide (e.g., the first antibody). In some embodiments, the second purified polypeptide comprises less than 1 ppm, less than 2 ppm, less than 3 ppm, less than 4 ppm, less than 5 ppm, less than 10 ppm, less than 20 ppm, less than 30 ppm, less than 40 ppm, less than 50 ppm, or less than 100 ppm of the first polypeptide.

[0104] In some embodiments, the methods of the present invention are used to reuse chromatographic materials used to purify a therapeutic polypeptide. In some embodiments, the polypeptide is an antagonist. In some embodiments, the polypeptide is an agonist. In some embodiments, the polypeptide is an antibody. In some embodiments, the polypeptide is tagged with an epitope. In some embodiments, the polypeptide retains biological and / or immunological activity. In some embodiments, the polypeptide is an antagonist. In some embodiments, the polypeptide initiates complement dependent cytotoxicity. In some embodiments, the polypeptide is an antibody or immunoadhesin.

[0105] In some embodiments, the polypeptide, first polypeptide, and / or second polypeptide has a molecular weight greater than about 5,000 daltons, about 10,000 daltons, about 15,000 daltons, about 25,000 daltons, about 50,000 daltons, about 75,000 daltons, about 100,000 daltons, about 125,000 daltons, or about 150,000 daltons. The polypeptide may have a molecular weight between about 50,000 daltons and about 200,000 daltons, or between about 100,000 daltons and about 200,000 daltons. Alternatively, the polypeptide for use herein may have a molecular weight of about 120,000 daltons or about 25,000 daltons.

[0106] pI is the isoelectric point, which is the pH at which a particular molecule or surface has no net electrical charge. In some embodiments of any of the methods described herein, the pI of a polypeptide, e.g., a first polypeptide and / or a second polypeptide, can be between about 6 and about 10, about 7 and about 9, or about 8 and about 9. In some embodiments, the polypeptide has a pI of about 6, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10.

[0107] Polypeptides purified using reusable chromatography materials purified by the methods described herein are generally produced using recombinant technology. Methods for producing recombinant proteins are described, for example, in U.S. Patent Nos. 5,534,615 and 4,816,567, which are incorporated herein by reference. In some embodiments, the protein of interest is produced in CHO cells (see, for example, WO 94 / 11026). When using recombinant technology, these polypeptides can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium.

[0108] Polypeptides purified using reusable chromatography materials purified by the methods described herein can be recovered from culture medium or host cell lysates. Cells used in polypeptide expression can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents. If the polypeptide is produced intracellularly, as a first step, particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describes a procedure for isolating polypolypeptides secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed for approximately 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. If the polypeptide is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available polypeptide concentration filter, e.g., an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0109] Examples of polypeptides that can be purified using reusable chromatography materials purified by the methods described herein include, but are not limited to, immunoglobulins, immunoadhesins, antibodies, enzymes, hormones, fusion proteins, Fc-containing proteins, immunoconjugates, cytokines, and interleukins. Examples of polypeptides include mammalian proteins such as renin; hormones; growth hormones, including human growth hormone and bovine growth hormone; growth hormone-releasing factor; parathyroid hormone; thyroid-stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle-stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors, such as factor VIIIC, factor IX, tissue factor, and von Willebrand factor; anticoagulants, such as protein C; atrial natriuretic factor; pulmonary surfactant; plasminogen activators, such as urokinase or human urinary or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factors; tumor necrosis factor-alpha and -beta; enkephalinase; RANTES (regulated on activation normally expressed and expressed by T-cells) secreted); human macrophage inflammatory protein (MIP-1-alpha); serum albumin, e.g., human serum albumin; Müllerian inhibitory factor; relaxin A chain; relaxin B chain; prorelaxin; mouse gonadotropin-related peptide; enzymes; microbial proteins, e.g., beta-lactamase; DNase; IgE; cytotoxic T lymphocyte-associated antigen (CTLA), e.g., CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; protein A or D; rheumatoid factor; neurotrophic factors, e.g., bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or nerve growth factors, e.g., NGF-b; platelet-derived growth factor (PDGF); fibroblast growth factors, e.g., aFGF and bFGF; epidermal growth factor (EGF);transforming growth factors (TGFs), e.g., TGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growth factors-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins (IGFBPs); cytokines; CD proteins, e.g., CD3, CD4, CD8, CD19, and CD20; erythropoietin; osteoinductive factors; immunotoxins; fusion polypeptides, i.e., polypeptides contained on two or more heterologous polypeptides or fragments thereof and encoded by a recombinant nucleic acid; Fc-containing polypeptides, e.g., fusion proteins comprising an immunoglobulin Fc region or a fragment thereof fused to a second polypeptide; immunoconjugates; bone morphogenetic proteins (BMPs); interferons, e.g., interferons These include, but are not limited to, feron-alpha, -beta, and -gamma; colony-stimulating factors (CSFs), such as M-CSF, GM-CSF, and G-CSF; interleukins (ILs), such as IL-1 through IL-10; superoxide dismutase; T-cell receptors; surface membrane proteins; decay-accelerating factors; viral antigens, such as portions of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; integrins, such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4, and VCAM; tumor-associated antigens, such as CA125 (ovarian cancer antigen) or HER2, HER3, or HER4 receptors; immunoadhesins; and fragments and / or variants of any of the above-listed proteins, as well as antibodies, including, for example, antibody fragments, that bind to proteins comprising any of the above-listed proteins.

[0110] (A) Antibody In some embodiments of any of the methods described herein, the polypeptide that can be purified using the reusable chromatography material purified by the methods described herein, e.g., the first polypeptide, the second polypeptide, or any subsequent polypeptide, is an antibody.

[0111] Molecular targets for antibodies include, for example, but are not limited to, CD proteins and their ligands: (i) CD3, CD4, CD8, CD19, CD11a, CD20, CD22, CD34, CD40, CD79α (CD79a), and CD79β (CD79b); (ii) members of the ErbB receptor family, such as EGF receptor, HER2, HER3, or HER4 receptor; (iii) cell adhesion molecules, such as LFA-1, Mac1, p150, 95, VLA-4, and ICAM-1. , VCAM, and αv / β3 integrins, including either their alpha or beta subunits (e.g., anti-CD11a, anti-CD18, or anti-CD11b antibodies); (iv) growth factors, such as VEGF; IgE; blood group antigens; flk2 / flt3 receptors; obesity (OB) receptors; mpl receptors; CTLA-4; protein C, BR3, c-met, tissue factor, β7, and the like; and (v) cell surface and transmembrane tumor-associated antigens (TAA), such as those described in U.S. Pat. No. 7,521,541.

[0112] Other exemplary antibodies include anti-estrogen receptor antibodies, anti-progesterone receptor antibodies, anti-p53 antibodies, anti-HER-2 / neu antibodies, anti-EGFR antibodies, anti-cathepsin D antibodies, anti-Bcl-2 antibodies, anti-E-cadherin antibodies, anti-CA125 antibodies, anti-CA15-3 antibodies, anti-CA19-9 antibodies, anti-c-erbB-2 antibodies, anti-P-glycoprotein antibodies, anti-CEA antibodies, anti-retinoblastoma protein antibodies, anti-ras oncoprotein antibodies, anti-Lewis antibodies, X antibody, anti-Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9 / p24 antibody, anti-CD10 antibody, anti-CD11a antibody, anti-CD11c antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD1 5 antibodies, anti-CD19 antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD23 antibodies, anti-CD30 antibodies, anti-CD31 antibodies, anti-CD33 antibodies, anti-CD34 antibodies, anti-CD35 antibodies, anti-CD38 antibodies, anti-CD41 antibodies, anti-LCA / CD45 antibodies, anti-CD45RO antibodies , anti-CD45RA antibody, anti-CD39 antibody, anti-CD100 antibody, anti-CD95 / Fas antibody, anti-CD99 antibody, anti-CD106 antibody, anti-ubiquitin antibody, anti-CD71 antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-vimentin antibody, anti-HPV protein antibody, anti-kappa light chain antibody, anti-lambda light chain antibody, anti-melanosome antibody, anti-prostate specific antigen antibody, anti-S-100 antibody, anti-tau antigen antibody, anti-fibrin antibody, anti-keratin antibody and anti-Tn-antigen antibody.

[0113] (i) Polyclonal antibodies In some embodiments, these antibodies are polyclonal. Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the appropriate antigen and adjuvant. The appropriate antigen is conjugated to a polypeptide that is immunogenic in the species being immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, such as maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine ​​residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R 1 N=C=NR, where R and R 1 It may be useful to conjugate using different alkyl groups.

[0114] Animals are immunized against the antigen, immunogenic conjugate, or derivative by combining, for example, 100 μg or 5 μg of the polypeptide or conjugate (for rabbits or mice, respectively) with 3 volumes of complete Freund's adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with 1 / 5 to 1 / 10 of the original amount of peptide or conjugate in complete Freund's adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. In some embodiments, the animals are boosted with a conjugate of the same antigen but conjugated to a different polypeptide and / or via a different cross-linking reagent. Conjugates can also be produced in recombinant cell culture as polypeptide fusions. Aggregating agents, such as alum, are also suitably used to enhance the immune response.

[0115] (ii) Monoclonal antibody In some embodiments, antibodies purified on reusable chromatographic materials purified by the methods of the invention are monoclonal antibodies. Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variants that arise during production of the monoclonal antibody, such variants generally being present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete or polyclonal antibodies.

[0116] For example, the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

[0117] In the hybridoma method, a mouse or other suitable host animal, e.g., a hamster, is immunized as described herein to elicit lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the polypeptide used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0118] The hybridoma cells thus prepared are seeded and grown in an appropriate culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if these parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

[0119] In some embodiments, these myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Of these, in some embodiments, the myeloma cell line is a myeloma cell line derived from a mouse myeloma line, such as MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California, USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland, USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0120] The culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen, hi some embodiments, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0121] The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem. 107:220 (1980).

[0122] After hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. Furthermore, these hybridoma cells can be grown in vivo as ascites tumors in animals.

[0123] The monoclonal antibodies secreted by these subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, polypeptide A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0124] DNA encoding monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). In some embodiments, these hybridoma cells serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector that is then transfected into host cells that do not otherwise produce immunoglobulin polypeptides, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol. 5:256-262 (1993) and Pluckthun, Immunol. Rev. 130:151-188 (1992).

[0125] In a further embodiment, antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature 348:552-554 (1990). Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology 10:779-783 (1992)) and combinatorial infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266 (1993)). These techniques are therefore viable alternatives to traditional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies.

[0126] The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851 (1984)), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0127] Typically, such a non-immunoglobulin polypeptide replaces the constant domains of an antibody, or such a non-immunoglobulin polypeptide replaces the variable domains of one antigen-binding site of an antibody to create a chimeric bivalent antibody comprising one antigen-binding site with specificity for one antigen and another antigen-binding site with specificity for a different antigen.

[0128] In some embodiments of any of the methods described herein, the antibody is IgA, IgD, IgE, IgG, or IgM. In some embodiments, the antibody is an IgG monoclonal antibody.

[0129] (iii) humanized antibody In some embodiments, the antibody is a humanized antibody. Methods for humanizing non-human antibodies have been described in the art. In some embodiments, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, and they are typically taken from an "import" variable domain. Humanization can be performed essentially according to the method of Winter and coworkers (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)) by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Such "humanized" antibodies are thus chimeric antibodies (U.S. Pat. No. 4,816,567), in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0130] The selection of human variable domains, both light and heavy, used in creating a humanized antibody is crucial to reducing antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence closest to that of the rodent is then accepted as the human framework region (FR) for the humanized antibody (Sims et al., J. Immunol. 151:2296 (1993); Chothia et al., J. Mol. Biol. 196:901 (1987)). Another method uses a specific framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chain variable regions. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993)).

[0131] It is further important that antibodies be humanized with high affinity for the antigen and retention of other favorable biological properties. To achieve this goal, in some embodiments of these methods, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, e.g., increased affinity for the target antigen(s), is achieved. In general, hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0132] (v) human antibody In some embodiments, the antibody is a human antibody. As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, the expression of antibody heavy chain joining regions (J) in chimeric and germline mutant mice can be expressed in vivo. H It has been described that homozygous deletion of the .gt; (Ig) gene results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice results in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggermann et al., Year in Immuno. 7:33 (1993); and U.S. Pat. Nos. 5,591,669; 5,589,369; and 5,545,807.

[0133] Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires derived from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either the major or minor coat polypeptide gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of B cells. Phage display can be performed in a variety of formats; for their reviews, see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V gene segments can be used for phage display. Clackson et al., Nature 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. Repertoires of V genes from unimmunized human donors can be constructed, and antibodies against a diverse array of antigens (including self-antigens) can be isolated essentially according to the techniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991) or Griffith et al., EMBO J. 12:725-734 (1993). See also U.S. Patent Nos. 5,565,332 and 5,573,905.

