Enhanced ultrafiltration / diafiltration process

Abstract

Disclosed herein are ultrafiltration / diafiltration (UF / DF) methods including the use of diafiltration buffer concentrates, and UF / DF methods in which a composition comprising a recombinant protein is concentrated by the ultrafiltration step to a recombinant protein concentration that is higher than the intended concentration after the subsequent diafiltration step.

Description

[0001] Cross-reference to related applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 565,068, filed March 14, 2024, which is hereby incorporated by reference in its entirety. Technical Field

[0002] This disclosure provides an ultrafiltration / percolation (UF / DF) method including the use of percolation buffer concentrate, and a UF / DF method wherein a composition containing recombinant protein is concentrated to a recombinant protein concentration higher than the desired concentration after a subsequent percolation step by an ultrafiltration step. Background Technology

[0003] Ultrafiltration and percolation (UF / DF) processes are commonly used in biomanufacturing to formulate active pharmaceutical ingredients (APIs) prior to large-volume storage or drug filling / final processing. During a UF / DF process, a composition containing recombinant proteins that has undergone one or more downstream purification processes can be concentrated and buffer-exchanged into a desired formulation matrix. For example, an incoming product pool can be concentrated to a target concentration via ultrafiltration using a semi-permeable membrane filter, buffer-exchanged via percolation, optionally further concentrated, and then recovered to obtain a UF / DF pool. In a typical UF / DF operation, the same percolation buffer is used for both buffer exchange and recovery.

[0004] These buffer exchange and final formulation steps can be time-consuming and resource-intensive, especially when used as part of an enhanced biomanufacturing process with high correlated titers. Specifically, UF / DF processes can be demanding in terms of percolation buffer consumption and associated buffer batch requirements, as a certain number of percolation volumes (typically as high as 7 to 10) are required during percolation for near-complete buffer exchange.

[0005] Therefore, there is a need in the art for enhanced ultrafiltration / percolation methods that maintain process performance while reducing resource requirements (including batch processing requirements for percolation buffer). Summary of the Invention

[0006] This disclosure provides a method for preparing a formulation containing a recombinant protein, the method comprising percolating a first composition containing the recombinant protein with a percolation buffer to obtain a second composition, wherein the percolation buffer is prepared by diluting a percolation buffer concentrate.

[0007] In some embodiments, the percolation buffer is prepared by diluting a percolation buffer concentrate with water (e.g., deionized water). In some embodiments, the percolation buffer is prepared by diluting a percolation buffer concentrate at a desired dilution ratio of 1:1. In some embodiments, the percolation buffer is prepared by diluting a percolation buffer concentrate with water (e.g., deionized water) at a desired dilution ratio of 1:1.

[0008] In some embodiments, dilution is performed using an online dilution system. In some embodiments, dilution is performed continuously using an online dilution system. In some embodiments, the pumps of the online dilution system have a relative dilution accuracy in the range of 90% to 110% of the expected dilution ratio.

[0009] In some embodiments, other steps of the method (e.g., UF / DF unit operation) do not use percolation buffer prepared by dilution of percolation buffer concentrate.

[0010] Another aspect of this disclosure provides a method for preparing a formulation comprising a recombinant protein, the method comprising: The ultrafiltration / percolation (UF / DF) membrane filter is equilibrated in a first percolation buffer to obtain a balanced UF / DF membrane filter; Using a balanced UF / DF membrane filter, the first composition containing the recombinant protein is concentrated by ultrafiltration to obtain a first concentrated composition; and The first concentrated composition is percolated with the first percolation buffer to obtain the second composition. The first percolation buffer is prepared by diluting a percolation buffer concentrate.

[0011] In some embodiments, the first percolation buffer is prepared by diluting a percolation buffer concentrate with water (e.g., deionized water). In some embodiments, the first percolation buffer is prepared by diluting a percolation buffer concentrate at a desired dilution ratio of 1:1. In some embodiments, the first percolation buffer is prepared by diluting a percolation buffer concentrate with water (e.g., deionized water) at a desired dilution ratio of 1:1.

[0012] In some embodiments, dilution is performed using an online dilution system. In some embodiments, dilution is performed continuously using an online dilution system. In some embodiments, the pumps of the online dilution system have a relative dilution accuracy in the range of 90% to 110% of the expected dilution ratio.

[0013] In some embodiments, other steps of the method (e.g., UF / DF unit operations, including, for example, a recovery process) do not use percolation buffer prepared by dilution of the percolation buffer concentrate.

[0014] This disclosure also provides a method for preparing a formulation containing a recombinant protein, the method comprising percolating a first composition containing the recombinant protein with a first percolation buffer and a second percolation buffer to obtain a second composition, wherein the first percolation buffer is prepared by diluting a percolation buffer concentrate; and the second percolation buffer is not prepared by diluting a percolation buffer concentrate.

[0015] In some embodiments, the second percolation buffer is prepared in batches according to its intended composition.

[0016] In some embodiments, the first percolation buffer and the second percolation buffer have substantially the same composition.

[0017] In some embodiments, the first percolation buffer and the second percolation buffer contain the same excipients, and each excipient in the first percolation buffer is present in the second percolation buffer in an amount ranging from 90% to 110% of its amount in the second percolation buffer.

[0018] In some embodiments, the first percolation buffer is prepared by diluting a percolation buffer concentrate with water (e.g., deionized water). In some embodiments, the first percolation buffer is prepared by diluting a percolation buffer concentrate at a desired dilution ratio of 1:1. In some embodiments, the first percolation buffer is prepared by diluting a percolation buffer concentrate with water (e.g., deionized water) at a desired dilution ratio of 1:1.

[0019] In some embodiments, dilution is performed using an online dilution system. In some embodiments, dilution is performed continuously using an online dilution system. In some embodiments, the pumps of the online dilution system have a relative dilution accuracy in the range of 90% to 110% of the expected dilution ratio.

[0020] In some embodiments, other steps of the method do not use percolation buffer prepared by dilution of percolation buffer concentrate.

[0021] Another aspect of this disclosure provides a method for preparing a formulation comprising a recombinant protein, the method comprising: The ultrafiltration / percolation (UF / DF) membrane filter is equilibrated in a first percolation buffer to obtain a balanced UF / DF membrane filter; Using a balanced UF / DF membrane filter, the first composition containing the recombinant protein is concentrated by ultrafiltration to obtain a first concentrated composition; and The first concentrated composition is percolated with the first percolation buffer and the second percolation buffer to obtain the second composition. in: The first percolation buffer was prepared by diluting a percolation buffer concentrate; This second percolation buffer was not prepared by diluting a percolation buffer concentrate; and The first percolation buffer and the second percolation buffer have essentially the same composition.

[0022] In some embodiments, the second percolation buffer is prepared in batches according to its intended composition.

[0023] In some embodiments, the first percolation buffer and the second percolation buffer contain the same excipients, and each excipient in the first percolation buffer is present in the second percolation buffer in an amount ranging from 90% to 110% of its amount in the second percolation buffer.

[0024] In some embodiments, the first percolation buffer is prepared by diluting a percolation buffer concentrate with water (e.g., deionized water). In some embodiments, the first percolation buffer is prepared by diluting a percolation buffer concentrate at a desired dilution ratio of 1:1. In some embodiments, the first percolation buffer is prepared by diluting a percolation buffer concentrate with water (e.g., deionized water) at a desired dilution ratio of 1:1.

[0025] In some embodiments, dilution is performed using an online dilution system. In some embodiments, dilution is performed continuously using an online dilution system. In some embodiments, the pumps of the online dilution system have a relative dilution accuracy in the range of 90% to 110% of the expected dilution ratio.

[0026] In some embodiments, other steps of the method (e.g., UF / DF unit operations, including, for example, a recovery process) do not use percolation buffer prepared by dilution of the percolation buffer concentrate.

[0027] Another aspect of this disclosure provides a method for preparing a formulation containing a recombinant protein, the method comprising percolating a first composition containing the recombinant protein with a first percolation buffer and a second percolation buffer to obtain a second composition, wherein the first percolation buffer is a concentrate of the second percolation buffer.

[0028] In some embodiments, the first percolation buffer is at least a 2X concentrate of the second percolation buffer.

[0029] In some embodiments, the method includes percolating 0.5 percolation volumes with a first percolation buffer, followed by percolating 6.5 to 9.5 percolation volumes with a second percolation buffer. In some embodiments, the method includes percolating one percolation volume with the first percolation buffer, followed by percolating 6 to 9 percolation volumes with the second percolation buffer. In some embodiments, the method includes percolating two percolation volumes with the first percolation buffer, followed by percolating 5 to 8 percolation volumes with the second percolation buffer. In some embodiments, the method includes percolating three percolation volumes with the first percolation buffer, followed by percolating 4 to 7 percolation volumes with the second percolation buffer.

[0030] Another aspect of this disclosure provides a method for preparing a formulation comprising a recombinant protein, the method comprising: The first composition containing the recombinant protein is concentrated by ultrafiltration to obtain a first concentrated composition; and The first concentrated composition is percolated to obtain a second composition, wherein the recombinant protein is present in the first concentrated composition at a concentration of at least 120% of the concentration of the recombinant protein in the second composition.

[0031] In some embodiments, the first concentrated composition is diluted with a percolation buffer prior to percolation. In some embodiments, the first concentrated composition is diluted with a percolation buffer prior to percolation such that the concentration of recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0032] This disclosure also provides a method for preparing a formulation containing a recombinant protein, the method comprising: The first composition containing the recombinant protein is concentrated by ultrafiltration to obtain a first concentrated composition; and The first concentrated composition is percolated with a first percolation buffer and a second percolation buffer to obtain a second composition, wherein: The first percolation buffer is a concentrate of the second percolation buffer; and The recombinant protein is present in the first concentrated composition at a concentration of at least 120% of the concentration of the recombinant protein in the second composition.

[0033] In some embodiments, prior to percolation, the first concentrated composition is diluted with a percolation buffer (e.g., a second percolation buffer). In some embodiments, prior to percolation, the first concentrated composition is diluted with a percolation buffer (e.g., a second percolation buffer) such that the concentration of recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0034] In some embodiments, the first percolation buffer is at least a 2X concentrate of the second percolation buffer.

[0035] In some embodiments, the method includes percolating 0.5 percolation volumes with a first percolation buffer, followed by percolating 6.5 to 9.5 percolation volumes with a second percolation buffer. In some embodiments, the method includes percolating one percolation volume with the first percolation buffer, followed by percolating 6 to 9 percolation volumes with the second percolation buffer. In some embodiments, the method includes percolating two percolation volumes with the first percolation buffer, followed by percolating 5 to 8 percolation volumes with the second percolation buffer. In some embodiments, the method includes percolating three percolation volumes with the first percolation buffer, followed by percolating 4 to 7 percolation volumes with the second percolation buffer. Attached Figure Description

[0036] Figure 1 A schematic diagram is provided showing the batch requirements related to different concentrations of percolation buffer (1X - 10X).

[0037] Figure 2 A schematic diagram is provided showing the relative delivery error associated with a 2% or 3% pump error rate at different percolation buffer concentrations (1X - 10X).

[0038] Figure 3 The relative change in predicted UF / DF cell osmolality is shown for the enhanced UF / DF process scenario described in Example 1, compared to a non-enhanced process in which the entire UF / DF process uses 1X percolation buffer.

[0039] Figure 4 The process capability (Ppk) of the osmotic molar concentration relative to the product specification limit is shown for the simulated scenario described in Example 1, assuming that the pump variability is uniformly distributed in the worst case.

[0040] Figure 5 The simulated concentrations of excipients under different target ultrafiltration concentrations / pre-diluted scenarios described in Example 2 are shown.

[0041] Figure 6 The simulated concentrations of excipients are shown in different accelerated buffer exchange scenarios described in Example 2. Detailed Implementation

[0042] This document discloses an ultrafiltration / percolation (UF / DF) method that includes using a percolation buffer concentrate. It also discloses a UF / DF method in which a composition containing recombinant protein is concentrated to a recombinant protein concentration higher than the desired concentration after a subsequent percolation step via an ultrafiltration step.