[0134] Human antibodies may also be generated by in vitro activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275).

[0135] (v)Antibody fragment In some embodiments, the antibody is an antibody fragment. Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, these antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv). See WO 93 / 16185; U.S. Pat. Nos. 5,571,894 and 5,587,458. The antibody fragment may also be a "linear antibody," e.g., as described in U.S. Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

[0136] In some embodiments, a fragment of an antibody described herein is provided. In some embodiments, the antibody fragment is an antigen-binding fragment. In some embodiments, the antigen-binding fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an scFv, an Fv, and a diabody.

[0137] (vi) Bispecific antibodies In some embodiments, the antibody is a bispecific antibody. A bispecific antibody is an antibody that has binding specificities for at least two different epitopes. An exemplary bispecific antibody can bind to two different epitopes. Alternatively, the binding arm of the bispecific antibody can be combined with an arm that binds to a trigger molecule on a leukocyte, such as a T cell receptor molecule (e.g., CD2 or CD3), or an Fc receptor for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), to focus cellular defenses on the cell. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0138] Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy-light chain pairs, with the two chains having different specificities (Millstein et al., Nature 305:537-539 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, usually by affinity chromatography steps, is rather cumbersome and the product yields are low. Similar procedures are disclosed in WO 93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0139] According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. In some embodiments, the fusion is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. In some embodiments, the first heavy-chain constant region (CH1), containing the site necessary for light-chain binding, is present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. This allows for great flexibility in adjusting the relative proportions of the three polypeptide fragments in embodiments where unequal ratios of the three polypeptide chains used in the construction provide optimal yields. However, if expression of at least two polypeptide chains in equal ratios results in high yields or if the ratios are not of particular importance, it is possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector.

[0140] In some embodiments of this approach, these bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure has been found to facilitate separation of the desired bispecific compound from undesired immunoglobulin chain combinations, because the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides an easy method of separation. This approach is disclosed in WO 94 / 04690. For further details on generating bispecific antibodies, see, e.g., Suresh et al., Methods in Enzymology 121:210 (1986).

[0141] According to another approach described in U.S. Patent No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. In some embodiments, the interface is located between the C and C regions of the antibody constant domains.H The method comprises at least a portion of domain 3 of the antibody. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). A compensatory "cavity" of identical or similar size to the large side chain(s) is created on the interface of a second antibody molecule by replacing the large amino acid side chain(s) with a smaller one (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

[0142] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, and the other to biotin. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980) and for the treatment of HIV infection (WO 91 / 00360, WO 92 / 200373, and EP 03089). Heteroconjugate antibodies can be made using any conventional cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with several cross-linking techniques.

[0143] Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985), describe a procedure in which intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The generated Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The provided bispecific antibodies can be used as agents for the selective immobilization of enzymes.

[0144] Various techniques for producing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). Leucine zipper peptides derived from Fos and Jun proteins were attached to the Fab' portions of two different antibodies by gene fusion. These antibody homodimers were reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for generating bispecific antibody fragments. These fragments comprise a light-chain variable domain (V) connected by a linker that is too short to allow pairing between the two domains on the same chain. L ) connected to the heavy chain variable domain (V H ) is included. Therefore, the V of one fragment H Domain and V L The domain is a complementary VL Domain and V H The Fv domains are forced to pair, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152:5368 (1994).

[0145] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

[0146] (vii) multivalent antibody In some embodiments, these antibodies are multivalent antibodies. Multivalent antibodies may be internalized (and / or catabolized) more quickly than bivalent antibodies by cells expressing the antigen to which the antibody binds. The antibodies provided herein may be multivalent antibodies (other than those of the IgM class) with three or more antigen-binding sites (e.g., tetravalent antibodies) that can be readily produced by recombinant expression of nucleic acids encoding the polypeptide chains of the antibody. The multivalent antibody may comprise a dimerization domain and three or more antigen-binding sites. A preferred dimerization domain comprises (or consists of) an Fc region or hinge region. In this scenario, the antibody comprises an Fc region and three or more antigen-binding sites amino-terminal to the Fc region. Preferred multivalent antibodies herein comprise (or consist of) three to about eight, but preferably four, antigen-binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains. For example, the polypeptide chain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc, where VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, the polypeptide chain(s) may comprise a VH-CH1-flexible linker-VH-CH1-Fc region chain or a VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. The multivalent antibody herein may comprise, for example, from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated herein comprise a light chain variable domain and, optionally, further comprise a CL domain.

[0147] In some embodiments, the antibody is a multispecific antibody. Examples of multispecific antibodies include V H V L The heavy chain variable domain (V H ) and the light chain variable domain (VL ) antibodies containing each V H V L Two or more V units bind to different epitopes L Domain and V H Examples of multispecific antibodies include, but are not limited to, antibodies having domains, antibodies having two or more single variable domains, where each single variable domain binds a different epitope, full-length antibodies, antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies, triabodies, trifunctional antibodies, and covalently or non-covalently linked antibody fragments. In some embodiments, the antibodies have polyepitopic specificity, e.g., the ability to specifically bind to two or more different epitopes on the same or different target(s). In some embodiments, these antibodies are monospecific, e.g., antibodies that bind to only one epitope. According to one embodiment, the multispecific antibody is an IgG antibody that binds to each epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0.1 μM to 0.001 pM.

[0148] (viii) Other antibody modifications It may be desirable to modify the antibodies provided herein with respect to effector function, for example, to enhance the antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively, or additionally, cysteine ​​residue(s) can be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, BJ, Immunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement-mediated lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989).

[0149] To increase the serum half-life of the antibody, amino acid alterations can be made in the antibody as described in US Patent Application Publication No. 2006 / 0067930, the entire contents of which are incorporated herein by reference.

[0150] (B) Polypeptide Variants and Modifications The amino acid sequence modification(s) of polypeptides, including antibodies, described herein may be used in reusable chromatographic materials purified by the methods described herein.

[0151] (i) mutant polypeptide By "polypeptide variant" is meant a polypeptide, preferably an active polypeptide, as defined herein, having at least about 80% amino acid sequence identity with the full-length native sequence of the polypeptide, with or without the signal peptide, the polypeptide sequence lacking the signal peptide, or the extracellular domain of the polypeptide. Such polypeptide variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N- or C-terminus of the full-length native amino acid sequence. Typically, a TAT polypeptide variant will have at least about 80% amino acid sequence identity, or at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the full-length native sequence polypeptide sequence, with or without the signal peptide, the polypeptide sequence lacking the signal peptide, or the extracellular domain of the polypeptide. Optionally, the variant polypeptide has no more than one conservative amino acid substitution compared to the native polypeptide sequence, or alternatively, no more than about two, no more than about three, no more than about four, no more than about five, no more than about six, no more than about seven, no more than about eight, no more than about nine, or no more than about ten conservative amino acid substitutions compared to the native polypeptide sequence.

[0152] The variant polypeptide may be truncated, for example, at the N-terminus or C-terminus, or may lack internal residues, when compared with the full-length native polypeptide. Certain variant polypeptides may lack amino acid residues that are not important for the desired biological activity. These variant polypeptides with truncations, deletions, and insertions may be prepared by any of several conventional techniques. The desired variant polypeptide may be chemically synthesized. Another suitable technique involves isolating and amplifying a nucleic acid fragment encoding the desired variant polypeptide by polymerase chain reaction (PCR). Oligonucleotides defining the desired fragment of the nucleic acid fragment are used as the 5' and 3' primers in the PCR. Preferably, the variant polypeptide shares at least one biological and / or immunological activity with the native polypeptide disclosed herein.

[0153] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or an antibody fused to a cytotoxic polypeptide. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme or a polypeptide which increases the serum half-life of the antibody.

[0154] For example, it may be desirable to improve the binding affinity and / or other biological properties of the polypeptide. Amino acid sequence variants of the polypeptide are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the polypeptide. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired properties. Amino acid changes can also alter post-translational processing of the polypeptide (e.g., antibody), for example, changing the number or location of glycosylation sites.

[0155] Guidance in determining which amino acid residues can be inserted, substituted, or deleted without adversely affecting a desired activity can be found by comparing the sequence of the polypeptide with the sequences of known homologous polypeptide molecules and minimizing the number of amino acid sequence changes made in areas of high homology.

[0156] A useful method for identifying specific residues or regions of a polypeptide (e.g., an antibody) that are preferred locations for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells, Science 244:1081-1085 (1989). In this method, a residue or group of target residues is identified (e.g., charged residues, e.g., Arg, Asp, His, Lys, and Glu) and replaced with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the antigen. Amino acid locations that demonstrate functional sensitivity to the substitution are then refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the sites for introducing amino acid sequence differences are predetermined, the nature of the mutation per se need not be. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is performed at the target codon or region, and the expressed antibody variants are screened for the desired activity.

[0157] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue. The most popular sites for substitutional mutagenesis include hypervariable regions, although FR alterations are also contemplated. Conservative substitutions are shown in Table 1 below under the heading of "preferred substitutions." If such substitutions result in a change in biological activity, more substantial changes, such as those referred to in Table 1 as "exemplary substitutions," or as further described below with reference to amino acid classes, can be introduced and the products screened. TIFF2025186245000002.tif184170

[0158] Substantial modifications in the biological properties of a polypeptide are achieved by selecting substitutions that differ significantly in their effect on (a) maintaining the structure of the polypeptide backbone in the region of the substitution, for example, as a sheet or helical conformation, (b) maintaining the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chain. Amino acids can be grouped according to similarities in the characteristics of their side chains (AL Lehninger, Biochemistry, 2nd ed., pp. 73-75, Worth Publishers, New York (1975)): (1) Non-polar: Ala(A), Val(V), Leu(L), Ile(I), Pro(P), Phe(F), Trp(W), Met(M) (2) Uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q) (3) Acidic: Asp(D), Glu(E) (4) Basic: Lys(K), Arg(R), His(H)

[0159] Alternatively, naturally occurring residues can be divided into groups based on common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0160] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0161] Any cysteine ​​residue not involved in maintaining the proper conformation of the antibody may also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine ​​bond(s) may be added to the polypeptide to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

[0162] A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient method for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent manner from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and target. Such contact and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, the panel of variants can be subjected to screening as described herein, and antibodies with superior properties in one or more appropriate assays can be selected for further development.

[0163] Another type of amino acid variant of a polypeptide alters the original glycosylation pattern of the antibody. The polypeptide may contain non-amino acid moieties. For example, the polypeptide may be glycosylated. Such glycosylation may occur naturally during expression of the polypeptide in a host cell or host organism, or may be an intentional modification resulting from human intervention. By altering is meant deleting one or more carbohydrate moieties found in the polypeptide and / or adding one or more glycosylation sites that are not present in the polypeptide.

[0164] Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0165] Addition of glycosylation sites to the polypeptide is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by adding, or substituting by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0166] Removal of carbohydrate moieties present on a polypeptide can be accomplished chemically or enzymatically, or by mutational substitution of codons encoding amino acid residues that serve as targets for glycosylation. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exoglycosidases.

[0167] Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains, acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0168] (ii) chimeric polypeptide The polypeptides described herein can be modified in a manner to form chimeric molecules comprising the polypeptide fused to another, heterologous polypeptide or amino acid sequence. In some embodiments, a chimeric molecule comprises a fusion of the polypeptide with a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino- or carboxyl-terminus of the polypeptide. The presence of such epitope-tagged forms of the polypeptide can be detected using an antibody against the tag polypeptide. Provision of the epitope tag also allows the polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.

[0169] In an alternative embodiment, the chimeric molecule may comprise a fusion of the polypeptide with an immunoglobulin or a particular region of an immunoglobulin. The bivalent form of the chimeric molecule is referred to as an "immunoadhesin."

[0170] As used herein, the term "immunoadhesin" designates antibody-like molecules that combine the binding specificity of a heterologous polypeptide with the effector functions of immunoglobulin constant domains. Structurally, these immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity that is other than the antigen recognition and binding site of an antibody (i.e., is "heterologous") with an immunoglobulin constant domain sequence. The adhesin portion of an immunoadhesin molecule is typically a contiguous amino acid sequence that includes at least the binding site of a receptor or ligand. The immunoglobulin constant domain sequence in an immunoadhesin can be obtained from any immunoglobulin, e.g., IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM.

[0171] These Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a polypeptide in place of at least one variable region within an Ig molecule. In one particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH2, and CH3, or the hinge, CH1, CH2, and CH3 regions of an IgG1 molecule.

[0172] (iii) Polypeptide conjugates Polypeptides for use in polypeptide formulations may be conjugated to a cytotoxic agent, e.g., a chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioactive isotope (i.e., a radioconjugate).

[0173] Chemotherapeutic agents useful in the production of such conjugates may be used. Additionally, enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes. A variety of radionuclides are available for the production of radioconjugated polypeptides. Examples include: 212 Bi, 131 I, 131 In, 90 Y and 186Conjugates of polypeptides and cytotoxic agents are prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutareldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotides to polypeptides.

[0174] Conjugates of a polypeptide and one or more small molecule toxins, such as a calicheamicin, a maytansinoid, a trichothene, and CC1065, and derivatives of these toxins that have toxin activity, are also contemplated herein.

[0175] Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata. Subsequently, it was discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters. Synthetic maytansinol and its derivatives and analogs are also contemplated. Numerous linking groups are known in the art for preparing polypeptide-maytansinoid conjugates, including, for example, those disclosed in U.S. Pat. No. 5,208,020. Linking groups include disulfide, thioether, acid-labile, photolabile, peptidase-labile, or esterase-labile groups, as disclosed in the above-identified patents; disulfide and thioether groups are preferred.

[0176] The linker can be attached to the maytansinoid molecule at various positions, depending on the type of bond. For example, an ester bond can be formed by reaction with a hydroxyl group using conventional coupling techniques. This reaction can occur at the C-3 position, which bears a hydroxyl group, the C-14 position, which is modified with a hydroxymethyl group, the C-15 position, which is modified with a hydroxyl group, and the C-20 position, which bears a hydroxyl group. In a preferred embodiment, the bond is formed at the C-3 position of maytansinol or a maytansinol analog.

[0177] Another conjugate of interest comprises a polypeptide conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics is capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of calicheamicin family conjugates, see, e.g., U.S. Patent No. 5,712,374. Structural analogs of calicheamicin that can be used include γ1 I , α2 I , α3 I , N-acetyl-γ1 I , PSAG and θ1 IOther antitumor drugs to which antibodies can be conjugated include, but are not limited to, QFA, an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these drugs via polypeptide (e.g., antibody)-mediated internalization greatly enhances their cytotoxic effects.

[0178] Other anti-tumor agents that can be conjugated to the polypeptides described herein include BCNU, streptozocin, vincristine and 5-fluorouracil, a family of drugs known collectively as LL-E33288 complexes, and esperamicin.

[0179] In some embodiments, the polypeptide may be conjugated between the polypeptide and a compound with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease, such as a deoxyribonuclease; DNase).

[0180] In yet another embodiment, the polypeptide (e.g., an antibody) can be conjugated to a "receptor" (such as streptavidin) for use in tumor pre-targeting; the polypeptide-receptor conjugate is administered to a patient, followed by removal of unbound conjugate from the circulation using a detergent, followed by administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionucleotide).

[0181] In some embodiments, the polypeptide may be conjugated to a prodrug-activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic agent) into an active anticancer drug. The enzyme component of the immunoconjugate includes any enzyme capable of acting on a prodrug in such a way as to convert it into its more active, cytotoxic form.

[0182] Useful enzymes include alkaline phosphatases, useful for converting phosphate-containing prodrugs to free drugs; arylsulfatases, useful for converting sulfate-containing prodrugs to free drugs; cytosine deaminases, useful for converting the non-toxic 5-fluorocytosine to the anticancer drug, 5-fluorouracil; proteases, such as Serratia protease, thermolysin, subtilisin, carboxypeptidase, and cathepsins (e.g., cathepsins B and L), useful for converting peptide-containing prodrugs to free drugs; These include, but are not limited to, D-alanylcarboxypeptidase, useful for converting prodrugs; carbohydrate-cleaving enzymes, such as β-galactosidase and neuraminidase, useful for converting glycosylated prodrugs to free drugs; β-lactamase, useful for converting β-lactam-derivatized drugs to free drugs; and penicillin amidase, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized with phenoxyacetyl or phenylacetyl groups, respectively, at their amine nitrogens to free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes," can be used to convert prodrugs to free active drugs.

[0183] (iv) Other Another type of covalent modification of a polypeptide involves coupling the polypeptide to one of a variety of nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. The polypeptide can also be entrapped in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared by coacervation techniques or by catalytic surface polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 18th ed., edited by Gennaro, AR (1990).

[0184] IV. Obtaining Polypeptides for Use in Formulations and Methods Polypeptides purified using reusable chromatography materials purified by the methods described herein can be obtained using methods well known in the art, including recombinant methods, and the following sections provide guidance regarding these methods.

[0185] (A) Polynucleotide "Polynucleotide" or "nucleic acid," when used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA.

[0186] Polynucleotides encoding polypeptides can be obtained from any source, including, but not limited to, cDNA libraries prepared from tissues believed to have polypeptide mRNA and express it at detectable levels. Thus, polynucleotides encoding polypeptides can conveniently be obtained from cDNA libraries prepared from human tissue. Polypeptide-encoding genes can also be obtained from genomic libraries or by known synthetic procedures (e.g., automated nucleic acid synthesis).

[0187] For example, the polynucleotide can encode an entire immunoglobulin molecule chain, e.g., a light chain or a heavy chain. A complete heavy chain includes the heavy chain variable region (V H ) as well as the heavy chain constant region (C H ), which typically also contains three constant domains: C H 1. C H 2 and C H 3; as well as a "hinge" region. In some circumstances, the presence of a constant region is desirable.

[0188] Other polypeptides that may be encoded by the polynucleotide include antigen-binding antibody fragments, such as single domain antibodies ("dAbs"), Fvs, scFvs, Fab's and F(ab')2s, and "minibodies." Minibodies are (typically) C H 1 and C K or C LMinibodies are bivalent antibody fragments with truncated domains. Because minibodies are smaller than conventional antibodies, they should achieve better tissue penetration in clinical / diagnostic uses, but because they are bivalent, they should retain higher binding affinity than monovalent antibody fragments such as dAbs. Thus, unless the context indicates otherwise, the term "antibody" as used herein encompasses not only whole antibody molecules but also antigen-binding antibody fragments of the types discussed above. Preferably, each framework region present in the encoded polypeptide contains at least one amino acid substitution compared to the corresponding human acceptor framework. Thus, for example, these framework regions can contain a total of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions compared to the acceptor framework region.

[0189] Suitably, the polynucleotides described herein may be isolated and / or purified. In some embodiments, the polynucleotides are isolated polynucleotides.

[0190] The term "isolated polynucleotide" is intended to indicate that the molecule has been removed or separated from its normal or natural environment, or has been produced in such a way that it is not present in its normal or natural environment. In some embodiments, these polynucleotides are purified polynucleotides. The term purified is intended to indicate that at least some contaminating molecules or substances have been removed.

[0191] Suitably, these polynucleotides are substantially purified such that the relevant polynucleotide constitutes the predominant (ie, most abundant) polynucleotide present in the composition.

[0192] (B) Expression of polynucleotides The following description primarily relates to producing polypeptides by culturing cells transformed or transfected with a vector containing a polypeptide-encoding polynucleotide. It is, of course, contemplated that alternative methods well known in the art can be used to prepare polypeptides. For example, the appropriate amino acid sequence or portions thereof can be produced by direct peptide synthesis using solid-phase techniques (see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963)). In vitro protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved using, for example, an Applied Biosystems Peptide Synthesizer (Foster City, Calif.) using the manufacturer's instructions. Various portions of a polypeptide can be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired polypeptide.

[0193] The polynucleotides described herein are inserted into an expression vector(s) for production of a polypeptide. The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences include, but are not limited to, promoters (e.g., naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences.

[0194] A polynucleotide is "operably linked" when it is placed into a functional relationship with another polynucleotide sequence. For example, nucleic acid for a presequence or secretory leader is operably linked to nucleic acid for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the nucleic acid sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[0195] For antibodies, the light and heavy chains can be cloned in the same or different expression vectors. The nucleic acid segments encoding the immunoglobulin chains are operably linked to regulatory sequences in the expression vector(s) that ensure the expression of immunoglobulin polypeptides.

[0196] Vectors containing polynucleotide sequences (e.g., variable heavy and / or variable light chain coding sequences and optional expression control sequences) can be transferred into host cells by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biolistic or viral-based transfection can be used for other cellular hosts (see generally Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989)). Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection. For the production of transgenic animals, transgenes can be microinjected into fertilized oocytes, or incorporated into the genome of embryonic stem cells, the nucleus of which can be transferred into an enucleated oocyte.

[0197] (C) Vector The term "vector" includes expression vectors and transformation vectors and shuttle vectors.

[0198] The term "expression vector" means a construct capable of in vivo or in vitro expression.

[0199] The term "transformation vector" refers to a construct that can be transferred from one entity to another, whether it is of the same species or a different species. If the construct can be transferred from one species to another, such as from an E. coli plasmid to a bacterium, such as a Bacillus, the transformation vector is sometimes called a "shuttle vector." The transformation vector can even be a construct that can be transferred from an E. coli plasmid to an Agrobacterium to a plant.

[0200] The vector can be transformed into a suitable host cell, as described below, to provide for expression of the polypeptide. A variety of vectors are publicly available. The vector can be in the form of, for example, a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence can be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Construction of suitable vectors containing one or more of these components employs standard ligation techniques known to those skilled in the art.

[0201] These vectors may be for example plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the polynucleotide and optionally a regulator of this promoter. Vectors may contain one or more selectable marker genes as are well known in the art.

[0202] These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA.

[0203] (D) Host cell The host cell can be, for example, a bacterium, a yeast or other fungal cell, an insect cell, a plant cell, or a mammalian cell.

[0204] Genetically engineered transgenic multicellular host organisms can be used to produce polypeptides, and can be, for example, transgenic mammalian organisms (e.g., transgenic goat or mouse strains).

[0205] Suitable prokaryotes include, but are not limited to, eubacteria, e.g., gram-negative or gram-positive organisms, e.g., Enterobacteriaceae, such as E. coli. Various strains of E. coli are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strains W3110 (ATCC 27,325) and K5772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae, e.g., Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans and Shigella, and Bacilli, e.g., B. subtilis and B. licheniformis (e.g., B. licheniformis 41P), Pseudomonas, e.g., P. aeruginosa, and Streptomyces. These examples are illustrative rather than limiting. Strain W3110 is one particularly preferred host or parent host because it is a common host strain for recombinant polynucleotide product fermentation. Preferably, the host cell secretes minimal amounts of proteolytic enzymes.For example, strain W3110 can be modified to introduce genetic mutations in genes encoding polypeptides endogenous to the host; examples of such hosts include E. coli W3110 strain 1A2, which has the complete tonA genotype; E. coli W3110 strain 9E4, which has the complete tonA ptr3 genotype; E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT kan'; E. coli W3110 strain 37D6, which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT rbs7 ilvG kan'; E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin-resistant degP deletion mutation; and E. coli strains with mutant periplasmic proteases. Alternatively, in vitro methods of cloning, such as PCR or other nucleic acid polymerase reactions, are suitable.

[0206] In these prokaryotic hosts, expression vectors can be made that typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, there are several well-known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or a promoter system derived from phage lambda. These promoters typically contain ribosome binding site sequences and the like to control expression, optionally with an operator sequence, and to initiate and complete transcription and translation.

[0207] Eukaryotic microorganisms can be used for expression. Eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for polypeptide-encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe; Kluyveromyces hosts, such as Kluyveromyces lactis (MW98-8C, CBS683, CBS4574), Kluyveromyces fragilis (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces wickeramii (ATCC 24,178), Kluyveromyces waltii (ATCC 56,500), and Kluyveromyces drosophilarum (ATCC 16,045). 36,906, Kluyveromyces thermotolerans and Kluyveromyces marxianus; Yarrowia (EP 402,226); Pichia pastoris; Candida; Trichoderma reesia; Neurospora crassa; Schwanniomyces, e.g., Schwanniomyces occidentalis. occidentalis); and filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.Methylotrophic yeasts are suitable herein, including, but not limited to, yeasts capable of growth on methanol selected from the genera Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. Saccharomyces is a preferred yeast host, and suitable vectors optionally contain expression control sequences (e.g., promoters), origins of replication, termination sequences, etc. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization, among others.

[0208] In addition to microorganisms, mammalian tissue cell cultures can also be used, and in some instances are preferred, to express and produce the polypeptides described herein (see Winnacker, From Genes to Clones, VCH Publishers, NY, NY (1987)). For some embodiments, eukaryotic cells may be preferred, as several suitable host cell lines capable of secreting heterologous polypeptides (e.g., intact immunoglobulins) have been developed in the art, including CHO cell lines, various Cos cell lines, HeLa cells, preferably myeloma cell lines, or transformed B cells or hybridomas. In some embodiments, the mammalian host cells are CHO cells.

[0209] In some embodiments, the host cell is a vertebrate host cell. Examples of useful mammalian host cell lines are SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO or CHO-DP-12 lines); mouse Sertoli cells; monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT) 060562, ATCC CCL51); TRI cells; MRC5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0210] V. Illustrative Embodiments In some embodiments, the present invention provides:

[0211] 1. A method of purifying a chromatography material for reuse, comprising the steps of: a) passing at least two material volumes of elution buffer through the material, wherein the elution buffer comprises about 0.15 M acetic acid and has a pH of about 2.9; b) statically holding the material in the elution buffer for a time ranging from about 10 minutes to about 30 minutes; c) passing at least two material volumes of elution buffer through the material; and d) passing at least two material volumes of regeneration buffer through the material, wherein the regeneration buffer comprises about 0.1 N NaOH and has a pH of about 13.