[0043] definition: The following definitions are provided to aid in understanding the scope of this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0044] In some embodiments, when used in conjunction with a measurable numerical variable, “about” refers to the indicated value of the variable and all values ​​of the variable within the experimental error of the indicated value (e.g., within a 95% confidence interval of the mean) or within ±10% of the indicated value (whichever is greater). In some embodiments, the numerical range includes numbers (i.e., endpoints) defining the range. When a composition contains the same components, wherein the amount of each component is within ±10% (e.g., ±5%; ±2.5%) of the amount in another component (as determined by the amount in a composition containing a higher amount of the component), the composition may be “substantially identical” to a reference composition.

[0045] Where a range of values ​​is provided, it should be understood that every intermediate value between the upper and lower limits of the range (unless otherwise explicitly stated in the context, to one-tenth of the lower limit unit), as well as any other stated or intermediate values ​​within the range, are included in this disclosure. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges also covered in this disclosure, subject to any specifically excluded limitations within the range. Where the range includes one or two limits, the range excluding any one or both of those included limits is also included in this disclosure.

[0046] As used herein, unless otherwise expressly indicated, the terms “a” and “an” mean one or more. Additionally, “one or more” and “at least one” are used interchangeably herein. Furthermore, unless the context requires otherwise, singular terms include plural terms, and plural terms include singular terms.

[0047] As used in this article, the term "acid precipitation" refers to a harvesting operation that lowers the pH of a cell culture to induce precipitation of one or more cell culture impurities.

[0048] As used herein, the term “affinity chromatography” (also known as “capture chromatography”) refers to a chromatographic operation that separates a biomolecule (e.g., a recombinant protein) from a mixture based on the selective interaction between the biomolecule and another substance (i.e., a ligand). Affinity chromatography is commonly used in biomanufacturing processes to separate and concentrate desired recombinant proteins from harvested cell culture media. In a typical affinity chromatography operation, biomolecules in the mobile phase selectively bind to or otherwise interact with the stationary phase, while the remainder of the mobile phase passes through the chromatographic material. The biomolecule is then eluted from the stationary phase by altering the conditions in a manner that reduces the affinity between the ligand and the biomolecule. Non-limiting examples of affinity chromatographic materials include protein A, protein G, protein A / G, and protein L materials. Additionally, immobilized metal affinity chromatography (IMAC) can be used to capture proteins that have an affinity for metal ions or are engineered to have an affinity for metal ions.

[0049] In some embodiments, protein A affinity chromatography can be used to capture the target recombinant protein. Protein A ligands exhibit high selectivity for a variety of proteins containing the antibody Fc region and provide robust removal of process-related impurities and high target protein yields. Commercially available protein A materials include, but are not limited to, MABSELECT™ SURE protein A, Protein A SepharoseFAST FLOW™, MABSELECT™ PrismA (Cytiva, Marburg, MAS), PROSEP-A™ (Merck Millipore, UK), TOYOPEARL™ HC-650F protein A (TosoHass Co., Philadelphia, PA), and AP Plus (Purolite, Kingsville, PA).

[0050] As used herein, the term “antigen-binding protein” refers to a protein or polypeptide that includes an antigen-binding region or antigen-binding portion that has an affinity for another molecule (antigen) to which it binds. Antigen-binding proteins include, but are not limited to, antibodies, fusion proteins, VH, VHH, VL, (s)dAb, Fv, light chains (VL-CL), Fd (VH-CH1), heavy chains, Fab, Fab', F(ab')2, or modified antigen-binding portions of "r IgG" (a "half-antibody" composed of heavy and light chains) or full-length antibodies, such as triple-chain antibody-like molecules, antibodies with only heavy chains, single-chain variable fragments (scFv), di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, single-chain Fab (scFab), Fab2, Fab3, biantibodies, single-chain biantibodies, tandem biantibodies (Tandab), tandem di-scFv, tandem tri-scFv, and "microantibodies" illustrated by the following structures: (VH-VL-CH3)2, (scFv-CH3)2, ((scFv)2-CH3+ CH3), ((scFv)2-CH3) or (scFv-CH3-scFv)2, multibody antibodies such as triabody or tetrabody, and single-domain antibodies (such as nanobodies or single variable domain antibodies containing only one variable region, which can be VHH, VH or VL, and which bind specifically to the antigen or target independently of other variable regions or domains).

[0051] As used in this article, the term "antibody" generally refers to a tetrameric immunoglobulin consisting of two light chain polypeptides (each approximately 25 kDa) and two heavy chain polypeptides (each approximately 50-70 kDa).

[0052] As used herein, the term "light chain" or "immunoglobulin light chain" refers to a polypeptide comprising a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL) from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus). The immunoglobulin light chain constant domain (CL) may be a human kappa (κ) or human lambda (λ) constant domain.

[0053] As used herein, the term "heavy chain" or "immunoglobulin heavy chain" refers to a polypeptide comprising a single immunoglobulin heavy chain variable region (VH), immunoglobulin heavy chain constant domain 1 (CH1), immunoglobulin hinge region, immunoglobulin heavy chain constant domain 2 (CH2), immunoglobulin heavy chain constant domain 3 (CH3), and optionally immunoglobulin heavy chain constant domain 4 (CH4) from the N-terminus (N-terminus) to the C-terminus (C-terminus). Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε) chains, which define antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. Antibodies in the IgG and IgA classes are further subdivided into several subclasses: IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2, respectively. The heavy chains of IgG, IgA, and IgD antibodies have three constant domains (CH1, CH2, and CH3), while the heavy chains of IgM and IgE antibodies have four constant domains (CH1, CH2, CH3, and CH4). These constant domains of the immunoglobulin heavy chain can originate from any immunoglobulin isotype, including subtypes. The antibody chains are linked together via interpeptide disulfide bonds between the CL and CH1 domains (i.e., between the light and heavy chains) and between the hinge regions of these two antibody heavy chains.

[0054] The variable regions of immunoglobulin chains typically exhibit the same overall structure, comprising relatively conserved framework regions (FRs) linked by three hypervariable regions (more commonly referred to as "complementarity-determining regions" or CDRs). The CDRs from each heavy and light chain pair are usually aligned via the framework regions to form a structure that specifically binds to a particular epitope on the target protein. From the N-terminus to the C-terminus, both naturally occurring light and heavy chain variable regions typically follow this order of elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A numbering system has been designed to assign numbers to the amino acids occupying positions in each of these domains. This numbering system is defined in the following literature: Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, National Institutes of Health (NIH), Bethesda, Maryland); or Chothia and Lesk, 1987. J. Mol. Biol. [ Molecular Biology Journal Zhi[196:901-917; Chothia et al., 1989, Nature 342:878-883.] The CDR and FR of a given antibody can be identified using this system. Other numbering systems for amino acids in the immunoglobulin chain include IMGT. ® (International ImMunoGeneTics Information System; Lefranc et al., Dev. Comp. Immunol. [Developmental and Comparative Immunology] 29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. [Journal of Molecular Biology] 309(3):657-670; 2001).

[0055] Antibody digestion with papain produces two identical antigen-binding proteins (called "Fab" fragments, each with a single antigen-binding site) and a residual "Fc" fragment containing all domains except the first domain of the immunoglobulin heavy chain constant region. The Fab fragment contains variable domains from both the light and heavy chains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Therefore, the "Fab fragment" consists of an immunoglobulin light chain (the light chain variable region (VL) and constant region (CL)) and the CH1 domain and variable region (VH) of the immunoglobulin heavy chain. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fd fragment" contains the VH and CH1 domains from the immunoglobulin heavy chain. The Fd fragment represents the heavy chain component of the Fab fragment.

[0056] An immunoglobulin's "Fc fragment" or "Fc region" typically contains two constant domains, namely a CH2 domain and a CH3 domain, and optionally includes a CH4 domain. The Fc region can be derived from IgG1, IgG2, IgG3, or IgG4 immunoglobulins. In some embodiments, the Fc region contains the CH2 and CH3 domains from human IgG1 or human IgG2 immunoglobulins. The Fc region can maintain effector functions such as C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), and phagocytosis. In other embodiments, the Fc region can be modified to reduce or eliminate effector functions.

[0057] "F(ab')2 fragment" is a divalent fragment consisting of two Fab' fragments connected by a disulfide bridge between the heavy chains in the hinge region.

[0058] The “Fv” fragment is the smallest fragment containing the complete antigen recognition and binding site from the antibody. This fragment consists of a tightly non-covalently associated dimer of an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL). It is in this conformation that the three CDRs of each variable region interact to confine the antigen binding site to the surface of the VH-VL dimer. A single light chain or heavy chain variable region (or just half of an Fv fragment containing only the three CDRs specific to the antigen) has the ability to recognize and bind the antigen, although its affinity is lower than that of the entire binding site containing both VH and VL.

[0059] A “single-chain variable fragment” or “scFv fragment” contains the VH and VL regions of an antibody, wherein these regions exist within a single polypeptide chain, and optionally includes a peptide linker between the VH and VL regions that enables the Fv to form the desired structure for antigen binding. See For example, Bird et al., Science, Vol. 242: 423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA, Vol. 85: 5879-5883, 1988.

[0060] "Nanobodies" are the variable regions of the heavy chain in heavy chain antibodies. These variable domains are the smallest fully functional antigen-binding fragments in such heavy chain antibodies, with a molecular weight of only 15 kDa. See Cortez-Retamozo et al. Cancer Research [ Cancer research[64:2853-57, 2004.] Functional heavy chain antibodies lacking the light chain are naturally found in certain animal species, such as nurse sharks, wobbeon sharks, and camelids, including camels, dromedary camels, alpacas, and llamas. In these animals, the antigen-binding site is reduced to a single domain, namely the VHH domain. These antibodies form the antigen-binding region using only the variable region of the heavy chain; that is, these functional antibodies are heavy chain homodimers possessing only the H2L2 structure (referred to as "heavy chain antibodies" or "HCAbs"). It has been reported that camelified VHHs recombine with constant regions of IgG2 and IgG3, which contain hinge, CH2, and CH3 domains but lack the CH1 domain. Camelized VHH domains have been found to bind to antigens with high affinity (Desmyter et al., J. Biol. Chem., Vol. 276: 26285-90, 2001) and exhibit high stability in solution (Ewert et al., Biochemistry, Vol. 41: 3628-36, 2002). Methods for generating antibodies with camelized heavy chains are described, for example, in U.S. Patent Publications 2005 / 0136049 and 2005 / 0037421. Alternative scaffolds could be made from human variable domains more closely resembling shark V-NAR scaffolds and could potentially provide a framework for long penetrating loop structures.

[0061] As used herein, the term "heavy-chain-only antibody" refers to an immunoglobulin protein composed of two heavy-chain polypeptides (e.g., heavy-chain polypeptides, each approximately 50-70 kDa). "Heavy-chain-only antibodies" lack the two light-chain polypeptides found in conventional antibodies. Heavy-chain antibodies account for approximately one-quarter of IgG antibodies produced by camelids (e.g., camels and llamas) (Hamers-Casterman C. et al.). Nature . [ nature [363, 446-448 (1993)]. These molecules are formed by two heavy chains but no light chains. Therefore, the variable antigen-binding moiety is called the VHH domain, and it represents the smallest naturally occurring, complete antigen-binding site, only about 120 amino acids in length (Desmyter, A. et al.). J. Biol. Chem . [ Journal of Biochemistry [276, 26285-26290 (2001)]. Immunization can generate heavy-chain antibodies with high specificity and affinity for multiple antigens (Vander Linden, RH et al.). Biochim. Biophys. Acta .[ Journal of Biochemistry and Biophysics [1431, 3746 (1999)], and the VHH motif can be easily cloned and expressed in yeast (Frenken, LGJ et al.) J. Biotechnol . [ Biotechnology Journal [78, 11-21(2000)]. Their expression levels, solubility, and stability are significantly higher than those of classic F(ab) or Fv fragments (Ghahroudi, MA et al.). FEBS Lett. [ European Federation of Biochemical Societies (EFSB) Newsletter [414, 521-526 (1997)]. It was also shown that sharks possess a single VH-like domain in their antibodies, termed VNAR. (Nuttall et al.) Eur.J. Biochem. [ European Biochemistry Magazine ] 270, 3543-3554 (2003); Nuttall et al. Function and Bioinformatics [ Function and biology Informatics ] 55, 187-197 (2004); Dooley et al., Molecular Immunology [ Molecular Immunology ] 40, 25-33 (2003)).