[0212] 2. A method of purifying a chromatography material for reuse, comprising the steps of: a) passing about 2 material volumes of elution buffer through the material, the elution buffer comprising about 0.15 M acetic acid and having a pH of about 2.9; b) statically holding the material in the elution buffer for about 30 minutes; c) passing about 2 material volumes of elution buffer through the material; and d) passing about 4 material volumes of regeneration buffer through the material, the regeneration buffer comprising about 0.1 N NaOH and having a pH of about 13.

[0213] 3. A method of purifying a chromatography material for reuse, comprising the steps of: a) passing about 2 material volumes of elution buffer through the material, the elution buffer comprising about 0.15 M acetic acid and having a pH of about 2.9; b) statically holding the material in the elution buffer for about 30 minutes; c) passing about 2 material volumes of elution buffer through the material; and d) passing about 2.5 material volumes of regeneration buffer through the material, the regeneration buffer comprising about 0.1 N NaOH and having a pH of about 13; e) statically holding the material in the regeneration buffer for about 30 minutes; f) passing about 2.5 material volumes of regeneration buffer through the material.

[0214] 4. A method of purifying a chromatographic material for reuse, comprising the steps of: a) passing about 2 material volumes of an equilibration buffer through the material, the equilibration buffer comprising about 25 mM Tris and about 25 mM NaCl, and having a pH of about 7.1; b) statically holding the material in the equilibration buffer for about 30 minutes; c) passing about 2 material volumes of the equilibration buffer through the material; d) passing about 2 material volumes of an elution buffer through the material, the elution buffer comprising about 0.5 mM Tris and about 25 mM NaCl, and having a pH of about 7.1. e) statically holding the material in the elution buffer for about 30 minutes; f) passing about 2 material volumes of the elution buffer through the material; g) passing about 2 material volumes of the regeneration buffer through the material, the regeneration buffer comprising 0.1 N NaOH and having a pH of 13; h) statically holding the material in the regeneration buffer for about 30 minutes; i) passing about 2 material volumes of the regeneration buffer through the material.

[0215] 5. A method of purifying a chromatography material for reuse, comprising the steps of: a) passing about 4 material volumes of equilibration buffer through the material, the re-equilibration buffer comprising about 25 mM Tris and about 25 mM NaCl and having a pH of 7.1; b) six cycles of: i) passing about 3 material volumes of elution buffer through the material, wherein the elution buffer comprises about 0.15 M acetic acid and has a pH of about 2.8; ii) statically holding the material in elution buffer for about 10 minutes; iii) passing about 1 material volume of elution buffer through the material; iv) passing about 3 material volumes of regeneration buffer through the material, wherein the regeneration buffer comprises about 0.1 N NaOH and has a pH of about 13; v) statically holding the material in regeneration buffer for about 10 minutes; and vi) passing about 1 material volume of regeneration buffer through the material. A method comprising the step of carrying out a step comprising:

[0216] 6. A method of purifying a chromatography material for reuse, comprising six cycles of: a) passing about 3 material volumes of elution buffer through the material, the elution buffer comprising about 0.15 M acetic acid and about pH 2.8; b) statically holding the material in elution buffer for about 15 minutes; c) passing about 1 material volume of elution buffer through the material; d) passing about 3 material volumes of regeneration buffer through the material, the regeneration buffer comprising about 0.1 N NaOH and about pH 2.8. e) statically holding the material in a regeneration buffer for about 15 minutes; f) passing about 1 material volume of regeneration buffer through the material; g) passing about 3 material volumes of storage buffer through the material, wherein the storage buffer comprises about 100 mM sodium acetate, about 2% benzyl alcohol, and has a pH of about 5.0; e) statically holding the material in a storage buffer for about 15 minutes; f) passing about 1 material volume of storage buffer through the material.

[0217] 7. The method of any one of embodiments 1 to 6, wherein the chromatographic material is present in a chromatographic column.

[0218] 8. The method of any one of embodiments 1 to 7, wherein the chromatographic material is an affinity material.

[0219] 9. The method of embodiment 8, wherein the affinity material is a Protein A affinity material.

[0220] 10. The method of embodiment 9, wherein said Protein A affinity material is a MAbSelect material, a MAbSelect SuRe material, or a MAbSelect SuRe LX material.

[0221] 11. The method of any one of embodiments 1 to 10, wherein the chromatography material is used for large-scale production of polypeptides.

[0222] 12. A method of purifying a chromatography material for reuse, comprising the steps of: a) passing about 3 material volumes of an equilibration buffer through the material, the equilibration buffer comprising about 40 mM sodium acetate and about pH 5.5; b) passing about 2 material volumes of about 0.5 N NaOH through the material; c) statically holding the material in about 0.5 N NaOH for about 10 minutes; d) passing about 1 material volume of about 0.5 N NaOH through the material; and e) statically holding the material in about 0.5 N NaOH for about 10 minutes; f) passing about 1 material volume of about 0.5 N NaOH through the material.

[0223] 13. The method of embodiment 12, wherein the chromatographic material is present in a chromatographic column.

[0224] 14. The method of embodiment 12 or 13, wherein the chromatographic material is an ion exchange material.

[0225] 15. The method of embodiment 14, wherein the ion exchange material is a cation exchange material.

[0226] 16. The method of embodiment 15, wherein the cation exchange material is a POROS HS50 material.

[0227] 17. The method of any one of embodiments 12 to 16, wherein the chromatographic material is used for large-scale production of antibodies.

[0228] 18. A method of purifying a chromatography material for reuse, comprising the steps of: a) passing about 3 material volumes of an equilibration buffer through the material, the equilibration buffer comprising about 50 mM Tris, 85 mM sodium acetate, and having a pH of about 8.8 and about 8.6 mS / cm; b) passing about 2 material volumes of about 0.5 N NaOH through the material; c) statically holding the material in about 0.5 N NaOH for about 10 minutes; d) passing about 1 material volume of about 0.5 N NaOH through the material; and e) statically holding the material in about 0.5 N NaOH for about 10 minutes; f) passing about 1 material volume of about 0.5 N NaOH through the material.

[0229] 19. The method of embodiment 18, wherein the chromatographic material is present in a chromatographic column.

[0230] 20. The method of embodiment 18 or 19, wherein the chromatographic material is an ion exchange material.

[0231] 21. The method of embodiment 20, wherein the ion exchange material is an anion exchange material.

[0232] 22. The method of embodiment 21, wherein the anion exchange material is a QSFF material.

[0233] 23. The method of any one of embodiments 18 to 22, wherein the chromatographic material is used for large-scale production of antibodies.

[0234] 24. The method of any one of embodiments 1 to 23, wherein the buffer is passed through the material at about 30 material volumes per hour, about 20 material volumes per hour, or about 15 material volumes per hour.

[0235] 25. The method of any one of embodiments 1 to 24, wherein the buffer passes through the material in a downflow or upflow direction.

[0236] 26. The method of any one of embodiments 1 to 25, wherein the purification of the chromatographic material is measured by performing a mock elution after purifying the chromatographic material.

[0237] 27. The method of embodiment 26, wherein the eluent of the mock elution contains one or more of: <0.25 mg / mL total protein, <1 ppm IgG fragments, <1 ppm leached Protein A, <1 μg / mL CZE LIF, <1 ppm CHOP, and <1 pg / mL CHO DNA, which are indicators of effective purification of the material for multi-product use.

[0238] 28. The method of any one of embodiments 1 to 27, wherein the chromatographic material is stable in alkali.

[0239] 29. The method of any one of embodiments 1 to 28, wherein the chromatography material is used to purify a polypeptide.

[0240] 30. The method of any one of embodiments 1 to 29, wherein the chromatography material is purified after purification of the first polypeptide, and after purification, the chromatography material is used to purify the second polypeptide.

[0241] 31. The method of embodiment 30, wherein the polypeptide is an antibody or immunoadhesin.

[0242] 32. The method of embodiment 31, wherein the polypeptide is an immunoadhesin.

[0243] 33. The method of embodiment 31, wherein the polypeptide is an antibody.

[0244] 34. The method of embodiment 33, wherein the antibody is a monoclonal antibody.

[0245] 35. The method of embodiment 34, wherein the monoclonal antibody is a chimeric, humanized, or human antibody.

[0246] 36. The method of embodiment 35, wherein the monoclonal antibody is an IgG monoclonal antibody.

[0247] 37. The method of embodiment 36, wherein the antibody is an antigen-binding fragment.

[0248] 38. The method of embodiment 37, wherein the antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFv, a di-scFv, a bi-scFv, a tandem (di, tri)-scFv, an Fv, an sdAb, a trifunctional antibody, a BiTE, a diabody, or a triabody.

[0249] 39. The method of embodiment 38, wherein the polypeptide is an enzyme, hormone, fusion protein, Fc-containing protein, immunoconjugate, cytokine or interleukin.

[0250] 40. The method of embodiment 30, wherein the first polypeptide is a first antibody or a first immunoadhesin, and the second polypeptide is a second antibody or a second immunoadhesin.

[0251] All of the features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0252] Further details of the present invention are provided by the following non-limiting examples, the disclosures of all references herein being expressly incorporated herein by reference. [Example]

[0253] The following examples are intended to be purely illustrative of the present invention and therefore should not be construed as limiting the present invention in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.

[0254] Example 1 Protein carryover This example describes an attempt to quantify protein carryover from sample to sample. A pre-cleanup test run was performed at laboratory scale using standard Protein A affinity material (0.66 x 20 cm). This run was designated a "mock run" because the process followed standard purification procedures, except that the load cycle was simulated with equilibration buffer. As with a typical Protein A process, the elution pool was collected and analyzed to determine the presence of protein. This analysis revealed that 20-30 ppm of protein carryover was present in the "mock elution" in the absence of any further column purification. Results were confirmed with a second run.

[0255] To determine safe carryover levels, a risk assessment was performed to determine acceptable immunoglobulin (IgG) and protein carryover levels in the mAb and to calculate the substance-specific Accepted Daily Exposure (ADE) for IgG. Comparison of the ADE with the EDI yields a "worst case" x-fold safety margin (see, e.g., OCTAGAM®; Product Approval Information Summary Basis of Approval OCTAGAM® 5%. OCTAPHARMA Pharmazeutika: Vienna, Austria. August 2002, available on the World Wide Web at fda.gov / downloads / BiologicsBloodVaccines / BloodBloodProducts / ApprovedProducts / LicensedProductsBLAs / FractionatedPlasmaProducts / ucm064955.pdf, accessed August 7, 2012). This "worst case" safety margin is the highest value of IgG carryover from the previous sample, which is set at 10 μg mAb A / ml mAb B or 1000 ppm. where mAb A is the carryover mAb and mAb B is the desired mAb of interest (Teschner, W. et al., Vox Sang. 2007, 92:42-55; Food and Drug Administration, HHS. Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers. Rockville, MD. July 2005, on the World Wide Web, accessed August 7, 2012, google.com / url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CE8QFjAA&url=http%3A%2F%2Fwww.fda.gov%2Fdownloads%2FDrugs%2F...%2FGuidances%2FUCM078932.European Medicines Agency. Impurities: Residual Solvents, Note for Guidance on Impurities: Residual Solvents (CPMP / ICH / 283 / 95). London, UK, September 1997, on the World Wide Web at ema.europa.eu / ema / index.jsp?curl=pages / regulation / general / general_content_000431.jsp&mid=WC0b01ac0580029593, accessed August 7, 2012; OCTAGAM®; Product Approval Information Summary Basis of Approval OCTAGAM® 5%. OCTAPHARMA Pharmazeutika: Vienna, Austria. August 2002, on the World Wide Web at fda.gov / downloads / BiologicsBloodVaccines / BloodBloodProducts / ApprovedProducts / LicensedProductsBLAs / FractionatedPlasmaProducts / ucm064955.pdf, accessed August 7, 2012.

[0256] Materials and Methods equipment AKTA Explorer 100 System: A standard AKTA Explorer 100 chromatography system from GE Healthcare (Uppsala, Sweden) was used for the experiments. A 0.66 cm diameter x 20 cm bed height column (Omnifit) packed with MabSelect™ SuRe (GE Healthcare) Protein A media was used for system evaluation. The system was controlled using UNICORN software (v5.11). Affinity Resin: MabSelect™ SuRe resin (GE Healthcare, Uppsala, Sweden) was used as the resin of choice in this project because it is composed of a rigid high-flow agarose matrix and an alkali-stabilized Protein A-derived ligand. This ligand offers greater stability than traditional Protein A-based media under the alkaline conditions used in clean-in-place (CIP) protocols. Clean-up can be performed using cost-effective reagents such as sodium hydroxide, which can aid in process economy.

[0257] Standard purification procedure ("mock elution"). Protein A cycles were performed using the following parameters: (a) MabSelect™ SuRe resin with a load capacity of 30 g / L resin, (b) harvested cell culture fluid (HCCF) was loaded onto a 0.66 x 20 cm column at 15°C (12-18°C) (all other phases at room temperature), (c) the pH of the pool was adjusted to pH 5.0 by the addition of 1.5 M Tris base. The buffer used was similar to that used in the batch process. The column was equilibrated with 25 mM Tris and 25 mM sodium chloride, washed with 0.4 M potassium phosphate, eluted with 0.1 N acetic acid (pH 2.9), and regenerated with 0.1 N sodium hydroxide for MabSelect™ SuRe (Fahrner, R.L. et al., Biotechnol. Genet. Eng. Rev. 2001, 18:301-327; Fahrner, R.L. et al., Biotechnol. Appl. Biochem. 1999, 30:121-128; B. Kelley, Biotechnol. Prog. 2007, 23:995-1008; Trexlar-Schmidt, M. et al., Biopharm. Intl. March 2, 2009).

[0258] buffer The following buffers were used: Elution buffer: 0.15 M acetic acid, pH 2.9 Renaturation buffer: 0.1 M NaOH, pH 13 Equilibration buffer: 25 mM Tris, 25 mM NaCl, pH 7.1 Storage buffer: 100 mM sodium acetate, 2% benzyl alcohol, pH 5.0

[0259] Purification strategy. The sanitization procedure is carried out at a flow rate of 20 CV / hr. The sanitization procedure was developed based on two factors: (a) pH cycling and (b) static hold time.

[0260] The pre-cleaning carryover results (20-30 ppm) obtained in a "mock run" without further purification were well below the established safety limit of 1000 ppm set by the risk assessment. However, because the limit for clinical manufacturing is lower than 1000 ppm, we decided to err on the side of caution. The goal of this project was to develop a purification procedure that could be transferred to clinical manufacturing; therefore, we identified a purification procedure to minimize carryover to less than 1 ppm. After careful optimization, the optimal purification strategy was based on (a) static hold and (b) pH cycling. The addition of a static hold step in the purification process allowed for additional residence time without the use of additional buffers. The increased residence time likely aided mass transfer, effectively extracting any remaining protein on the column into the buffer. Alternating between acidic and basic buffers, known as pH cycling, enhanced protein extraction and thus efficiently cleaned the column. The optimal purification conditions included the buffers already used as the elution and regeneration buffers. The "elution buffer" was 0.15 M AcOH (pH 2.9), and the "regeneration buffer" for purification was 0.1 N NaOH (pH 13). The choice of buffers was based on their respective properties. For example, the "elution buffer" (0.15 M AcOH, pH 2.9) was used to wash bound IgG from the Protein A resin. Sodium hydroxide solubilizes proteins and nucleic acids (all components of the production process) by denaturing and cleaving proteins into small fragments. Additionally, sodium hydroxide destroys endotoxins and regenerates the resin. Since none of these conditions was incompatible with the resin and was not already used for in-house mAb purification, their use was also economical.

[0261] Resin Selection MabSelect™ SuRe Protein A affinity resin was chosen for optimization because it has a large working pH range (pH 3-12), is stable under basic conditions without loss of binding capacity, and was therefore compatible with the current elution (0.15 M AcOH) and regeneration (0.1 N NaOH) buffers for purification.

[0262] Other resins, such as ProSep® vA, have been previously investigated. Briefly, several different cleaning agents were investigated to purify the ProSep® vA column. However, the majority of conditions showed similar performance. Screening of various buffers followed by sequential "mock runs," as outlined in the standard purification procedure, resulted in reduced protein carryover from sample to sample. Increasing elution flow rates also efficiently purified the column. A key finding of this study was that static hold and pH cycling contributed significantly more to the reduction of protein carryover compared to the other variables tested. While some of the purification procedures reduced protein carryover on ProSep® vA, this reduction was not sufficient to warrant its use at pilot or larger scales. Nevertheless, the results from the pH cycling and static hold experiments proved useful in optimizing the purification procedure with MabSelect™ SuRe resin.

[0263] Analysis method The antibody concentration of HCCF was determined using an Agilent 1100 HPLC (Agilent Technologies, Santa Clara, CA) with a 2.1 x 30 cm POROS column (Applied Biosystems, Foster City, CA). Buffer A (100 mM sodium phosphate, 250 mM sodium chloride, pH 6.3), buffer B (2% acetic acid, 100 mM glycine), and buffer C (0.1 M phosphate, 20% ACE (20% acetonitrile)) were used, with a total run time of 4.5 min. Protein concentration in the purified pool was measured at 280 nm using an Agilent 8453 (Agilent Technologies, Santa Clara, CA) spectrophotometer. Multi-product enzyme-linked immunosorbent assay (ELISA) was used for analysis of CHOP and leached ProA. TaqMan polymerase chain reaction was used for CHO DNA analysis. Total protein was measured using a capillary zone electrophoresis / laser-induced fluorescence detection (CZE-LIF) assay. Intact and fragmented antibody moieties were measured using a general ELISA assay. SDS / PAGE was performed on 18% Tris-HCl gels.

[0264] Quantifying protein carryover without purification The experimental protocol for determining mAb carryover is as follows: First, 18 load cycles of mAb were loaded onto a Protein A affinity column (0.66 × 20 cm, volume = 6.8 mL) at 30 g / L, and the sample was eluted. The Protein A affinity column was subsequently cleaned according to one of the column cleanup procedures outlined in Table 2. After cleanup, a "mock run" was performed. To determine the level of protein or impurity carryover, analytical samples were taken at specific time points during the "mock run" or during the column wash procedure. The collected analytical samples were adjusted to pH 5-5.5 (1.5 M Tris-base buffer) and then treated with detergent (0.1% polysorbate, 0.05% sodium azide) to prevent protein surface adhesion, which would result in false-negative results.

[0265] In the first experiment, carryover in the elution pool was first determined for three different mAbs (mAbA, mAbB, and mAbC) sequentially purified on a MabSelect™ SuRe Protein A column without intermittent purification. Three purification cycles were loaded onto a Protein A affinity column (0.66 × 20 cm, volume = 6.8 mL) at 30 g / L, and the results are shown in (Figure 1). The data are graphed as the amount of intact IgG protein carried over from the previous run (ng carryover / mg product) as a function of elution. According to the graph without intermittent purification, the highest carryover of the three loading cycles was 30-40 ppm (Figure 1). These results clearly indicated that additional purification cycles were required to recycle the column to keep protein carryover below 1 ppm.

[0266] Optimization of the MabSelect™ SuRe Purification Procedure (CP) In an attempt to simplify the purification procedure by reducing buffer consumption and purification time, different combinations of buffer and run time were investigated (Table 2, entries 1-3). Because carryover levels never dropped below the 1 ppm limit, it was clear that a more stringent purification procedure needed to be identified for lab-scale recycle on the MabSelect™ SuRe column. More stringent purification conditions included the addition of a static hold, in which the column was held in buffer for a specified period and run at zero flow (Table 2, entries 4-5). It was found that static hold efficiently washed more protein off the resin than buffer flushing. Static hold effectively increased the amount of intact IgG washed off the column after the elution buffer static hold by 5-fold, and intact IgG was not detected after the regeneration buffer static hold (Figure 2). Furthermore, the amount of carryover is significantly reduced in the "mock elution" to less than 10 ppm intact IgG for entry 4 (Table 2), and less than 1 ppm intact IgG is carried over for entry 5 (Table 2). TIFF2025186245000003.tif255170TIFF2025186245000004.tif30170

[0267] Increasing the number of purifications with static hold time was investigated in entries 6-7 (Table 2). Clearly, static hold time with additional purification cycles purified the resin more efficiently than all other previously investigated conditions. As a result, after performing a "mock run," carryover of less than 3 ppm for entry 6 (Table 2) and less than 0.3 ppm for entry 7 (Table 2) was detected (Table 2, Figure 3). Increasing the pH cycles eluted more protein during sharp pH transitions. However, aggregation time in 0.1 N NaOH was increased by increasing the number of cycles, which, along with a long static hold, may be detrimental to resin binding capacity. Since the majority of the protein eluted after the first cycle for both the 30- and 10-minute static hold times (Figure 3), additional static hold time had limited additional benefit. However, shorter static hold times with increased cycles were preferable to longer hold times. In addition to the ELISA assay, capillary electrophoresis-sodium dodecyl sulfate analysis (CE-SDS) was performed to confirm fragment clearance through the purification cycles. CE-SDS analysis of the "mock elution" after MabSelect™ SuRe column cleanup using the procedure outlined in entry 7 (Table 2) for the 94 ng / mL "mock elution" sample revealed that the isolated mAb was >90% completely intact (Figure 4).

[0268] As a "proof of concept" using the purification procedure of entry 7 (Table 2), a "mock elution" Äkta chromatogram (generated during the purification run) suggested that efficient purification of the column was achieved when a shift from the elution buffer (0.15 M acetic acid) to the regeneration buffer (0.1 N NaOH) was performed. This was evident by a series of spikes in UV intensity during this pH cycling for each of the six cycles (Figure 5). Taken together, pH cycling and static hold provide an ideal purification procedure.

[0269] Scaling of the lab-scale optimized cleanup procedure (entry 7, Table 2) for purification of mAbA on a MabSelect™ SuRe Omnifit column (0.66 × 20 cm, volume = 6.7 mL, 18 cycles of HCCF at 30 g / L) indeed minimized protein carryover (Figure 6). These results clearly demonstrate that after cleaning the column using six cycles of elution buffer (0.15 M acetic acid), significantly less intact IgG or Fc fragments were detected after each cycle, resulting in less than 5 ppm by cycle 6 (Figure 6). Similarly, even less intact IgG and Fc fragments (<10 ppm) were detected after each of six wash cycles with regeneration buffer (0.1 M NaOH, Figure 6). Furthermore, by the time a "mock elution" was performed (after pre-elution, pre-regeneration, and pre-equilibration), less than 1 ppm of protein carryover was detected in the "mock elution" sample (Figure 6).

[0270] During the course of optimizing the purification procedure, a small amount of protein came off the column after a period of storage in the storage buffer (100 mM sodium acetate, 2% benzyl alcohol, pH 5.0). This observation suggested that perhaps the storage buffer could also function as an efficient purification buffer for the MabSelect™ SuRe resin. However, a subsequent "mock run" after intermittent column purification with the storage buffer resulted in less efficient column purification than the previously optimized conditions (entry 8, Table 2; Figure 7). Furthermore, the addition of this purification buffer in the process made the overall process longer without any additional benefit. Therefore, it was decided to proceed with the existing optimized purification procedure.

[0271] The optimized purification procedure (entry 7, Table 2) was then carried out at pilot scale (14 x 20, volume = 3.23 L) for the purification of mAbZ as a final test before scaling up this procedure to the mAb of interest. The results were promising, as expected, with decreasing intact IgG and Fc detected after each purification, resulting in less than 1 ppm protein carryover detected in the "mock elution" of the "mock run." This particular pilot run was performed with mAbZ on a MabSelect™ SuRe column previously used for nine purification cycles (Figure 8).

[0272] This purification procedure was so effective that a total of five pilot-scale columns were subsequently purified. Measurements of leached protein A (leached protein A assay) (Zhu-Shimoni, J. et al., J Immunol. Methods. 2009, 341:59-67), other proteins (CZE LIF-total protein assay; D. Michels, (in preparation)), Chinese hamster ovary protein (CHOP assay; Fahrner, R.L. et al., Biotechnol. Appl. Biochem. 1999, 30:121-128), and DNA (CHO DNA assay; TaqMan polymerase chain reaction used for CHO DNA analysis), antibody Fc fragments (human Fc ELISA; intact and fragmented antibody moieties were measured using a common in-house developed sandwich ELISA), and total antibody (intact human IgG). Further analysis of all these pilot-scale samples to determine the amount of other impurities, such as by ELISA (intact antibody and fragmented antibody moieties were measured using a common in-house developed sandwich ELISA), was also performed to verify that the process performed similarly at pilot scale (Table 3). As expected, all detected impurities were well below the accepted limits, and this cleanup procedure can be used for mAb purification (entry 6, Table 3). TIFF2025186245000005.tif63170

[0273] result The previously optimized extended purification conditions (entry 7, Table 2) were slightly modified for further study, incorporating a 15-minute static hold time instead of a 10-minute static hold time (Figure 8). The entire resin purification process took 4.5 hours at a flow rate of 20 column volumes (CV) / hour and was carried out over six cycles (six times). These conditions included pH cycling and static holds between the elution buffer and regeneration buffer to efficiently wash the column. Briefly, this procedure is detailed in Figure 9. The entire process was carried out for a total of six cycles to thoroughly purify the resin. Finally, the resin was washed with equilibration buffer (3 CV) and then stored in storage buffer (5 CV, storage buffer). To effectively monitor resin purification, samples were collected after the 15-minute hold time to analyze carryover in each cycle and determine how much protein was removed from the resin at each step and each cycle (Figure 9).