[0062] In some embodiments, a "heavy-chain-only antibody" is a dimeric antibody comprising a VH antigen-binding domain and constant CH2 and CH3 domains but lacking a CH1 domain. In some embodiments, a heavy-chain-only antibody comprises a variable region antigen-binding domain comprising frames 1, CDR1, 2, CDR2, 3, CDR3, and 4. In some embodiments, a heavy-chain-only antibody comprises an antigen-binding domain, at least a portion of a hinge region, and CH2 and CH3 domains. In some embodiments, a heavy-chain-only antibody comprises an antigen-binding domain, at least a portion of a hinge region, and a CH2 domain. In some embodiments, a heavy-chain-only antibody comprises an antigen-binding domain, at least a portion of a hinge region, and a CH3 domain. This document also includes heavy-chain-only antibodies with truncated CH2 and / or CH3 domains. The heavy-chain-only antibodies described herein may belong to the IgG subclass, but heavy-chain-only antibodies belonging to other subclasses such as IgM, IgA, IgD, and IgE are also included herein. In some embodiments, the heavy-chain-only antibody may belong to the IgG1, IgG2, IgG3, or IgG4 subtypes, such as IgG1 or IgG4 subtypes. In some embodiments, the heavy-chain-only antibody is an IgG1 or IgG4 subtype, wherein one or more CH domains are modified to alter the effector function of the antibody. In some embodiments, the heavy-chain-only antibody is an IgG4 subtype, wherein one or more CH domains are modified to alter the effector function of the antibody. In some embodiments, the heavy-chain-only antibody is an IgG1 subtype, wherein one or more CH domains are modified to alter the effector function of the antibody. Modifications of the CH domains that alter effector function are further described herein. Non-limiting examples of heavy-chain-only antibodies are described, for example, in WO 2018 / 039180, the disclosure of which is incorporated herein by reference in its entirety.

[0063] As used herein, the term "triple-chain antibody-like molecule" or "TCA" refers to an antibody-like molecule comprising, substantially composed of, or composed of three polypeptide subunits, wherein two polypeptide subunits comprise, substantially composed of, or composed of, an antigen-binding fragment of such antibody chain comprising an antigen-binding region and at least one CH domain. This heavy / light chain pair is specific for binding to a first antigen. The third polypeptide subunit comprises, substantially composed of, or composed of, an antibody consisting only of a heavy chain, which includes an Fc portion containing CH2 and / or CH3 and / or CH4 domains but lacking a CH1 domain, and one or more antigen-binding domains (e.g., two antigen-binding domains) binding to epitopes of a second antigen or different epitopes of a first antigen, wherein such binding domains originate from or have sequence identity with a variable region of the antibody heavy or light chain. A portion of such a variable region may be derived from V Hand / or V L Gene fragments, D and J H Gene fragments or J L Gene segments encode. Variable regions can be composed of rearranged V... H DJ H V L DJ H V H J L or V L J L Gene segment encoding.

[0064] As used herein, the term "cell culture" or "culture" refers to the growth and reproduction of cells outside a multicellular organism or tissue. Suitable culture conditions for mammalian cells and bacterial cells are known in the art. See For example, *Animal Cell Culture: A Practical Approach*, edited by D. Rickwood, Oxford University Press, New York (1992). Mammalian cells can be cultured in suspension or attached to a solid substrate. In some embodiments, fluidized bed bioreactors, hollow fiber bioreactors, roller flasks, shake flasks, and / or stirred tank bioreactors (with or without microcarriers) can be used for cell culture. In some embodiments, bioreactors of 500 L to 2000 L are used for cell culture (e.g., as part of a seed chain). In some embodiments, bioreactors of 1000 L to 2000 L are used for cell culture (e.g., as part of a seed chain).

[0065] As used herein, the term “connected” in relation to unit operations refers to a direct connection or mechanism that allows continuous flow between one or more unit operations in a single operation cycle.

[0066] As used in this article, the term "continuous" in relation to unit operations refers to a direct connection or mechanism that allows for continuous flow between one or more unit operations.

[0067] As used herein, the term “dynamic binding capacity” for chromatographic materials refers to the amount of product (e.g., peptide) that the material will bind under actual flow conditions before a significant breakthrough occurs in the binding of the product.

[0068] As used herein, the term "expression vector" or "expression construct" refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid control sequences necessary for the expression of an operably linked coding sequence in a specific host cell (e.g., a mammalian host cell). Vectors may include viral vectors, non-attachment mammalian vectors, plasmids, and other non-viral vectors. Expression vectors may include sequences that affect or control transcription, translation, and, if introns, affect the splicing of coding regions to which they are operably linked. "Operably linked" means that the components to which the term applies are in a relationship that allows them to perform their inherent functions. For example, the arrangement of control sequences (e.g., promoters) in a vector that is "operably linked" to a protein-coding sequence such that normal activity of the control sequence leads to transcription of the protein-coding sequence, resulting in recombinant expression of the encoded protein.

[0069] As used herein, a "fusion protein" is a protein containing at least one polypeptide fused to or linked to a heterologous polypeptide. Typically, fusion proteins are expressed from a fusion gene, wherein a nucleotide sequence encoding a polypeptide sequence from one protein is attached within a frame to a nucleotide sequence encoding a polypeptide sequence from a different protein, and optionally separated by a linker. The fusion gene can then be expressed in a recombinant host cell to produce the fusion protein. Fusion proteins may contain fragments from immunoglobulins, such as Fc regions, fused to or linked to ligand polypeptides, receptor polypeptides, hormones, cytokines, growth factors, enzymes, or other polypeptides that are not components of immunoglobulins.

[0070] As used herein, the term “polypeptide” refers to a polymer containing at least 50 amino acids, such as an amino acid polymer containing at least 100 amino acids.

[0071] As used herein, the term "purification chromatography" refers to a chromatographic operation performed after capture or affinity chromatography to remove residual impurities and obtain compositions and / or recombinant proteins of higher purity. Common impurities removed in purification steps include, but are not limited to, product-related impurities (e.g., HMW and LMW substances), host cell proteins, DNA, leached protein A, viral contaminants, and endotoxins. Additionally, typical chromatographic techniques used for purification include, but are not limited to, ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and multimode (or mixed-mode) chromatography (MMC).

[0072] Anion exchange chromatography (AEX) is a form of ion exchange chromatography performed on a positively charged solid-phase medium (such as a resin or membrane) capable of exchanging anions with anions passing through or through an aqueous solution in the solid phase. For example, AEX chromatography is used for virus removal and impurity removal. Commercially available anion exchange media include, but are not limited to, sulfopropyl (SP) immobilized on agarose (e.g., Source 15 Q, Capto™ Q, Q-SEPHAROSE FAST FLOW™ (Stopvan), FRACTOGEL TMAE™, FRACTOGEL EDM DEAE™ (EMD Merck), TOYOPEARL Super Q® and TOYOPEARL NH2-750F (Tosoh Bioscience), POROS HQ™ and POROS XQ™ (Thermo Fisher Scientific)).

[0073] Cation exchange chromatography (CEX) is a form of ion exchange chromatography performed on a negatively charged solid-phase medium (such as a resin or membrane) capable of exchanging cations with cations in an aqueous solution passing through or across the solid phase. The charge can be provided by attaching one or more charged ligands to the solid phase (e.g., via covalent bonds). Alternatively or additionally, the charge can be an inherent property of the solid phase (e.g., silica, which has a generally negative charge). CEX chromatography is typically used to remove high molecular weight (HMW) contaminants, process-related impurities, and / or viral contaminants. Commercially available cation exchange media include, but are not limited to, sulfopropyl (SP) immobilized on agarose (e.g., SPSPPHARSE FAST FLOW™, SP-SEPHAROSE FAST FLOW XL™ or SP-SEPHAROSE HIGH PERFORMANCE™, CAPTO S™, CAPTO SPImpRes™, CAPTO S ImpAct™ (Stopfan), FRACTOGEL-SO3™, FRACTOGEL-SE HICAP™ and FRACTOPREP™ (EMD Merck, Darmstadt, Germany), TOYOPEARL® XS, TOYOPEARL® HS (Tosoh Biosciences, Kingsville, Prussia, Pennsylvania), UNOsphere™ (BioRad, Hercules, California), S Ceramic Hyper™ DF (Pall, Port Washington, NY), POROS™ (Thermo Fisher Scientific, Waltham, Massachusetts), ESHMUNO® CSP and ESHMUNO® CP-FT (Millipore Sigma, Darmstadt, Germany).

[0074] Hydrophobic interaction chromatography (HIC) refers to chromatography performed on a solid-phase medium, utilizing the interaction between hydrophobic ligands and hydrophobic residues on the surface of a solute. Commercially available hydrophobic interaction chromatography media include, but are not limited to, Phenyl Sephrose. TM (Stopvan Company), TOYOPEARL Hexyl (Tosoh Biotechnology Co., Ltd.) and Capto TM Phenyl (Situofan Company).

[0075] Mixed-mode or multi-mode chromatography (MMC) refers to chromatography that utilizes more than one type of interaction between the stationary phase and the analyte to achieve separation. MMC differs from single-mode chromatography in that two or more interaction types (e.g., electrostatic, hydrogen bonding, and / or hydrophobic interactions) significantly contribute to the retention of the solute. Commercially available multi-mode chromatographic media include, but are not limited to, Capto.TM Adhere, Capto™ MMC Impress, Capto MMC (Stopfan), PPAHypercel, MEP Hypercell, HEA Hypercell (Pall Corporation, Port Washington, NY), Eshmuno™ HCX (Merck Millipore), and Toyopearl™ MX-Trp-650M (Tosoh Biotechnology).

[0076] Purification chromatography unit operations utilize materials containing reagents that can operate in multiple modes, such as binding-elution and flow-through. In binding-elution chromatography, the target biomolecule is typically loaded onto the chromatographic material to maximize the dynamic binding capacity, and then washing and elution conditions are used to maximize the purity of the product in the eluent. In contrast, in flow-through chromatography, the loading conditions allow impurities to bind to the chromatographic material while allowing the target biomolecule to pass through. Flow-through chromatography allows for higher loading densities of many biomolecules compared to binding-elution chromatography.

[0077] Besides the two most common modes, weak partition chromatography, overload chromatography, and frontier chromatography can also be used in purification processes. In weak partition chromatography (isocratic separation method), the flow pattern is altered by identifying solution conditions that promote weak binding of biomolecules to the resin, in addition to the binding of one or more impurities; the low product partition coefficient ranges from 0.1 to 20. In overload chromatography, the target biomolecule is loaded onto the chromatographic material beyond the material's dynamic binding capacity. Additionally, frontier chromatography allows for continuous, high-density feeds (containing the target protein and at least one impurity) to the chromatographic medium. In frontier chromatography, the separation of the target protein from impurities and contaminants is driven by the binding affinity of the components in the feed to the chromatographic medium. The amount of target protein that can be loaded and bound to the chromatographic medium in frontier mode typically depends on the amount of higher-charged impurities / contaminants (such as product-related impurities) in the feed. Initially, all components in the feed will bind to the chromatographic medium. The separation of the target product from impurities / contaminants is driven by affinity for the chromatographic medium. When the chromatographic medium reaches saturation binding, components in the feed that have a greater affinity for the medium (typically product-related impurities such as HMW substances) will displace proteins with a weaker affinity (e.g., the target product), leading to the separation of these weaker proteins from the chromatographic medium. These proteins are expelled from the column in the load flow-through. As loading continues, bound proteins are successively replaced in order of increasing affinity for the chromatographic medium until the column reaches or nearly reaches saturation with proteins having a greater affinity than the target protein.

[0078] As used herein, the term "partition factor" or "product partition factor" (K) p () refers to the molar concentration of the product (e.g., recombinant protein) bound to the stationary phase during the chromatographic step divided by the molar concentration of the product in the mobile phase.