[0274] After purging the resin, a "mock run" was performed to verify protein carryover (Figure 9). This "mock elution" was collected and assayed to determine the amount of carryover and the presence of other impurities (Zhu-Shimoni, J. et al., J Immunol. Methods. 2009, 341:59-67; Fahrner, R.L. et al., Biotechnol. Appl. Biochem. 1999, 30:121-128). Total protein was measured using a capillary zone electrophoresis / laser-induced fluorescence (CZE-LIF) assay (D. Michaels et al., in press). TaqMan polymerase chain reaction was used for CHO DNA analysis. Intact antibody and fragmented antibody moieties were measured using a standard sandwich ELISA. These other impurities include host cell components, proteins, viruses, or DNA. These assays include testing for intact human immunoglobulin (IgG) using an ELISA, testing for human Fc fragments in another ELISA, testing for any other protein using a capillary zone electrophoresis / laser-induced fluorescence detection assay (CZE / LIF), testing for Chinese hamster ovary protein in a CHOP assay (Fahrner, R.L. et al., Biotechnol. Appl. Biochem. 1999, 30:121-128), and leached Protein A assays (Zhu-Shimoni, J. et al., J. Immunol. Methods. 2009, 341:59-67). The "intact human IgG ELISA" and "human Fc ELISA" quantify the amount of whole antibody or antibody fragment on the column; the former binds both the fragment antigen-binding region (Fab) and the fragment crystallizable (Fc) region, while the latter binds only the human Fc region. The CZE-LIF assay can confirm these results by quantifying the total amount of protein in the sample.Finally, it is known that Protein A can leach from the resin during run-through or harsh purification, negatively affecting binding capacity, and therefore it is important to determine the amount of leached Protein A (Fahrner, R.L. et al., Biotechnol. Appl. Biochem. 1999, 30:121-128; Kelley, B., Biotechnol. Prog. 2007, 23995-1008; D. Michaels, in press; Fahrner, R.L. et al., Biotechnol. Genet. Eng. Rev. 2001, 18:301-327).

[0275] To test the efficiency of the purification procedure, the mAb of interest, mAbC, was purified on a MabSelect™ SuRe column (0.66 × 20 cm, volume = 6.8 mL) on a laboratory scale using an AKTA Explorer 100 (as described in 2.2). Protein carryover was measured during and after several purification cycles, and the results demonstrated that protein carryover decreased after each purification cycle (Figure 10). In a subsequent "mock run," the intact IgG protein and Fc fragment carryover of mAbC decreased from 25 ng / mg intact IgG and 35 ng / mg Fc fragment in the first cycle with elution buffer to less than 5 ng / mg for both after the sixth cycle with elution buffer (Figure 10). During and after regeneration (after elution), significantly more intact IgG and Fc fragment were washed off the column until the sixth cycle, at which point the levels cumulatively decreased to less than 5 ng / mg carryover. To test for carryover, a "mock run" (a run after the entire purification cycle) was performed in which additional IgG and Fc fragments were washed off the column in the pre-regeneration; by the time the "mock elution" process began (when the second mAb was expected to come off in the reuse process), less than 1 ppm of IgG and Fc fragments were detected (Figure 10). Taken together, these results confirm that these conditions are valid purification conditions, and that the total amount of protein carried over from a previous run is less than 1 ppm when the resin is reused after using this purification procedure.

[0276] To get a better idea of ​​what types of protein fragments were present per cycle wash, samples from each cycle were run on a 10% Tris-HCl gel (mAbC) (Figure 11) (Trexlar-Schmidt, M. et al., Biopharm. Intl. March 2, 2009). From cycle 1 to cycle 6, less protein was observed in each successive cycle (decreasing band intensity in each lane, Figure 11). Furthermore, the lane containing the sample from the static hold cycle was more concentrated than between cycles 1-6, and more protein was removed after each static hold cycle. These results demonstrate that an extended residence time helps remove residual protein from the column.

[0277] This optimized purification process was extended to the purification of mAbX at pilot scale on a MabSelect™ SuRe column (13.8 x 20, volume = 3 L). The results were similar to those previously seen at laboratory scale (Figure 6). As seen in the laboratory-scale run, protein impurities are removed from the resin in the initial purification step, and their overall concentration decreases after each elution and regeneration cycle until the sixth cycle is reached (Figure 12). During resin regeneration, the amount of protein initially washed off the column is significantly higher than after the sixth cycle, such that by the sixth regeneration cycle, less than 1 ppm of protein impurities are detected (Figure 12).

[0278] Purification of mAbY on a MabSelect™ SuRe column (20 x 20, volume = 6.28 L) and subsequent column cleanup using the above cleanup protocol (Figure 9), followed by a "mock run," resulted in less than 1 ppm leached Protein A, less than 0.25 mg / mL (limit of quantification) CZE-LIF, less than 0.5 ppm CHOP, and less than 1.0 pg / mL CHO DNA after the sixth regeneration cycle in the mock elution (Table 4). All impurities were comparable to historical data and within acceptable limits. TIFF2025186245000006.tif78170

[0279] In a final test of the robustness of the optimized purification protocol (Figure 9), the mAb of interest was purified using 6.28 L of MabSelect™ SuRe resin that had previously undergone 153 multiproduct load cycles. Carryover from the previously used resin during a "mock run" (entry 1, Table 5) was compared to the carryover observed from three other different MabSelect™ SuRe resins. These results are summarized in Table 5. Briefly, the older MabSelect™ SuRe large-scale multiproduct resin (entries 1 and 2, Table 5) performed comparably to the new mAb-specific (non-multiproduct) MabSelect™ SuRe resin (entry 3, Table 5) and the new mAb-specific lab-scale MabSelect™ SuRe resin (entry 4, Table 5). According to these results, all Protein A resins provided mAb in greater than 90% yield with comparable CHOP, percent aggregates, and leached Protein A (ng / mg). In summary, the multi-product resin has no negative impact on product impurity profile or process yield. Pilot-scale and laboratory-scale results are also comparable (entry 4, Table 5).

[0280] In addition to the work presented, several more mAbs have been purified on multi-product resins purified using this procedure. The results were all very similar and reproducible, with total protein levels below 0.25 ppm (assay detection limit) (Table 6). These results suggest that the optimized purification procedure is an efficient, reproducible, and robust method for purifying, regenerating, reusing, and recycling multi-product MabSelect™ SuRe Protein A resins. Use of the MSSCCP purification procedure for intermittent Protein A resin purification reduces protein carryover to well below established safety margins. TIFF2025186245000007.tif109170

[0281] A highly effective MabSelect™ SuRe purification method has been developed that allows MabSelect™ SuRe Protein A resin to be used for multi-product purification without impact on product purity and / or loss of resin binding capacity. Data from laboratory and pilot-scale experiments suggest that a purification protocol involving six cycles of 0.15 M acetic acid (elution buffer) and 0.1 N sodium hydroxide (regeneration buffer) washes and a 15-minute hold time purifies MabSelect™ SuRe resin to less than 5 ppm protein carryover in this first mAb purification step. This process was successfully carried out on a multi-product Protein A resin (MabSelect™ SuRe) at pilot scale, lending further credence to the utility of this strategy. TIFF2025186245000008.tif63170

[0282] Example 2 Evaluation of ion-exchange columns for multiproduct use. A study was conducted to determine whether a similar cleanup process could be developed for ion exchange chromatography. MAbA and MAbB from the ProA pool were loaded onto a cation exchange column (POROS) or an anion exchange column (QSFF). After normal elution, the columns were subjected to a "mock elution." Fractions were analyzed for the presence of MAbA and / or MabB using the MabSelectSure assay, with a detection limit of 0.82 ng / mL. As can be seen in Figure 13, the mock elution results indicated the need for further cleanup of these columns.

[0283] The following clean-in-place (CIP) procedures were tested: I. 3 CV of equilibration buffer II. 2 CV of 0.5N NaOH 10 minutes static hold III. 1 CV of 0.5 N NaOH 10 minutes static hold IV. 1 CV of 0.5N NaOH V. Post-cleanup mock run

[0284] The samples were conditioned with a low concentration of detergent (0.1% polysorbate 20, 0.05% sodium azide) to prevent the sample from sticking to the container walls. The samples were adjusted to a neutral pH before loading onto the columns. MAbA and MAbB were loaded onto a cation exchange column (POROS) or an anion exchange column (QSFF). After normal elution, the columns were cleaned using the protocol described above. A second set of columns was loaded with MAbA or MAbB, but after elution, these columns were not cleaned using the protocol described above. All columns were then subjected to a "mock elution." The mock eluate was analyzed for the presence of intact IgG and analyzed by ELISA.

[0285] As shown in Figure 14, this purification method significantly reduced protein carryover of MAbA on the POROS column (Panel A) and significantly reduced protein carryover of MAbB on the QSFF column (Panel B). Almost no MAbA carryover was observed on the QSFF column, even without a CIP step (Panel A), and almost no MAbB carryover was observed on the POROS column, even without a CIP step (Panel B).

[0286] A third antibody, MAbC, was applied to a POROS column and a QSFF column, and the amount of intact IgG eluted from the column at the end of selected steps in the cleanup protocol was measured (Figure 15), showing that by the end of the CIP cycle, the amount of protein carryover was less than 0.1 ppm. Protein carryover in the mock elution pool was also less than 0.1 ppm.

[0287] To determine whether the cleanup protocol would be effective on large-scale ion exchange chromatography columns, the cleanup protocol was performed on pilot-scale columns previously loaded with MAbD. These columns were a 7.22 L POROS HS50 column and a 1.57 L QSFF column. After loading and elution of MAbD, the columns were mock-equilibrated without cleanup and then mock-eluted. The columns were then cleaned according to the CIP protocol described above, followed by additional mock equilibrations and mock elutions. Samples from each step were removed and analyzed for intact IgG as described above. The results are shown in Figure 16. For both the POROS HS50 column and the QSFF column, protein carryover was less than approximately 0.1 ppm.

[0288] Example 3 Evaluation of ProSep A columns for multi-product use. Different clarification solutions were evaluated on a small scale to assess which solution was most effective at reducing product carryover. Several different categories of solutions were tested, including acids, chaotropes, salts, and organic solvents. This study was designed to follow a standard Protein A antibody process to best mimic typical process conditions, although actual processing conditions may vary depending on the specific product used.

[0289] Flow was directed in a downflow direction through the column for all processes except the purification cycle. During the purification cycle, flow was directed in an upward direction through the column, hoping to create the best purification scenario. Because the feedstock is directed downward through the column during the loading cycle, the top of the Protein A column is theoretically nearly clogged. The theory is that by directing flow upward in the purification cycle, carryover and other impurities that build up at the top of the column do not have to traverse the entire column length before eluting. At the time of this study, it was unclear whether upflow was more beneficial than downflow in purifying the column. For the purposes of this section, all experiments were consistent in using upflow, so comparisons can still be made between different purification solutions despite this finding.

[0290] Materials and Methods Protein A Chromatography Processing Protein A chromatography was performed using an AKTA Explorer 100 chromatography system (Amersham Pharmacia Biotech) and Unicorn 5.10 control software (GE Healthcare). ProSep A resin was used instead of ProSep vA resin. The performance of the two resins has previously been shown to be equivalent. This resin was packed to a bed height of 14 cm in a 0.66 cm diameter Omnifit glass column. Naive resin was packed for all runs. All experiments were performed at room temperature (20-30°C). Harvested cell culture fluid (HCCF) of mAb1 and mAb2 was used. A standard Protein A antibody process was maintained with a flow rate of 30 CV / h, a loading capacity of 14 g / L resin, and pooling from 0.5 OD to a final volume of 2 CV based on UV absorbance at 280 nm.

[0291] A pre-cycle of elution and regeneration was followed by a loading cycle consisting of equilibration, antibody load, three washes, elution / pooling, and regeneration. Nine loading cycles were performed sequentially to fully clog the column. This was followed by a purification cycle consisting of regeneration, equilibration, 10 CV of the tested purifying agent, and extended regeneration. This purification cycle was performed in the upflow direction using a slower flow rate of 10 CV / h. 1 CV fractions were collected through the purifying solution block. The column was then stored before a series of mock runs (carryover cycles) were performed to assess product carryover. An integrity check consisting of a pre-cycle, a normal loading cycle, and storage followed the mock runs to ensure that protein yields were not reduced. Each experiment was performed in duplicate.

[0292] A mock run was defined as a run followed by the normal phases of each process step except for the load phase, during which no protein was loaded onto the column. Instead, phosphate-buffered saline (PBS) was loaded onto the column (mock load) to simulate the volume, pH, and conductivity of the normal load pool containing protein. A carryover sample pool (mock pool) was collected from the mock run at the same starting volume as the normal protein elution pool was collected.