[0079] As used herein, when used in connection with a composition, the term "purified" refers to a composition in which at least one impurity is present at a lower concentration relative to the composition prior to one or more unit operations. Additionally, "purified" recombinant proteins (e.g., purified antibodies) refer to recombinant proteins whose purity has been increased, such that they exist in a purer form than when they were synthesized and / or amplified in their natural environment and / or initially under laboratory conditions. Purity is a relative term and does not necessarily refer to absolute purity.

[0080] As used herein, the term "recombinant protein" refers to a heterologous protein produced by host cells transfected with nucleic acids encoding the protein when the host cells are cultured in a cell culture.

[0081] As used herein, the term "unit operation" refers to a functional step performed as part of a process for purifying a recombinant protein for a specific purpose. Unit operations can be designed to achieve a single or multiple objectives, such as capture and virus inactivation steps. Unit operations may also include storage or preservation steps between processing steps.

[0082] As used herein, the term "viral pre-filter" refers to a filter upstream of and in fluid communication with a viral filter that can bind one or more biological process impurities to increase the flux and / or throughput of the viral filter.

[0083] As used herein, the term "concentrate" refers to a composition containing the same components as a reference composition, wherein each component is present at a higher concentration than the same component in the reference composition, and further wherein the amount of each component in the concentrate is substantially the same as the amount in the reference composition. In some embodiments, the concentrate can be prepared by concentrating the reference composition. In other embodiments, the concentrate can be prepared by batch processing, for example, without a concentration step, by adding an additional amount of each component to the solution relative to the amount added during the preparation of the reference composition (e.g., 2X concentrate is twice the amount of the reference composition).

[0084] As used herein, the terms “ultrafiltration”, “UF”, and “ultrafiltration” refer to the use of a semi-permeable membrane to separate different molecules or concentrate similar molecules. In some embodiments, a force is applied to drive the filtration process. Many types of semi-permeable membranes are commercially available and can be used in certain UF processes, including but not limited to regenerated cellulose, Pellicon™ (Millibos Sigma, Danvers, MA), stabilized cellulose, Sartocon® Slice, Sartocon® ECOHydrosart® (Sartorius, Göttingen, Germany), and polyethersulfone (PES) membranes, and Omega (Pall, Port Washington, NY).

[0085] As used herein, the terms “diafiltering,” “diafiltration,” and “DF” refer to the removal, replacement, or reduction of the concentration of certain components (e.g., salts) in a solution or mixture using a semipermeable membrane. In some embodiments, diafiltration achieves buffer exchange by adding fresh buffer to the retentate from the ultrafiltration process (i.e., the composition containing molecules too large to pass through the membrane that remain on the retentate side of the membrane).

[0086] As used herein, the term “percolation volume” is defined as “the total buffer volume introduced during percolation [divided by the initial retentate volume]” (i.e., the volume equivalent relative to the initial retentate volume used during percolation).

[0087] Non-restrictive instance features Without limitation, some example embodiments / features disclosed herein include: E1. A method for preparing a formulation comprising a recombinant protein, the method comprising: The ultrafiltration / percolation (UF / DF) membrane filter is equilibrated in a first percolation buffer to obtain a balanced UF / DF membrane filter; Using a balanced UF / DF membrane filter, the first composition containing the recombinant protein is concentrated by ultrafiltration to obtain a first concentrated composition; and The first concentrated composition is percolated with the first percolation buffer to obtain the second composition. The first percolation buffer is prepared by diluting a percolation buffer concentrate.

[0088] E2. The method as described in E1, wherein the percolation comprises percolating the first concentrated composition with 6 to 10 (e.g., 7 to 10; 6 to 9; 6; 7; 8; 9; 10) percolation volumes of the first percolation buffer.

[0089] E3. The method as described in E1 or E2, wherein the dilution is performed using an online dilution system.

[0090] E4. The method as described in any one of E1-E3, wherein the dilution is performed continuously using an online dilution system.

[0091] E5. The method as described in E3 or E4, wherein the pump of the online dilution system has a relative dilution accuracy in the range of 90% to 110% of the expected dilution ratio.

[0092] E6. The method of any one of E1-E5, wherein the first percolation buffer is prepared by diluting a percolation buffer concentrate with water (e.g., deionized water).

[0093] E7. The method of any one of E1-E6, wherein the first percolation buffer is prepared by diluting the percolation buffer concentrate at a desired dilution ratio of 1:1.

[0094] E8. The method of any one of E1-E7, wherein the first percolation buffer is prepared by diluting the percolation buffer concentrate with water (e.g., deionized water) at a desired dilution ratio of 1:1.

[0095] E9. The method of any one of E1-E8, wherein the first percolation buffer is prepared by diluting the percolation buffer concentrate with water (e.g., deionized water) at a desired dilution ratio of 1:1 using an online dilution system.

[0096] E10. The method of any one of E1-E9, wherein the first percolation buffer is continuously prepared by diluting the percolation buffer concentrate with water (e.g., deionized water) at a desired dilution ratio of 1:1 using an online dilution system.

[0097] E11. The method of any one of E1-E10, wherein the first percolation buffer is continuously prepared by diluting the percolation buffer concentrate with water (e.g., deionized water) at a desired dilution ratio of 1:1 using an online dilution system, wherein the pump of the online dilution system has a relative dilution accuracy in the range of 90% to 110% of the desired dilution ratio.

[0098] E12. The method of any one of E1-E11, wherein the recombinant protein is present in the first concentrated composition at a concentration of at least 100% of the concentration of the recombinant protein in the second composition, and optionally the first concentrated composition is diluted with a percolation buffer prior to the percolation, optionally wherein the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0099] E13. The method of any one of E1-E12, wherein the recombinant protein is present in the first concentrated composition at a concentration of at least 120% of the concentration of the recombinant protein in the second composition, and optionally the first concentrated composition is diluted with a percolation buffer prior to the percolation, optionally wherein the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0100] E14. The method of any one of E1-E13, wherein the recombinant protein is present in the first concentrated composition at a concentration of at least 140% of the concentration of the recombinant protein in the second composition, and optionally the first concentrated composition is diluted with a percolation buffer prior to the percolation, optionally wherein the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0101] E15. The method of any one of E1-E14, wherein the recombinant protein is present in the first concentrated composition at a concentration in the range of 100% to 150% of the concentration of the recombinant protein in the second composition (e.g., 110% to 140%; 120% to 140%; 105%; 110%; 115%; 120%; 125%; 130%; 135%; 140%; 145%; 150%), and optionally the first concentrated composition is diluted with a percolation buffer prior to the percolation, optionally wherein the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0102] E16. The method as described in any one of E1-15, wherein the other steps of the method do not use a percolation buffer prepared by dilution of a percolation buffer concentrate.

[0103] E17. The method as described in any one of E1-E16, wherein the method further comprises: The second composition is concentrated to obtain a second concentrated composition; The balanced UF / DF membrane filter was rinsed with a second percolation buffer to obtain the recovered composition, wherein the second percolation buffer was not prepared by diluting a percolation buffer concentrate; and The recycled composition is combined with the second concentrated composition to obtain a third composition.

[0104] E18. The method as described in E17, wherein concentrating the second composition includes ultrafiltration using a balanced UF / DF membrane filter.

[0105] E19. The method as described in any one of E1-E16, wherein the method further comprises: The balanced UF / DF membrane filter was rinsed with a second percolation buffer to obtain the recovered composition, wherein the second percolation buffer was not prepared by diluting a percolation buffer concentrate; and The recycled composition is combined with the second composition to obtain the third composition.

[0106] E20. The method of any one of E17-E19, wherein the first percolation buffer and the second percolation buffer contain the same excipients, and each excipient in the first percolation buffer is present in the second percolation buffer in an amount ranging from 90% to 110% of its amount in the second percolation buffer.

[0107] E21. The method as described in any one of E17-E20, wherein the first percolation buffer and the second percolation buffer have substantially the same composition.

[0108] E22. The method of any one of E17-E21, wherein the second percolation buffer is prepared in batches according to its intended composition.

[0109] E23. A method for preparing a formulation comprising a recombinant protein, the method comprising percolating a first composition comprising the recombinant protein with a first percolation buffer and a second percolation buffer to obtain a second composition, wherein the first percolation buffer is prepared by diluting a percolation buffer concentrate and the second percolation buffer is not prepared by diluting a percolation buffer concentrate.

[0110] E24. A method for preparing a formulation comprising a recombinant protein, the method comprising: The ultrafiltration / percolation (UF / DF) membrane filter is equilibrated in a first percolation buffer to obtain a balanced UF / DF membrane filter; Using a balanced UF / DF membrane filter, the first composition containing the recombinant protein is concentrated by ultrafiltration to obtain a first concentrated composition; and The first concentrated composition is percolated with the first percolation buffer and the second percolation buffer to obtain the second composition. in: The first percolation buffer was prepared by diluting a percolation buffer concentrate; and This second percolation buffer was not prepared by diluting a percolation buffer concentrate.

[0111] E25. The method as described in E23 or E24, wherein the first percolation buffer and the second percolation buffer contain the same excipients, and each excipient in the first percolation buffer is present in the second percolation buffer in an amount ranging from 90% to 110% of its amount in the second percolation buffer.

[0112] E26. The method of any one of E23-E25, wherein the first percolation buffer and the second percolation buffer have substantially the same composition.

[0113] E27. The method of any one of E23-E21, wherein the second percolation buffer is prepared in batches according to its intended composition.

[0114] E28. The method of any one of E23-E27, wherein the perfiltration comprises perfiltration of 6 to 6.5 perfiltration volumes with the first perfiltration buffer and then perfiltration of 0.5 to 1 perfiltration volume with the second perfiltration buffer.

[0115] E29. The method of any one of E23-E28, wherein the perfiltration comprises perfiltration of 6 to 6.5 perfiltration volumes with the first perfiltration buffer, followed by perfiltration of 0.5 to 1 perfiltration volume with the second perfiltration buffer, wherein the total number of perfiltration volumes used during the perfiltration is 7.

[0116] E30. The method of any one of E23-E29, wherein the perfiltration comprises perfiltration of 6.5 perfiltration volumes with the first perfiltration buffer and then perfiltration of 0.5 perfiltration volumes with the second perfiltration buffer.

[0117] E31. The method of any one of E23-E29, wherein the perfiltration comprises perfiltration of 6 perfiltration volumes with the first perfiltration buffer and then perfiltration of 1 perfiltration volume with the second perfiltration buffer.

[0118] E32. The method as described in any one of E23-E31, wherein the dilution is performed using an online dilution system.

[0119] E33. The method as described in any one of E23-E32, wherein the dilution is performed continuously using an online dilution system.

[0120] E34. The method as described in E32 or E33, wherein the pump of the online dilution system has a relative dilution accuracy in the range of 90% to 110% of the expected dilution ratio.

[0121] E35. The method of any one of E23-E34, wherein the first percolation buffer is prepared by diluting a percolation buffer concentrate with water (e.g., deionized water).

[0122] E36. The method of any one of E23-E35, wherein the first percolation buffer is prepared by diluting the percolation buffer concentrate at a desired dilution ratio of 1:1.

[0123] E37. The method of any one of E23-E36, wherein the first percolation buffer is prepared by diluting the percolation buffer concentrate with water (e.g., deionized water) at a desired dilution ratio of 1:1.

[0124] E38. The method of any one of E23-E37, wherein the first percolation buffer is prepared by diluting the percolation buffer concentrate with water (e.g., deionized water) at a desired dilution ratio of 1:1 using an online dilution system.

[0125] E39. The method of any one of E23-E38, wherein the first percolation buffer is continuously prepared by diluting the percolation buffer concentrate with water (e.g., deionized water) at a desired dilution ratio of 1:1 using an online dilution system.

[0126] E40. The method of any one of E23-E39, wherein the first percolation buffer is continuously prepared by diluting the percolation buffer concentrate with water (e.g., deionized water) at a desired dilution ratio of 1:1 using an online dilution system, wherein the pump of the online dilution system has a relative dilution accuracy in the range of 90% to 110% of the desired dilution ratio.