[0293] The overall process flow is summarized below: Pre-cycle Elution 3CV Play 3CV Loading cycle (x9) Equilibration 4CV HCCF loading 14g / L Wash 1 3CV Wash 2 3CV Wash 3 3CV Elution / Pool 3CV Play 3CV Purification Cycle Play 7CV Equilibration 5CV Purifying agent 10CV Play 10CV Reserve 5CV Pre-cycle Mock execution Loading cycle (PBS load) Integrity Check Loading cycle (HCCF load) storage

[0294] The buffer components are shown in Table 7. TIFF2025186245000009.tif54170

[0295] The composition of the cleaning agent is shown in Table 8. TIFF2025186245000010.tif75170

[0296] Post-processing of the Protein A pool Within 3 hours of elution, the clarified fractions and carryover pool were conditioned with polysorbate 20 and sodium azide to final concentrations of 0.1% polysorbate 20 and 0.05% sodium azide. Polysorbate 20 is a detergent that prevents low levels of protein from adsorbing onto the sample container walls, and sodium azide is a preservative that prevents inhibition of bacterial growth. The Protein A pool (both protein and mock) was adjusted to pH 5.0, and the clarified fraction was adjusted to between pH 5.0 and 7.0 using 1.5 M Tris base. All samples were stored at 4°C until analyzed for product (intact IgG).

[0297] analytical theory Protein pool enrichment was determined using a UV spectrophotometer (Shimadzu) at absorbance of 280 nm. Purified and mock pool samples were submitted in either duplicate or triplicate and analyzed for product using an intact human IgG ELISA.

[0298] The results from the intact human IgG ELISA were converted to carryover values ​​using the following calculation: TIFF2025186245000011.tif17170

[0299] To represent worst case carryover, the lowest product concentration from the protein pool sample in each experiment was used in the carryover calculations (Table 9). TIFF2025186245000012.tif128170

[0300] result Carryover results from the sanitizing solution screening are summarized in Figure 17. For comparison, two runs were performed in which all of the processing steps were the same, but no sanitizing cycle was performed. All of the columns exposed to the sanitizing solution showed a significant reduction in carryover when compared to the unsanitized column. The average carryover was reduced between 65-93% compared to the column not exposed to the sanitizing cycle.

[0301] The equilibration buffer was included as a negative control for the cleanup solution because proteins typically do not elute from Protein A columns when exposed to the equilibration buffer. However, this equilibration buffer performed just as well as any of the other solutions in reducing carryover. This suggested that the additional flow-through of buffer through the column helped to remove carryover, regardless of the actual solution composition.

[0302] High run-to-run variability was observed for many of the different cleanup solutions. This variability may be due in part to the use of two different raw materials throughout the experiment. MAb1 HCCF was used for one of two runs from each of the following samples: no cleanup, equilibration buffer, 6 M guanidine HCl, 1% v / v phosphoric acid, 19% ethanol, 0.1 M imidazole / 19% ethanol; mAb2 HCCF was used for all other runs. Limited raw material availability prevented consistent raw material use throughout all of the runs. However, because the 2 M potassium phosphate sample showed high variability (56.5% RSD) despite the use of mAb2 HCCF for both runs, it is possible that inconsistent raw materials do not explain all of the variability observed.

[0303] An additional load cycle was performed after the carryover cycle of each run to evaluate the column performance after clarification. Product yields were consistent with those obtained during the load cycle before exposure to the clarification agent (results not shown).

[0304] Fractions were collected over the purification block of the purification cycle to assess the amount of antibody appearing in the purification (Figure 18). Because some of the purification agents may have had a denaturing effect on the protein, these results were used to establish trends rather than to find absolute amounts. All of the purification agents released stabilized amounts of antibody to low, nearly constant levels well within 10 CV. In contrast, carryover eluted during the mock cycle remained at significantly higher concentrations. Even with buffers such as the equilibration buffer, where proteolysis was not expected, higher levels of carryover were observed, despite the low levels of antibody released from the purification. These findings suggest that simply reducing or extending the purification duration does not significantly affect carryover levels when this purification procedure is used.

[0305] Several sequential mock runs were performed after the cleanup cycle for each solution tested. The UV 280 nm signals from five sequential mock runs of the 0.1 M acetic acid cleanup are shown overlaid on the chromatogram in Figure 19. The sharp peak in the pooling region gradually decreased with every sequential mock run. This decrease in carryover with each sequential cycle was not limited to this particular column. As can be seen in Figure 20, the same trend occurred with nearly every cleanup solution screened. Even when no cleanup was performed on the column, carryover decreased significantly with each additional mock run. In Figure 21, the 6 M guanidine HCl sample lacks data from the second carryover, but the same trend was observed. The 20% hexylene glycol sample also showed the same trend up to the fifth carryover. It remains to be determined whether the high fifth carryover reflects actual carryover or human error in sample collection.

[0306] The decrease in carryover with each successive mock run suggested that carryover could be reduced using standard Protein A buffers instead of more specialized purification agents such as those being screened. The use of existing buffers makes these purification procedures easier to perform in a purification pilot plant, as less preparation time is required for buffer metering and less uncertainty exists regarding the chemical's effect on the column.

[0307] More importantly, these results also suggested pulsing mock run buffer as an alternative purification procedure for Protein A columns. Because carryover was reduced more from sequential mock runs than from sequential exposure to 0.1 M acetic acid or 1% v / v phosphoric acid, the low pH elution and regeneration buffers during the mock runs cannot fully explain the improved purification. Instead, the transition from high to low pH during the mock runs may have been responsible for the reduced carryover.

[0308] Figure 21 shows that the 2 M arginine HCl sample did not follow the carryover reduction trend. Power to the Akta system was interrupted, and the column was held in buffer after and possibly during the third mock run. The increased carryover after extended durations of buffer exposure indicated that a single carryover result may not fully represent how much protein remains on the column. The possibility of further carryover elution presents a significant obstacle, even after initial results suggest the column has been "cleaned."

[0309] To identify a suitable purification strategy to reduce carryover on a Protein A column packed with ProSep A, 10 different purification agents were screened. All of these purification solutions helped reduce carryover; however, due to variability issues, most of these agents performed similarly. The analyzed carryover pools from sequential mock runs showed a trend of reduced carryover with each additional mock run.

[0310] Example 4 Pulsing from a high pH to a low pH buffer A study was conducted to investigate pulsing of mock running buffer as a means of reducing product carryover. After identifying a basic strategy, optimization was performed using a larger, small-scale column. Optimization parameters included analysis of flow directionality, flow rate, and static immersion.

[0311] Materials and Methods Preliminary analysis of product elution Protein A chromatography was performed according to the method described in Example 2 using mAb3 HCCF loaded at 15°C. Nine loading cycles were performed, followed by storage, pre-cycles, and two mock runs. No cleanup was performed. 1 CV fractions were collected over each mock run and conditioned with Tris base, polysorbate 20, and sodium azide, as previously described. Fractions were analyzed by intact human IgG ELISA.

[0312] pH Pulse Oscillation and Optimization All experiments were performed using mAb3 HCCF on a 1.6 cm diameter column. The bed height remained at 14 cm. Nine loading cycles were performed, followed by a purification cycle consisting of 3 CV equilibration buffer and 3 CV regeneration buffer. Ten purification cycles were performed, followed by storage, pre-cycles, and a series of mock runs. 1 CV fractions were collected throughout the purification process. Purification fractions and mock pools were conditioned and analyzed as previously described.

[0313] The default purification procedure was performed in the downflow direction at 30 CV / hr for 10 cycles. Optimization studies involved modifying the flow direction and flow rate, testing static soak conditions, and reducing the purification duration. For flow direction optimization, purification cycles were performed in the upflow direction. For flow rate optimization, purification cycles were performed at a flow rate of 15 CV / hr. Static soak was tested by holding the column in either equilibration buffer or regeneration buffer for 3 hours during the fourth purification cycle. For static soak optimization, the column was held in equilibration buffer for 3 hours during four of the ten purification cycles. For purification duration optimization, the purification cycle was reduced to 2 CV of equilibration buffer and 2 CV of regeneration buffer. Additionally, static soak in equilibration buffer was performed for five of the ten purification cycles. The optimization parameters are summarized in Table 10. TIFF2025186245000013.tif78170

[0314] result Fractions were collected over the first two mock run cycles of the clogged column to investigate when the product actually eluted during the mock run cycle. Chromatogram and carryover results are shown in Figure 22. The majority of the product eluted at the transition from high to low pH, where the equilibration buffer was replaced by the elution buffer. The product eluted to a lesser extent at the next pH drop, where the regeneration buffer replaced the elution buffer. These observations were repeated in the second carryover cycle. The carryover results confirmed that simply holding the column at low pH did not effectively clear the column. If low pH were the only requirement, the peak during the second carryover cycle would have been smaller, and more protein would have been released during the entire elution and regeneration block of the first carryover cycle.

[0315] Comparison of elution from the pH drop with further elution during the second mock run strongly supported pulsing from high to low pH as a potential purification strategy. Protein elution peaked within 3 CV of elution buffer, so buffer duration was set to 3 CV for each of the high and low pH buffers. Equilibration buffer (pH 7.1) was selected as the high pH buffer, and renaturation buffer (pH 1.7) was selected as the low pH buffer.

[0316] pH pulse vibration Product elution through equilibration and regeneration buffers for 10 pulsing cycles is shown in Figure 23. A peak of IgG elution from the column occurred with each transition from high to low pH of the purification. With each subsequent cycle, the amount of protein eluting decreased.

[0317] Several sequential mock runs were performed after pulse purification. The carryover amount from five sequential mock runs is shown in Figure 24. For comparison, the sequential carryover from the unpurified column is also shown. Ten cycles of pH pulse purification resulted in an 86% reduction in carryover from the column, from 484 ng IgG / mg product to 68 ng IgG / mg product. Although purification resulted in a significant reduction in carryover, additional mock runs still resulted in carryover. Optimization was required to further reduce carryover.

[0318] Optimizing pulsed purification Upflow and downflow of buffer through the column during the purification cycles were compared. Carryover for five sequential mock runs is shown in Figure 25. Upflow resulted in a 54% increase in initial carryover when compared to downflow (105 ng IgG / mg product vs. 68 ng IgG / mg product). All subsequent carryovers showed similar increases in carryover. Because polysorbate 20 and sodium azide were erroneously not added to the sample, carryover during the fourth mock run was negligible. Figure 26 shows product elution throughout the duration of the purification for both the upflow and downflow columns. Upflow resulted in more mock run carryover, but caused more protein to elute from the column throughout the 10 purification cycles, which purified the column more thoroughly and efficiently. Because a significant amount of protein still eluted from the column during the final purification cycle, the upflow run could arguably be extended for more cycles to remove more of the product eluting as carryover.

[0319] The effect of buffer flow rate during purification on mock run carryover levels is shown in Figure 27. Reducing the flow rate in half, from 30 CV / hr to 15 CV / hr, reduced the initial carryover by nearly 50% (from 68 ng IgG / mg product to 35 ng IgG / mg product). A slower flow rate effectively cut the carryover in half, but at the expense of doubling the purification time.

[0320] Results from static soaking of the column in buffer during the purification cycle are shown in Figure 28. The column was held in either equilibration buffer or regeneration buffer for 3 hours to assess carryover. Soaking the column in equilibration buffer was superior to soaking the column in regeneration buffer (19 ng IgG / mg product vs. 28 ng IgG / mg product). Both static soaks were a notable improvement over the 68 ng IgG / mg product from the normal pulse purification of the column. The penalty of increased purification time with reduced flow rate had to be weighed against the benefit of reduced carryover.

[0321] Static soaking of the column in buffer was further evaluated by comparing a single 3-hour hold to multiple 3-hour holds in equilibration buffer. The column was held in the static soak for 4 of 10 pulsed oscillation cycles and evaluated for carryover (FIG. 29). With multiple static soaks, carryover was reduced from 19 ng IgG / mg product to 8 ng IgG / mg product.

[0322] Fractions collected throughout the purification cycle for each optimization run were evaluated for product elution to determine the optimal purification duration (Figures 23, 26, 30-32). The majority of purification cycles showed little or no product elution during the third CV of both equilibration buffer and regeneration buffer. As a result, the duration of each purification cycle was reduced to 2 CV of equilibration buffer and 2 CV of regeneration buffer. This reduction in purification duration resulted in less buffer and shorter purification time; however, product carryover either remained the same or slightly increased. To further reduce carryover while maintaining a smaller buffer volume, the number of static soaks was increased from 4 to 5. Figure 33 shows that these changes slightly reduced carryover, from 7.8 ng IgG / mg product to 6.7 ng IgG / mg product. Product elution throughout the column purification is shown in Figure 34. By the end of the 10th purification cycle, 5.0 ng IgG / mg product eluted in the last column volume of regeneration buffer.