[0127] E41. The method of any one of E23-E40, wherein prior to the percolation, the recombinant protein is present in the first composition or the first concentrated composition at a concentration of at least 100% of the concentration of the recombinant protein in the second composition, and optionally prior to the percolation, the first composition or the first concentrated composition is diluted with a percolation buffer, optionally wherein the first composition or the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0128] E42. The method of any one of E23-E41, wherein prior to the percolation, the recombinant protein is present in the first composition or the first concentrated composition at a concentration of at least 120% of the concentration of the recombinant protein in the second composition, and optionally prior to the percolation, the first composition or the first concentrated composition is diluted with a percolation buffer, optionally wherein the first composition or the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0129] E43. The method of any one of E23-E42, wherein prior to the percolation, the recombinant protein is present in the first composition or the first concentrated composition at a concentration of at least 140% of the concentration of the recombinant protein in the second composition, and optionally prior to the percolation, the first composition or the first concentrated composition is diluted with a percolation buffer, optionally wherein the first composition or the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0130] E44. The method of any one of E23-E41, wherein prior to the percolation, the recombinant protein is present in the first composition or the first concentrated composition at a concentration in the range of 100% to 150% of the concentration of the recombinant protein in the second composition (e.g., 110% to 140%; 120% to 140%; 105%; 110%; 115%; 120%; 125%; 130%; 135%; 140%; 145%; 150%), and optionally prior to the percolation, the first composition or the first concentrated composition is diluted with a percolation buffer, optionally wherein the first composition or the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0131] E45. The method as described in any one of E23-E44, wherein other steps of the method do not use a percolation buffer prepared by dilution of a percolation buffer concentrate.

[0132] E46. The method as described in any one of E23-E45, wherein the method further comprises: The balanced UF / DF membrane filter was rinsed with a third percolation buffer to obtain the recovered composition, wherein the third percolation buffer was not prepared by diluting a percolation buffer concentrate; and The recycled composition is combined with the second composition to obtain the third composition.

[0133] E47. The method of any one of E23-E45, wherein the method further comprises: The second composition is concentrated to obtain a second concentrated composition; The balanced UF / DF membrane filter was rinsed with a third percolation buffer to obtain the recovered composition, wherein the third percolation buffer was not prepared by diluting a percolation buffer concentrate; and The recycled composition is combined with the second concentrated composition to obtain a third composition.

[0134] E48. The method as described in E47, wherein concentrating the second composition includes ultrafiltration using a balanced UF / DF membrane filter.

[0135] E49. The method of any one of E46-E48, wherein the first percolation buffer and the third percolation buffer contain the same excipients, and each excipient in the first percolation buffer is present in the third percolation buffer in an amount ranging from 90% to 110% of its amount in the third percolation buffer.

[0136] E50. The method as described in any one of E46-E49, wherein the first percolation buffer and the third percolation buffer have substantially the same composition.

[0137] E51. The method of any one of E46-E50, wherein the first percolation buffer, the second percolation buffer, and the third percolation buffer all have substantially the same composition.

[0138] E52. A method for preparing a formulation comprising a recombinant protein, the method comprising percolating a first composition comprising the recombinant protein with a first percolation buffer and a second percolation buffer to obtain a second composition, wherein the first percolation buffer is a concentrate of the second percolation buffer.

[0139] E53. The method as described in E52, wherein the first percolation buffer is at least 2X (e.g., at least 2.5X; at least 3X; 2X; 2.25X; 2.5X; 3X; 3.25X; 3.5X; 3.75X; 4X; 4.25X; 4.5X; 4.75X; 5X; 2X to 3X; 2X to 4X; 2X to 5X) concentrate of the second percolation buffer.

[0140] E54. The method as described in E52 or E43, wherein the first percolation buffer is a 2X concentrate of the second percolation buffer.

[0141] E55. The method of any one of E52-E54, wherein the perfiltration comprises perfiltration of 0.5 to 3 perfiltration volumes with the first perfiltration buffer, followed by perfiltration of 6.5 to 9.5 perfiltration volumes with the second perfiltration buffer.

[0142] E56. The method of any one of E52-E55, wherein the perfiltration comprises perfiltration of 0.5 to 3 perfiltration volumes with the first perfiltration buffer, followed by perfiltration of 6.5 to 9.5 perfiltration volumes with the second perfiltration buffer, wherein the total number of perfiltration volumes used during the perfiltration is at most 10.

[0143] E57. The method of any one of E52-E56, wherein the perfiltration comprises perfiltration of 0.5 to 3 perfiltration volumes with the first perfiltration buffer, followed by perfiltration of 6.5 to 9.5 perfiltration volumes with the second perfiltration buffer, wherein the total number of perfiltration volumes used during the perfiltration is 10.

[0144] E58. The method of any one of E52-E57, wherein the perfiltration comprises perfiltration of 0.5 perfiltration volumes with the first perfiltration buffer and then perfiltration of 6.5 to 9.5 perfiltration volumes with the second perfiltration buffer.

[0145] E59. The method of any one of E52-E57, wherein the perfiltration comprises perfiltration of one perfiltration volume with the first perfiltration buffer, followed by perfiltration of 6 to 9 perfiltration volumes with the second perfiltration buffer.

[0146] E60. The method of any one of E52-E57, wherein the perfiltration comprises perfiltration of two perfiltration volumes with the first perfiltration buffer, followed by perfiltration of 5 to 8 perfiltration volumes with the second perfiltration buffer.

[0147] E61. The method of any one of E52-E57, wherein the perfiltration comprises perfiltration of three perfiltration volumes with the first perfiltration buffer, followed by perfiltration of four to seven perfiltration volumes with the second perfiltration buffer.

[0148] E62. The method of any one of E52-E61, wherein the recombinant protein is present in the first composition at a concentration of at least 100% of the concentration of the recombinant protein in the second composition.

[0149] E63. The method of any one of E52-E62, wherein the recombinant protein is present in the first composition at a concentration of at least 120% of the concentration of the recombinant protein in the second composition.

[0150] E64. The method of any one of E52-E63, wherein the recombinant protein is present in the first composition at a concentration of at least 140% of the concentration of the recombinant protein in the second composition.

[0151] E65. The method of any one of E52-E62, wherein the recombinant protein is present in the first composition at a concentration in the range of 100% to 150% of the concentration of the recombinant protein in the second composition (e.g., 110% to 140%; 120% to 140%; 105%; 110%; 115%; 120%; 125%; 130%; 135%; 140%; 145%; 150%).

[0152] E66. A method for preparing a formulation comprising a recombinant protein, the method comprising: The ultrafiltration / percolation (UF / DF) membrane filter is equilibrated in a first percolation buffer to obtain a balanced UF / DF membrane filter; Using a balanced UF / DF membrane filter, the first composition containing the recombinant protein is concentrated by ultrafiltration to obtain a first concentrated composition; and The first concentrated composition is percolated with a second percolation buffer and a third percolation buffer to obtain a second composition, wherein the second percolation buffer is a concentrate of the third percolation buffer.

[0153] E67. The method as described in E66, wherein the first percolation buffer and the third percolation buffer contain the same excipients, and each excipient in the first percolation buffer is present in the third percolation buffer in an amount ranging from 90% to 110% of its amount in the third percolation buffer.

[0154] E68. The method as described in E66 or E67, wherein the first percolation buffer and the third percolation buffer have substantially the same composition.

[0155] E69. The method of any one of E66-E68, wherein the second percolation buffer is a concentrate of the third percolation buffer at least 2X (e.g., at least 2.5X; at least 3X; 2X; 2.25X; 2.5X; 3X; 3.25X; 3.5X; 3.75X; 4X; 4.25X; 4.5X; 4.75X; 5X; 2X to 3X; 2X to 4X; 2X to 5X).

[0156] E70. The method as described in any one of E66-E69, wherein the second percolation buffer is a 2X concentrate of the third percolation buffer.

[0157] E71. The method of any one of E66-E70, wherein the perfiltration comprises perfiltration of 0.5 to 3 perfiltration volumes with the second perfiltration buffer, followed by perfiltration of 6.5 to 9.5 perfiltration volumes with the third perfiltration buffer.

[0158] E72. The method of any one of E66-E71, wherein the perfiltration comprises perfiltration of 0.5 to 3 perfiltration volumes with the second perfiltration buffer, followed by perfiltration of 6.5 to 9.5 perfiltration volumes with the third perfiltration buffer, wherein the total number of perfiltration volumes used during the perfiltration is at most 10.

[0159] E73. The method of any one of E66-E72, wherein the perfiltration comprises perfiltration of 0.5 to 3 perfiltration volumes with the second perfiltration buffer, followed by perfiltration of 6.5 to 9.5 perfiltration volumes with the third perfiltration buffer, wherein the total number of perfiltration volumes used during the perfiltration is 10.

[0160] E74. The method of any one of E66-E73, wherein the perfiltration comprises perfiltration of 0.5 perfiltration volumes with the second perfiltration buffer, followed by perfiltration of 6.5 to 9.5 perfiltration volumes with the third perfiltration buffer.

[0161] E75. The method of any one of E66-E73, wherein the perfiltration comprises perfiltration of one perfiltration volume with the second perfiltration buffer, followed by perfiltration of 6 to 9 perfiltration volumes with the third perfiltration buffer.

[0162] E76. The method of any one of E66-E73, wherein the percolation comprises percolating two percolation volumes with the second percolation buffer and then percolating 5 to 8 percolation volumes with the third percolation buffer.

[0163] E77. The method of any one of E66-E73, wherein the perfiltration comprises perfiltration of three perfiltration volumes with the second perfiltration buffer, followed by perfiltration of four to seven perfiltration volumes with the third perfiltration buffer.

[0164] E78. The method of any one of E66-E77, wherein the recombinant protein is present in the first concentrated composition at a concentration of at least 100% of the concentration of the recombinant protein in the second composition.

[0165] E79. The method of any one of E66-E78, wherein the recombinant protein is present in the first concentrated composition at a concentration of at least 120% of the concentration of the recombinant protein in the second composition.

[0166] E80. The method of any one of E66-E79, wherein the recombinant protein is present in the first concentrated composition at a concentration of at least 140% of the concentration of the recombinant protein in the second composition.

[0167] E81. The method of any one of E66-E78, wherein the recombinant protein is present in the first concentrated composition at a concentration in the range of 100% to 150% of the concentration of the recombinant protein in the second composition (e.g., 110% to 140%; 120% to 140%; 105%; 110%; 115%; 120%; 125%; 130%; 135%; 140%; 145%; 150%).

[0168] E82. A method for preparing a formulation comprising a recombinant protein, the method comprising: The first composition containing the recombinant protein is concentrated by ultrafiltration to obtain a first concentrated composition; and The first concentrated composition is percolated to obtain a second composition, wherein the recombinant protein is present in the first concentrated composition at a concentration of at least 120% of the concentration of the recombinant protein in the second composition.

[0169] E83. A method for preparing a formulation comprising a recombinant protein, the method comprising: The first composition containing the recombinant protein is concentrated by ultrafiltration to obtain a first concentrated composition; and The first concentrated composition is percolated with a first percolation buffer and a second percolation buffer to obtain a second composition, wherein: The first percolation buffer is a concentrate of the second percolation buffer; and The recombinant protein is present in the first concentrated composition at a concentration of at least 120% of the concentration of the recombinant protein in the second composition.

[0170] E84. The method as described in E83, wherein the first percolation buffer is at least 2X (e.g., at least 2.5X; at least 3X; 2X; 2.25X; 2.5X; 3X; 3.25X; 3.5X; 3.75X; 4X; 4.25X; 4.5X; 4.75X; 5X; 2X to 3X; 2X to 4X; 2X to 5X) concentrate of the second percolation buffer.

[0171] E85. The method as described in E83 or E84, wherein the first percolation buffer is a 2X concentrate of the second percolation buffer.

[0172] E86. The method of any one of E83-E85, wherein the perfiltration comprises perfiltration of 0.5 to 3 perfiltration volumes with the first perfiltration buffer and then perfiltration of 6.5 to 9.5 perfiltration volumes with the second perfiltration buffer.

[0173] E87. The method of any one of E83-E86, wherein the perfiltration comprises perfiltration of 0.5 to 3 perfiltration volumes with the first perfiltration buffer, followed by perfiltration of 6.5 to 9.5 perfiltration volumes with the second perfiltration buffer, wherein the total number of perfiltration volumes used during the perfiltration is at most 10.