[0323] Example 5 Large-scale purification performance. The most promising pH pulse purification procedures identified during the optimization study were applied to a previously used pilot-scale column. Columns for this study were selected based on molecule, size, and number of previous protein contacts. Because only the Pharmacia skid allowed for automatic pause duration, columns were selected that did not exceed the skid's flow rate limit of 2 L / min.

[0324] Materials and Methods Previously used mAb4 and mAb5 Protein A columns were obtained from cold room storage at the pilot plant. The mAb4 column was 20 cm in diameter with a 13.5 cm bed height and had been previously used for 28 cycles. Bioprocess skid 1538 (Amersham Biosciences Pharmacia) was used for purification, and skid 1050 (Millipore) was used for mock runs. The mAb5 column (Pharmacia Index) was 14 cm in diameter with a 15 cm bed height and had been previously used for 20 cycles. Bioprocess skid 1076 (Amersham Pharmacia Biotech) was used for all mAb5 processes.

[0325] Ten cleanup cycles were performed, consisting of 2 CV equilibration buffer and 2 CV regeneration buffer. The column was held in a 3-hour static soak in equilibration buffer for 5 of the 10 pulsed cycles. Cleanup was followed by column storage, skid sanitization, and a mock run. For the mAb5 column, sanitization was performed immediately followed by an additional mock run without the column to obtain a value for system carryover.

[0326] Mock run parameters were based on parameters previously used in Protein A processing of each particular molecule. For the mAb5 column, the wash 3 step was extended to 4 CV for a typical mAb5 Protein A chromatography. A summary of the mock run parameters and the original run used for reference are listed in Table 11. TIFF2025186245000014.tif30170

[0327] Product carryover is shown in Table 12. After clarification, the aIGF1R column had a carryover of 48.2 ng IgG / mg product, while the antiAbeta column had a carryover of 8.6 ng IgG / mg product. Previous studies have shown that carryover values ​​increase with increased protein contact. Although the mAb4 column was exposed to eight more protein contact cycles than the mAb5 column, the increased number of contacts alone may not be significantly sufficient to explain the large disparity in carryover values. TIFF2025186245000015.tif33170

[0328] By the end of the final purification cycle, product was still eluting with the purification buffer for both columns. Compared to the 5.0 ng IgG / mg product from the final purification CV observed during small-scale optimization, product elution at pilot scale was higher than expected (12.7 ng IgG / mg product and 62.9 ng IgG / mg product for the aIGF1R and anti-Abeta columns, respectively).

[0329] System carryover was analyzed by performing a mock run without the column in place after skid sanitization. System carryover indicates the amount of carryover attributable to the skid and not to the Prosep A resin. The skid sanitization procedure and sanitization should have eliminated all system carryover, but 0.97 ng IgG / mg product was still detected in the system mock pool during the mAb5 run. These results indicate that the system's contribution to carryover is minimal, but further sanitization of the skid itself may be required to ensure the absolute absence of product carryover from one run to the next.

[0330] Application of the cleanup procedure used during small-scale optimization to pilot scale resulted in higher carryover levels with significant variability. Product continued to elute through the end of the cleanup cycle at higher levels than previously observed at small scale as well. The system was found to contribute small levels of carryover even when the column was not in place.

[0331] Pulsing the buffer from high to low pH has been found to be an effective means of reducing carryover on a small scale. Using a 1.6 cm diameter column, carryover was reduced to 6.7 ng IgG / mg product. However, application of the purification procedure to the actual column used during the previous pilot run resulted in a significantly higher carryover value of 48.2 ng IgG / mg product, the highest value measured to date. While carryover levels are expected to vary somewhat between columns due to differences in column usage, the completed purification procedure should ubiquitously eliminate carryover, regardless of the details of any given column. Furthermore, it is important to evaluate additional parameters such as long-term column performance and minimum carryover detection limits.

Claims

1. 1. A method for purifying chromatography material for reuse, comprising: a) passing at least two material volumes of an elution buffer through the material, the elution buffer comprising about 0.15 M acetic acid and having a pH of about 2.9; b) statically holding the material in the elution buffer for a time ranging from about 10 minutes to about 30 minutes; c) passing at least about 2 material volumes of elution buffer through the material; and d) passing about 2 or more material volumes of regeneration buffer through the material, the regeneration buffer comprising about 0.1 N NaOH and having a pH of about 13. A method comprising:

2. 1. A method for purifying chromatography material for reuse, comprising: a) passing about 2 material volumes of an elution buffer through the material, the elution buffer comprising about 0.15 M acetic acid and having a pH of about 2.9; b) statically holding the material in elution buffer for about 30 minutes; c) passing about 2 material volumes of elution buffer through the material; and d) passing the material through about 4 material volumes of a regeneration buffer, the regeneration buffer comprising about 0.1 N NaOH and having a pH of about 13; A method comprising:

3. 1. A method for purifying chromatography material for reuse, comprising: a) passing about 2 material volumes of an elution buffer through the material, the elution buffer comprising about 0.15 M acetic acid and having a pH of about 2.9; b) statically holding the material in elution buffer for about 30 minutes; c) passing about 2 material volumes of elution buffer through the material; and d) passing about 2.5 material volumes of a regeneration buffer through the material, the regeneration buffer comprising about 0.1 N NaOH and having a pH of about 13; e) statically holding the material in the renaturation buffer for about 30 minutes; f) passing the material through about 2.5 material volumes of regeneration buffer. A method comprising:

4. 1. A method for purifying chromatography material for reuse, comprising: a) passing about 2 material volumes of an equilibration buffer through the material, the equilibration buffer comprising about 25 mM Tris and about 25 mM NaCl, and having a pH of about 7.1; b) statically holding the material in equilibration buffer for about 30 minutes; c) passing about 2 material volumes of equilibration buffer through the material; d) passing about 2 material volumes of elution buffer through the material, the elution buffer comprising about 0.15 M acetic acid and having a pH of about 2.8; e) statically holding the material in elution buffer for about 30 minutes; f) passing about 2 material volumes of elution buffer through the material; g) passing the material through about 2 material volumes of a regeneration buffer, the regeneration buffer comprising 0.1 N NaOH and having a pH of 13; h) statically holding the material in the renaturation buffer for about 30 minutes; i) passing the material through about 2 material volumes of regeneration buffer; A method comprising:

5. 1. A method for purifying chromatography material for reuse, comprising: a) passing about 4 material volumes of an equilibration buffer through the material, the equilibration buffer comprising about 25 mM Tris and about 25 mM NaCl, at a pH of 7.1; b) 6 cycles of i) passing about 3 material volumes of elution buffer through the material, wherein the elution buffer comprises about 0.15 M acetic acid and has a pH of about 2.8; ii) statically holding the material in elution buffer for approximately 10 minutes; iii) passing about 1 material volume of elution buffer through the material; iv) passing about 3 material volumes of regeneration buffer through the material, wherein the regeneration buffer comprises about 0.1 N NaOH and has a pH of about 13; v) statically holding the material in the renaturation buffer for about 10 minutes; vi) passing the material through about 1 material volume of regeneration buffer; carrying out a step including A method comprising:

6. 1. A method for purifying chromatography material for reuse, comprising six cycles of: a) passing about 3 material volumes of an elution buffer through the material, the elution buffer comprising about 0.15 M acetic acid and having a pH of about 2.8; b) statically holding the material in elution buffer for about 15 minutes; c) passing about 1 material volume of elution buffer through the material; d) passing the material through about 3 material volumes of a regeneration buffer, the regeneration buffer comprising about 0.1 N NaOH and having a pH of about 13; e) statically holding the material in the renaturation buffer for about 15 minutes; f) passing the material through about 1 material volume of regeneration buffer; g) passing the material through about 3 material volumes of a storage buffer, the storage buffer comprising about 100 mM sodium acetate, about 2% benzyl alcohol, and at about pH 5.0; e) statically holding the material in storage buffer for about 15 minutes; f) passing the material through about 1 material volume of storage buffer. A method comprising:

7. 7. The method of claim 1, wherein the chromatographic material is present in a chromatographic column.

8. 8. The method of claim 1, wherein the chromatographic material is an affinity material.

9. 9. The method of claim 8, wherein the affinity material is a Protein A affinity material.

10. 10. The method of claim 9, wherein the Protein A affinity material is a MAbSelect material, a MAbSelect SuRe material, or a MAbSelect SuRe LX material.

11. 11. The method of claim 1, wherein the chromatography material is used for large-scale production of the polypeptide.

12. 1. A method for purifying chromatography material for reuse, comprising: a) passing about 3 material volumes of an equilibration buffer through the material, the equilibration buffer comprising about 40 mM sodium acetate and having a pH of about 5.5; b) passing about 2 material volumes of about 0.5 N NaOH through the material; c) statically holding the material in about 0.5 N NaOH for about 10 minutes; d) passing about 1 material volume of about 0.5 N NaOH through the material; and e) statically holding the material in about 0.5 N NaOH for about 10 minutes; f) passing about 1 material volume of about 0.5 N NaOH through the material. A method comprising:

13. 13. The method of claim 12, wherein the chromatographic material is present in a chromatographic column.

14. 14. The method of claim 12 or 13, wherein the chromatographic material is an ion exchange material.

15. 15. The method of claim 14, wherein the ion exchange material is a cation exchange material.

16. 16. The method of claim 15, wherein the cation exchange material is a POROS HS50 material.

17. 17. The method of any one of claims 12 to 16, wherein the chromatographic material is used for large-scale production of antibodies.

18. 1. A method for purifying chromatography material for reuse, comprising: a) passing about 3 material volumes of an equilibration buffer through the material, the equilibration buffer comprising about 50 mM Tris, 85 mM sodium acetate, at about pH 8.8 and about 8.6 mS / cm; b) passing about 2 material volumes of about 0.5 N NaOH through the material; c) statically holding the material in about 0.5 N NaOH for about 10 minutes; d) passing about 1 material volume of about 0.5 N NaOH through the material; and e) statically holding the material in about 0.5 N NaOH for about 10 minutes; f) passing about 1 material volume of about 0.5 N NaOH through the material. A method comprising:

19. 20. The method of claim 18, wherein the chromatographic material is present in a chromatographic column.

20. 20. The method of claim 18 or 19, wherein the chromatographic material is an ion exchange material.

21. 21. The method of claim 20, wherein the ion exchange material is an anion exchange material.

22. 22. The method of claim 21, wherein the anion exchange material is a QSFF material.

23. 23. The method of any one of claims 18 to 22, wherein the chromatographic material is used for large-scale production of antibodies.

24. 24. The method of any one of claims 1 to 23, wherein the buffer is passed through the material at about 30 material volumes per hour, about 20 material volumes per hour, or about 15 material volumes per hour.

25. 25. The method of any one of claims 1 to 24, wherein the buffer is passed through the material in a downflow or upflow direction.

26. 26. The method of any one of claims 1 to 25, wherein the purification of the chromatographic material is measured by performing a mock elution after purifying the chromatographic material.

27. 27. The method of claim 26, wherein the mock elution eluent contains one or more of <0.25 mg / mL total protein, <1 ppm IgG fragments, <1 ppm leached Protein A, <1 μg / mL CZE LIF, <1 ppm CHOP, and <1 pg / mL CHO DNA, which are indicators of effective purification of the material for multi-product use.

28. 28. The method of any one of claims 1 to 27, wherein the chromatographic material is stable in alkali.

29. 29. The method of any one of claims 1 to 28, wherein the chromatographic material is used to purify the polypeptide.

30. 30. The method of any one of claims 1 to 29, wherein the chromatographic material is purified after purification of the first polypeptide, and after purification, the chromatographic material is used to purify a second polypeptide.

31. 31. The method of claim 30, wherein the polypeptide is an antibody or an immunoadhesin.

32. 32. The method of claim 31, wherein the polypeptide is an immunoadhesin.

33. 32. The method of claim 31 , wherein the polypeptide is an antibody.

34. 34. The method of claim 33, wherein the antibody is a monoclonal antibody.

35. 35. The method of claim 34, wherein the monoclonal antibody is a chimeric antibody, a humanized antibody, or a human antibody.

36. 36. The method of claim 35, wherein the monoclonal antibody is an IgG monoclonal antibody.

37. 37. The method of claim 36, wherein the antibody is an antigen-binding fragment.

38. The antigen-binding fragment may be a Fab fragment, a Fab' fragment, or a F(ab') ２ The method of claim 37, wherein the antibody is a fragment, scFv, di-scFv, bi-scFv, tandem (di, tri)-scFv, Fv, sdAb, trifunctional antibody, BiTE, diabody or triabody.

39. 39. The method of claim 38, wherein the polypeptide is an enzyme, a hormone, a fusion protein, an Fc-containing protein, an immunoconjugate, a cytokine, or an interleukin.

40. 31. The method of claim 30, wherein the first polypeptide is a first antibody or a first immunoadhesin and the second polypeptide is a second antibody or a second immunoadhesin.

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