[0174] E88. The method of any one of E83-E87, wherein the perfiltration comprises perfiltration of 0.5 to 3 perfiltration volumes with the first perfiltration buffer, followed by perfiltration of 6.5 to 9.5 perfiltration volumes with the second perfiltration buffer, wherein the total number of perfiltration volumes used during the perfiltration is 10.

[0175] E89. The method of any one of E83-E88, wherein the perfiltration comprises perfiltration of 0.5 perfiltration volumes with the first perfiltration buffer and then perfiltration of 6.5 to 9.5 perfiltration volumes with the second perfiltration buffer.

[0176] E90. The method as described in any one of E83-E88, wherein the perfiltration comprises perfiltration of one perfiltration volume with the first perfiltration buffer, followed by perfiltration of 6 to 9 perfiltration volumes with the second perfiltration buffer.

[0177] E91. The method of any one of E83-E88, wherein the perfiltration comprises perfiltration of two perfiltration volumes with the first perfiltration buffer, followed by perfiltration of 5 to 8 perfiltration volumes with the second perfiltration buffer.

[0178] E92. The method of any one of E83-E88, wherein the perfiltration comprises perfiltration of three perfiltration volumes with the first perfiltration buffer, followed by perfiltration of four to seven perfiltration volumes with the second perfiltration buffer.

[0179] E93. The method as described in any one of E82-E92, wherein the first concentrated composition is diluted with a percolation buffer prior to the percolation.

[0180] E94. The method of any one of E82-E93, wherein the first concentrated composition is diluted with a percolation buffer such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0181] E95. The method of any one of E82-E94, wherein the recombinant protein is present in the first concentrated composition at a concentration of at least 140% of the concentration of the recombinant protein in the second composition, and optionally the first concentrated composition is diluted with a percolation buffer prior to the percolation, preferably wherein the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0182] E96. The method of any one of E82-E93, wherein the recombinant protein is present in the first concentrated composition at a concentration in the range of 120% to 150% of the concentration of the recombinant protein in the second composition (e.g., 120% to 140%; 120%; 125%; 130%; 135%; 140%; 145%; 150%), and optionally the first concentrated composition is diluted with a percolation buffer prior to the percolation, preferably wherein the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0183] E97. The method as described in any one of E1-E96, wherein the recombinant protein is an antibody.

[0184] E98. The method as described in any one of E1-E96, wherein the recombinant protein is a human antibody.

[0185] E99. The method as described in any one of E1-E96, wherein the recombinant protein is an IgG1, IgG2 or IgG4 antibody.

[0186] E100. The method as described in any one of E1-E96, wherein the recombinant protein is a human IgG1, IgG2 or IgG4 antibody.

[0187] E101. The method as described in any one of E1-E96, wherein the recombinant protein is an antibody fragment.

[0188] E102. The method as described in any one of E1-E97, wherein the recombinant protein is a fusion protein.

[0189] Ultrafiltration / percolation process This article provides a method for preparing formulations containing recombinant proteins, the method comprising: The ultrafiltration / percolation (UF / DF) membrane filter is equilibrated in a first percolation buffer to obtain a balanced UF / DF membrane filter; Using a balanced UF / DF membrane filter, the first composition containing the recombinant protein is concentrated by ultrafiltration to obtain a first concentrated composition; and The first concentrated composition is percolated with the first percolation buffer to obtain the second composition. The first percolation buffer is prepared by diluting a percolation buffer concentrate.

[0190] In some embodiments, perfiltration includes perfiltration of the first concentrated composition with 6 to 10 (e.g., 7 to 10; 6 to 9; 6; 7; 8; 9; 10) perfiltration volumes of a first perfiltration buffer. In some embodiments, perfiltration includes perfiltration of the first concentrated composition with 7 to 10 perfiltration volumes of a first perfiltration buffer. In some embodiments, perfiltration includes perfiltration of the first concentrated composition with 8 to 10 perfiltration volumes of a first perfiltration buffer.

[0191] In some embodiments, perfiltration includes perfiltration of the first concentrated composition with 6 perfiltration volumes of a first perfiltration buffer. In some embodiments, perfiltration includes perfiltration of the first concentrated composition with 7 perfiltration volumes of a first perfiltration buffer. In some embodiments, perfiltration includes perfiltration of the first concentrated composition with 8 perfiltration volumes of a first perfiltration buffer. In some embodiments, perfiltration includes perfiltration of the first concentrated composition with 9 perfiltration volumes of a first perfiltration buffer. In some embodiments, perfiltration includes perfiltration of the first concentrated composition with 10 perfiltration volumes of a first perfiltration buffer.

[0192] In some embodiments, dilution is performed using an online dilution system. In some embodiments, the pumps of the online dilution system have a relative dilution accuracy in the range of 90% to 110% of the expected dilution ratio (e.g., 95% to 105%; 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99%; 100%; 101%; 102%; 103%; 104%; 105%; 106%; 107%; 108%; 109%; 110%).

[0193] In some embodiments, dilution is performed continuously using an online dilution system. In some embodiments, the pumps of the online dilution system have a relative dilution accuracy in the range of 90% to 110% of the expected dilution ratio (e.g., 95% to 105%; 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99%; 100%; 101%; 102%; 103%; 104%; 105%; 106%; 107%; 108%; 109%; 110%).

[0194] In some embodiments, the first percolation buffer is prepared by diluting a percolation buffer concentrate with water (e.g., deionized water). In some embodiments, the first percolation buffer is prepared by diluting a percolation buffer concentrate at a desired dilution ratio of 1:1. In some embodiments, the first percolation buffer is prepared by diluting a percolation buffer concentrate with water (e.g., deionized water) at a desired dilution ratio of 1:1.

[0195] In some embodiments, the first percolation buffer is continuously prepared by diluting the percolation buffer concentrate with water (e.g., deionized water) at a desired dilution ratio of 1:1 using an online dilution system. In some embodiments, the first percolation buffer is continuously prepared by diluting the percolation buffer concentrate with water (e.g., deionized water) at a desired dilution ratio of 1:1 using an online dilution system, wherein the pump of the online dilution system has a relative dilution accuracy in the range of 90% to 110% of the desired dilution ratio (e.g., 95% to 105%; 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99%; 100%; 101%; 102%; 103%; 104%; 105%; 106%; 107%; 108%; 109%; 110%).

[0196] In some embodiments, the recombinant protein is present in the first concentrated composition at a concentration of at least 100% (e.g., at least 105%; at least 110%; at least 115%; at least 120%; at least 125%; at least 130%; at least 135%; at least 140%; 100%; 105%; 110%; 115%; 120%; 125%; 130%; 135%; 140%; 145%; 150%) of the concentration of the recombinant protein in the second composition, and the first concentrated composition is optionally diluted with a percolation buffer prior to percolation. In some embodiments, the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0197] In some embodiments, the recombinant protein is present in the first concentrated composition at a concentration of at least 120% of the concentration of the recombinant protein in the second composition, and the first concentrated composition is optionally diluted with a percolation buffer prior to percolation. In some embodiments, the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0198] In some embodiments, the recombinant protein is present in the first concentrated composition at a concentration of at least 140% of the concentration of the recombinant protein in the second composition, and the first concentrated composition is optionally diluted with a percolation buffer prior to percolation. In some embodiments, the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0199] In some embodiments, the recombinant protein is present in the first concentrated composition at a concentration in the range of 100% to 150% of the concentration of the recombinant protein in the second composition (e.g., 110% to 140%; 120% to 140%; 105%; 110%; 115%; 120%; 125%; 130%; 135%; 140%; 145%; 150%), and the first concentrated composition is optionally diluted with a percolation buffer prior to percolation. In some embodiments, the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0200] In some embodiments, other steps of the method do not use percolation buffer prepared by dilution of percolation buffer concentrate.

[0201] In some embodiments, the method further includes: The second composition is concentrated to obtain a second concentrated composition; The balanced UF / DF membrane filter was rinsed with a second percolation buffer to obtain the recovered composition, wherein the second percolation buffer was not prepared by diluting a percolation buffer concentrate; and The recovered composition is combined with the second concentrated composition to obtain a third composition.

[0202] In some embodiments, concentrating the second composition includes ultrafiltration using a balanced UF / DF membrane filter.

[0203] In some embodiments, the first percolation buffer and the second percolation buffer contain the same excipients, and each excipient in the first percolation buffer is present in the second percolation buffer in an amount ranging from 90% to 110% of its amount in the second percolation buffer (e.g., 95% to 105%; 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99%; 100%; 101%; 102%; 103%; 104%; 105%; 106%; 107%; 108%; 109%; 110%).

[0204] In some embodiments, the first percolation buffer and the second percolation buffer have substantially the same composition.

[0205] In some embodiments, the second percolation buffer is prepared in batches according to its intended composition.

[0206] In other embodiments, the method further includes: The balanced UF / DF membrane filter was rinsed with a second percolation buffer to obtain the recovered composition, wherein the second percolation buffer was not prepared by diluting a percolation buffer concentrate; and The recycled composition is combined with the second composition to obtain the third composition.

[0207] In some embodiments, the first percolation buffer and the second percolation buffer contain the same excipients, and each excipient in the first percolation buffer is present in the second percolation buffer in an amount ranging from 90% to 110% of its amount in the second percolation buffer (e.g., 95% to 105%; 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99%; 100%; 101%; 102%; 103%; 104%; 105%; 106%; 107%; 108%; 109%; 110%).

[0208] In some embodiments, the first percolation buffer and the second percolation buffer have substantially the same composition.

[0209] In some embodiments, the second percolation buffer is prepared in batches according to its intended composition.

[0210] In some embodiments, the recombinant protein is an antigen-binding protein. In some embodiments, the recombinant protein is an antibody or an antibody fragment. In some embodiments, the recombinant protein is an antibody. In some embodiments, the recombinant protein is an antibody fragment.

[0211] In some embodiments, the recombinant protein is an IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is an IgG1 antibody. In some embodiments, the recombinant protein is an IgG2 antibody. In some embodiments, the recombinant protein is an IgG4 antibody.

[0212] In some embodiments, the recombinant protein is a human antibody. In some embodiments, the recombinant protein is a human IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is a human IgG1 antibody. In some embodiments, the recombinant protein is a human IgG2 antibody. In some embodiments, the recombinant protein is a human IgG4 antibody.

[0213] In some embodiments, the recombinant protein is a fragment of an IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is a fragment of an IgG1 antibody. In some embodiments, the recombinant protein is a fragment of an IgG2 antibody. In some embodiments, the recombinant protein is a fragment of an IgG4 antibody.

[0214] In some embodiments, the recombinant protein is a fragment of a human antibody. In some embodiments, the recombinant protein is a fragment of a human IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is a fragment of a human IgG1 antibody. In some embodiments, the recombinant protein is a fragment of a human IgG2 antibody. In some embodiments, the recombinant protein is a fragment of a human IgG4 antibody.

[0215] In some embodiments, the recombinant protein is a fusion protein.

[0216] This document also provides a method for preparing a formulation containing a recombinant protein, the method comprising percolating a first composition containing the recombinant protein with a first percolation buffer and a second percolation buffer to obtain a second composition, wherein the first percolation buffer is prepared by diluting a percolation buffer concentrate, and the second percolation buffer is not prepared by diluting a percolation buffer concentrate. For example, in some embodiments, the second percolation buffer is prepared in batches according to its intended composition.

[0217] In some embodiments, the first percolation buffer and the second percolation buffer contain the same excipients, and each excipient in the first percolation buffer is present in the second percolation buffer in an amount ranging from 90% to 110% of its amount in the second percolation buffer (e.g., 95% to 105%; 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99%; 100%; 101%; 102%; 103%; 104%; 105%; 106%; 107%; 108%; 109%; 110%).

[0218] In some embodiments, the first percolation buffer and the second percolation buffer have substantially the same composition.

[0219] In some embodiments, perfiltration includes perfiltration of 6 to 6.5 perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 0.5 to 1 perfiltration volume with a second perfiltration buffer.

[0220] In some embodiments, perfiltration includes perfiltration of 6 to 6.5 perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 0.5 to 1 perfiltration volume with a second perfiltration buffer, wherein the total number of perfiltration volumes used during perfiltration is 7. In some embodiments, perfiltration includes perfiltration of 6 perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 1 perfiltration volume with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of 6.5 perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 0.5 perfiltration volumes with a second perfiltration buffer.

[0221] In some embodiments, dilution is performed using an online dilution system. In some embodiments, the pumps of the online dilution system have a relative dilution accuracy in the range of 90% to 110% of the expected dilution ratio (e.g., 95% to 105%; 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99%; 100%; 101%; 102%; 103%; 104%; 105%; 106%; 107%; 108%; 109%; 110%).

[0222] In some embodiments, dilution is performed continuously using an online dilution system. In some embodiments, the pumps of the online dilution system have a relative dilution accuracy in the range of 90% to 110% of the expected dilution ratio (e.g., 95% to 105%; 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99%; 100%; 101%; 102%; 103%; 104%; 105%; 106%; 107%; 108%; 109%; 110%).

[0223] In some embodiments, the first percolation buffer is prepared by diluting a percolation buffer concentrate with water (e.g., deionized water). In some embodiments, the first percolation buffer is prepared by diluting a percolation buffer concentrate at a desired dilution ratio of 1:1. In some embodiments, the first percolation buffer is prepared by diluting a percolation buffer concentrate with water (e.g., deionized water) at a desired dilution ratio of 1:1.

[0224] In some embodiments, the first percolation buffer is continuously prepared by diluting the percolation buffer concentrate with water (e.g., deionized water) at a desired dilution ratio of 1:1 using an online dilution system. In some embodiments, the first percolation buffer is continuously prepared by diluting the percolation buffer concentrate with water (e.g., deionized water) at a desired dilution ratio of 1:1 using an online dilution system, wherein the pump of the online dilution system has a relative dilution accuracy in the range of 90% to 110% of the desired dilution ratio (e.g., 95% to 105%; 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99%; 100%; 101%; 102%; 103%; 104%; 105%; 106%; 107%; 108%; 109%; 110%).

[0225] In some embodiments, prior to percolation, the recombinant protein is present in the first concentrated composition at a concentration of at least 100% (e.g., at least 105%; at least 110%; at least 115%; at least 120%; at least 125%; at least 130%; at least 135%; at least 140%; 100%; 105%; 110%; 115%; 120%; 125%; 130%; 135%; 140%; 145%; 150%) of the concentration of the recombinant protein in the second composition, and optionally, the first concentrated composition is diluted with percolation buffer prior to percolation. In some embodiments, the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0226] In some embodiments, prior to percolation, the recombinant protein is present in the first composition at a concentration of at least 120% of the concentration of the recombinant protein in the second composition, and optionally, the first composition is diluted with percolation buffer prior to percolation. In some embodiments, the first composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0227] In some embodiments, prior to percolation, the recombinant protein is present in the first composition at a concentration of at least 140% of the concentration of the recombinant protein in the second composition, and optionally, the first composition is diluted with percolation buffer prior to percolation. In some embodiments, the first composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0228] In some embodiments, prior to percolation, the recombinant protein is present in the first composition at a concentration in the range of 100% to 150% of the concentration of the recombinant protein in the second composition (e.g., 110% to 140%; 120% to 140%; 105%; 110%; 115%; 120%; 125%; 130%; 135%; 140%; 145%; 150%), and optionally the first composition is diluted with percolation buffer prior to percolation. In some embodiments, the first composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0229] In some embodiments, other steps of the method do not use percolation buffer prepared by dilution of percolation buffer concentrate.

[0230] In some embodiments, the method further includes: The balanced UF / DF membrane filter was rinsed with a third percolation buffer to obtain the recovered composition, wherein the third percolation buffer was not prepared by diluting a percolation buffer concentrate; and The recycled composition is combined with the second composition to obtain the third composition.

[0231] In other embodiments, the method further includes: The second composition is concentrated to obtain a second concentrated composition; The balanced UF / DF membrane filter was rinsed with a third percolation buffer to obtain the recovered composition, wherein the third percolation buffer was not prepared by diluting a percolation buffer concentrate; and The recovered composition is combined with the second concentrated composition to obtain a third composition.

[0232] In some embodiments, concentrating the second composition includes ultrafiltration using a balanced UF / DF membrane filter.

[0233] In some embodiments, the first percolation buffer and the third percolation buffer contain the same excipients, and each excipient in the first percolation buffer is present in the third percolation buffer in an amount ranging from 90% to 110% of its amount in the third percolation buffer (e.g., 95% to 105%; 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99%; 100%; 101%; 102%; 103%; 104%; 105%; 106%; 107%; 108%; 109%; 110%).

[0234] In some embodiments, the first percolation buffer and the third percolation buffer have substantially the same composition. In some embodiments, the first percolation buffer, the second percolation buffer, and the third percolation buffer all have substantially the same composition.

[0235] In some embodiments, the recombinant protein is an antigen-binding protein. In some embodiments, the recombinant protein is an antibody or an antibody fragment. In some embodiments, the recombinant protein is an antibody. In some embodiments, the recombinant protein is an antibody fragment.

[0236] In some embodiments, the recombinant protein is an IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is an IgG1 antibody. In some embodiments, the recombinant protein is an IgG2 antibody. In some embodiments, the recombinant protein is an IgG4 antibody.

[0237] In some embodiments, the recombinant protein is a human antibody. In some embodiments, the recombinant protein is a human IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is a human IgG1 antibody. In some embodiments, the recombinant protein is a human IgG2 antibody. In some embodiments, the recombinant protein is a human IgG4 antibody.

[0238] In some embodiments, the recombinant protein is a fragment of an IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is a fragment of an IgG1 antibody. In some embodiments, the recombinant protein is a fragment of an IgG2 antibody. In some embodiments, the recombinant protein is a fragment of an IgG4 antibody.

[0239] In some embodiments, the recombinant protein is a fragment of a human antibody. In some embodiments, the recombinant protein is a fragment of a human IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is a fragment of a human IgG1 antibody. In some embodiments, the recombinant protein is a fragment of a human IgG2 antibody. In some embodiments, the recombinant protein is a fragment of a human IgG4 antibody.

[0240] In some embodiments, the recombinant protein is a fusion protein.

[0241] This article also provides a method for preparing a formulation containing a recombinant protein, the method comprising: The ultrafiltration / percolation (UF / DF) membrane filter is equilibrated in a first percolation buffer to obtain a balanced UF / DF membrane filter; Using a balanced UF / DF membrane filter, the first composition containing the recombinant protein is concentrated by ultrafiltration to obtain a first concentrated composition; and The first concentrated composition is percolated with the first percolation buffer and the second percolation buffer to obtain the second composition. in: The first percolation buffer was prepared by diluting a percolation buffer concentrate; and The second percolation buffer was not prepared by diluting the percolation buffer concentrate.

[0242] In some embodiments, the second percolation buffer is prepared in batches according to its intended composition.

[0243] In some embodiments, the first percolation buffer and the second percolation buffer contain the same excipients, and each excipient in the first percolation buffer is present in the second percolation buffer in an amount ranging from 90% to 110% of its amount in the second percolation buffer (e.g., 95% to 105%; 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99%; 100%; 101%; 102%; 103%; 104%; 105%; 106%; 107%; 108%; 109%; 110%).

[0244] In some embodiments, the first percolation buffer and the second percolation buffer have substantially the same composition.

[0245] In some embodiments, perfiltration includes perfiltration of 6 to 6.5 perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 0.5 to 1 perfiltration volume with a second perfiltration buffer.

[0246] In some embodiments, perfiltration includes perfiltration of 6 to 6.5 perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 0.5 to 1 perfiltration volume with a second perfiltration buffer, wherein the total number of perfiltration volumes used during perfiltration is 7. In some embodiments, perfiltration includes perfiltration of 6 perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 1 perfiltration volume with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of 6.5 perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 0.5 perfiltration volumes with a second perfiltration buffer.

[0247] In some embodiments, dilution is performed using an online dilution system. In some embodiments, the pumps of the online dilution system have a relative dilution accuracy in the range of 90% to 110% of the expected dilution ratio (e.g., 95% to 105%; 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99%; 100%; 101%; 102%; 103%; 104%; 105%; 106%; 107%; 108%; 109%; 110%).

[0248] In some embodiments, dilution is performed continuously using an online dilution system. In some embodiments, the pumps of the online dilution system have a relative dilution accuracy in the range of 90% to 110% of the expected dilution ratio (e.g., 95% to 105%; 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99%; 100%; 101%; 102%; 103%; 104%; 105%; 106%; 107%; 108%; 109%; 110%).

[0249] In some embodiments, the first percolation buffer is prepared by diluting a percolation buffer concentrate with water (e.g., deionized water). In some embodiments, the first percolation buffer is prepared by diluting a percolation buffer concentrate at a desired dilution ratio of 1:1. In some embodiments, the first percolation buffer is prepared by diluting a percolation buffer concentrate with water (e.g., deionized water) at a desired dilution ratio of 1:1.

[0250] In some embodiments, the first percolation buffer is continuously prepared by diluting the percolation buffer concentrate with water (e.g., deionized water) at a desired dilution ratio of 1:1 using an online dilution system. In some embodiments, the first percolation buffer is continuously prepared by diluting the percolation buffer concentrate with water (e.g., deionized water) at a desired dilution ratio of 1:1 using an online dilution system, wherein the pump of the online dilution system has a relative dilution accuracy in the range of 90% to 110% of the desired dilution ratio (e.g., 95% to 105%; 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99%; 100%; 101%; 102%; 103%; 104%; 105%; 106%; 107%; 108%; 109%; 110%).

[0251] In some embodiments, prior to percolation, the recombinant protein is present in the first concentrated composition at a concentration of at least 100% (e.g., at least 105%; at least 110%; at least 115%; at least 120%; at least 125%; at least 130%; at least 135%; at least 140%; 100%; 105%; 110%; 115%; 120%; 125%; 130%; 135%; 140%; 145%; 150%) of the concentration of the recombinant protein in the second composition, and optionally, the first concentrated composition is diluted with percolation buffer prior to percolation. In some embodiments, the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0252] In some embodiments, prior to percolation, the recombinant protein is present in the first concentrated composition at a concentration of at least 120% of the concentration of the recombinant protein in the second composition, and optionally, the first concentrated composition is diluted with percolation buffer prior to percolation. In some embodiments, the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0253] In some embodiments, prior to percolation, the recombinant protein is present in the first concentrated composition at a concentration of at least 140% of the concentration of the recombinant protein in the second composition, and optionally, the first concentrated composition is diluted with percolation buffer prior to percolation. In some embodiments, the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0254] In some embodiments, prior to percolation, the recombinant protein is present in the first concentrated composition at a concentration in the range of 100% to 150% of the concentration of the recombinant protein in the second composition (e.g., 110% to 140%; 120% to 140%; 105%; 110%; 115%; 120%; 125%; 130%; 135%; 140%; 145%; 150%), and optionally, the first concentrated composition is diluted with percolation buffer prior to percolation. In some embodiments, the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0255] In some embodiments, other steps of the method do not use percolation buffer prepared by dilution of percolation buffer concentrate.

[0256] In some embodiments, the method further includes: The balanced UF / DF membrane filter was rinsed with a third percolation buffer to obtain the recovered composition, wherein the third percolation buffer was not prepared by diluting a percolation buffer concentrate; and The recycled composition is combined with the second composition to obtain the third composition.

[0257] In other embodiments, the method further includes: The second composition is concentrated to obtain a second concentrated composition; The balanced UF / DF membrane filter was rinsed with a third percolation buffer to obtain the recovered composition, wherein the third percolation buffer was not prepared by diluting a percolation buffer concentrate; and The recovered composition is combined with the second concentrated composition to obtain a third composition.

[0258] In some embodiments, concentrating the second composition includes ultrafiltration using a balanced UF / DF membrane filter.

[0259] In some embodiments, the first percolation buffer and the third percolation buffer contain the same excipients, and each excipient in the first percolation buffer is present in the third percolation buffer in an amount ranging from 90% to 110% of its amount in the third percolation buffer (e.g., 95% to 105%; 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99%; 100%; 101%; 102%; 103%; 104%; 105%; 106%; 107%; 108%; 109%; 110%).

[0260] In some embodiments, the first percolation buffer and the third percolation buffer have substantially the same composition. In some embodiments, the first percolation buffer, the second percolation buffer, and the third percolation buffer all have substantially the same composition.

[0261] In some embodiments, the recombinant protein is an antigen-binding protein. In some embodiments, the recombinant protein is an antibody or an antibody fragment. In some embodiments, the recombinant protein is an antibody. In some embodiments, the recombinant protein is an antibody fragment.

[0262] In some embodiments, the recombinant protein is an IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is an IgG1 antibody. In some embodiments, the recombinant protein is an IgG2 antibody. In some embodiments, the recombinant protein is an IgG4 antibody.

[0263] In some embodiments, the recombinant protein is a human antibody. In some embodiments, the recombinant protein is a human IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is a human IgG1 antibody. In some embodiments, the recombinant protein is a human IgG2 antibody. In some embodiments, the recombinant protein is a human IgG4 antibody.

[0264] In some embodiments, the recombinant protein is a fragment of an IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is a fragment of an IgG1 antibody. In some embodiments, the recombinant protein is a fragment of an IgG2 antibody. In some embodiments, the recombinant protein is a fragment of an IgG4 antibody.

[0265] In some embodiments, the recombinant protein is a fragment of a human antibody. In some embodiments, the recombinant protein is a fragment of a human IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is a fragment of a human IgG1 antibody. In some embodiments, the recombinant protein is a fragment of a human IgG2 antibody. In some embodiments, the recombinant protein is a fragment of a human IgG4 antibody.

[0266] In some embodiments, the recombinant protein is a fusion protein.

[0267] This article also provides a method for preparing a formulation containing a recombinant protein, the method comprising percolating a first composition containing the recombinant protein with a first percolation buffer and a second percolation buffer to obtain a second composition, wherein the first percolation buffer is a concentrate of the second percolation buffer.

[0268] In some embodiments, the first percolation buffer is at least a 2X (e.g., at least 2.5X; at least 3X; 2X; 2.25X; 2.5X; 3X; 3.25X; 3.5X; 3.75X; 4X; 4.25X; 4.5X; 4.75X; 5X; 2X to 3X; 2X to 4X; 2X to 5X) concentrate of the second percolation buffer. In some embodiments, the first percolation buffer is a 2X concentrate of the second percolation buffer. In some embodiments, the first percolation buffer is a 3X concentrate of the second percolation buffer. In some embodiments, the first percolation buffer is a 4X concentrate of the second percolation buffer. In some embodiments, the first percolation buffer is a 5X concentrate of the second percolation buffer.

[0269] In some embodiments, perfiltration includes perfiltration of 0.5 to 3 perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 6.5 to 9.5 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of 0.5 to 3 perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 6.5 to 9.5 perfiltration volumes with a second perfiltration buffer, wherein the total number of perfiltration volumes used during perfiltration is at most 10. In some embodiments, perfiltration includes perfiltration of 0.5 to 3 perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 6.5 to 9.5 perfiltration volumes with a second perfiltration buffer, wherein the total number of perfiltration volumes used during perfiltration is 10.

[0270] In some embodiments, perfiltration includes perfiltration of 0.5 perfiltration volume with a first perfiltration buffer, followed by perfiltration of 6.5 to 9.5 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of 0.5 perfiltration volume with a first perfiltration buffer, followed by perfiltration of 6.5 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of 0.5 perfiltration volume with a first perfiltration buffer, followed by perfiltration of 7.5 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of 0.5 perfiltration volume with a first perfiltration buffer, followed by perfiltration of 8.5 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of 0.5 perfiltration volume with a first perfiltration buffer, followed by perfiltration of 9.5 perfiltration volumes with a second perfiltration buffer.

[0271] In some embodiments, perfiltration includes perfiltration of one perfiltration volume with a first perfiltration buffer, followed by perfiltration of 6 to 9 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of one perfiltration volume with a first perfiltration buffer, followed by perfiltration of 6 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of one perfiltration volume with a first perfiltration buffer, followed by perfiltration of 7 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of one perfiltration volume with a first perfiltration buffer, followed by perfiltration of 8 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of one perfiltration volume with a first perfiltration buffer, followed by perfiltration of 9 perfiltration volumes with a second perfiltration buffer.

[0272] In some embodiments, perfiltration includes perfiltration of two perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 5 to 8 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of two perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 5 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of two perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 6 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of two perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 7 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of two perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 8 perfiltration volumes with a second perfiltration buffer.

[0273] In some embodiments, perfiltration includes perfiltration of three perfiltration volumes with a first perfiltration buffer, followed by perfiltration of four to seven perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of three perfiltration volumes with a first perfiltration buffer, followed by perfiltration of four perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of three perfiltration volumes with a first perfiltration buffer, followed by perfiltration of five perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of three perfiltration volumes with a first perfiltration buffer, followed by perfiltration of six perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of three perfiltration volumes with a first perfiltration buffer, followed by perfiltration of seven perfiltration volumes with a second perfiltration buffer.

[0274] In some embodiments, prior to percolation, the recombinant protein is present in the first composition at a concentration of at least 100% (e.g., at least 105%; at least 110%; at least 115%; at least 120%; at least 125%; at least 130%; at least 135%; at least 140%; 100%; 105%; 110%; 115%; 120%; 125%; 130%; 135%; 140%; 145%; 150%) of the concentration of the recombinant protein in the second composition, and optionally the first composition is diluted with percolation buffer prior to percolation. In some embodiments, the first composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0275] In some embodiments, prior to percolation, the recombinant protein is present in the first composition at a concentration of at least 120% of the concentration of the recombinant protein in the second composition, and optionally, the first composition is diluted with percolation buffer prior to percolation. In some embodiments, the first composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0276] In some embodiments, prior to percolation, the recombinant protein is present in the first composition at a concentration of at least 140% of the concentration of the recombinant protein in the second composition, and optionally, the first composition is diluted with percolation buffer prior to percolation. In some embodiments, the first composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0277] In some embodiments, prior to percolation, the recombinant protein is present in the first composition at a concentration in the range of 100% to 150% of the concentration of the recombinant protein in the second composition (e.g., 110% to 140%; 120% to 140%; 105%; 110%; 115%; 120%; 125%; 130%; 135%; 140%; 145%; 150%), and optionally the first composition is diluted with percolation buffer prior to percolation. In some embodiments, the first composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0278] In some embodiments, the recombinant protein is an antigen-binding protein. In some embodiments, the recombinant protein is an antibody or an antibody fragment. In some embodiments, the recombinant protein is an antibody. In some embodiments, the recombinant protein is an antibody fragment.

[0279] In some embodiments, the recombinant protein is an IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is an IgG1 antibody. In some embodiments, the recombinant protein is an IgG2 antibody. In some embodiments, the recombinant protein is an IgG4 antibody.

[0280] In some embodiments, the recombinant protein is a human antibody. In some embodiments, the recombinant protein is a human IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is a human IgG1 antibody. In some embodiments, the recombinant protein is a human IgG2 antibody. In some embodiments, the recombinant protein is a human IgG4 antibody.

[0281] In some embodiments, the recombinant protein is a fragment of an IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is a fragment of an IgG1 antibody. In some embodiments, the recombinant protein is a fragment of an IgG2 antibody. In some embodiments, the recombinant protein is a fragment of an IgG4 antibody.

[0282] In some embodiments, the recombinant protein is a fragment of a human antibody. In some embodiments, the recombinant protein is a fragment of a human IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is a fragment of a human IgG1 antibody. In some embodiments, the recombinant protein is a fragment of a human IgG2 antibody. In some embodiments, the recombinant protein is a fragment of a human IgG4 antibody.

[0283] In some embodiments, the recombinant protein is a fusion protein.

[0284] This article also provides a method for preparing a formulation containing a recombinant protein, the method comprising: The ultrafiltration / percolation (UF / DF) membrane filter is equilibrated in a first percolation buffer to obtain a balanced UF / DF membrane filter; Using a balanced UF / DF membrane filter, the first composition containing the recombinant protein is concentrated by ultrafiltration to obtain a first concentrated composition; and The first concentrated composition is percolated with a second percolation buffer and a third percolation buffer to obtain a second composition, wherein the second percolation buffer is a concentrate of the third percolation buffer.

[0285] In some embodiments, the first percolation buffer and the third percolation buffer contain the same excipients, and each excipient in the first percolation buffer is present in the third percolation buffer in an amount ranging from 90% to 110% of its amount in the third percolation buffer (e.g., 95% to 105%; 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99%; 100%; 101%; 102%; 103%; 104%; 105%; 106%; 107%; 108%; 109%; 110%).

[0286] In some embodiments, the first percolation buffer and the third percolation buffer have substantially the same composition.

[0287] In some embodiments, the second percolation buffer is at least a 2X (e.g., at least 2.5X; at least 3X; 2X; 2.25X; 2.5X; 3X; 3.25X; 3.5X; 3.75X; 4X; 4.25X; 4.5X; 4.75X; 5X; 2X to 3X; 2X to 4X; 2X to 5X) concentrate of the third percolation buffer. In some embodiments, the second percolation buffer is a 2X concentrate of the third percolation buffer. In some embodiments, the second percolation buffer is a 3X concentrate of the third percolation buffer. In some embodiments, the second percolation buffer is a 4X concentrate of the third percolation buffer. In some embodiments, the second percolation buffer is a 5X concentrate of the third percolation buffer.

[0288] In some embodiments, perfiltration includes perfiltration of 0.5 to 3 perfiltration volumes with a second perfiltration buffer, followed by perfiltration of 6.5 to 9.5 perfiltration volumes with a third perfiltration buffer. In some embodiments, perfiltration includes perfiltration of 0.5 to 3 perfiltration volumes with a second perfiltration buffer, followed by perfiltration of 6.5 to 9.5 perfiltration volumes with a third perfiltration buffer, wherein the total number of perfiltration volumes used during perfiltration is at most 10. In some embodiments, perfiltration includes perfiltration of 0.5 to 3 perfiltration volumes with a second perfiltration buffer, followed by perfiltration of 6.5 to 9.5 perfiltration volumes with a third perfiltration buffer, wherein the total number of perfiltration volumes used during perfiltration is 10.

[0289] In some embodiments, perfiltration includes perfiltration of 0.5 perfiltration volume with a second perfiltration buffer, followed by perfiltration of 6.5 to 9.5 perfiltration volumes with a third perfiltration buffer. In some embodiments, perfiltration includes perfiltration of 0.5 perfiltration volume with a second perfiltration buffer, followed by perfiltration of 6.5 perfiltration volumes with a third perfiltration buffer. In some embodiments, perfiltration includes perfiltration of 0.5 perfiltration volume with a second perfiltration buffer, followed by perfiltration of 7.5 perfiltration volumes with a third perfiltration buffer. In some embodiments, perfiltration includes perfiltration of 0.5 perfiltration volume with a second perfiltration buffer, followed by perfiltration of 8.5 perfiltration volumes with a third perfiltration buffer. In some embodiments, perfiltration includes perfiltration of 0.5 perfiltration volume with a second perfiltration buffer, followed by perfiltration of 9.5 perfiltration volumes with a third perfiltration buffer.

[0290] In some embodiments, perfiltration includes perfiltration of one perfiltration volume with a second perfiltration buffer, followed by perfiltration of 6 to 9 perfiltration volumes with a third perfiltration buffer. In some embodiments, perfiltration includes perfiltration of one perfiltration volume with a second perfiltration buffer, followed by perfiltration of 6 perfiltration volumes with a third perfiltration buffer. In some embodiments, perfiltration includes perfiltration of one perfiltration volume with a second perfiltration buffer, followed by perfiltration of 7 perfiltration volumes with a third perfiltration buffer. In some embodiments, perfiltration includes perfiltration of one perfiltration volume with a second perfiltration buffer, followed by perfiltration of 8 perfiltration volumes with a third perfiltration buffer. In some embodiments, perfiltration includes perfiltration of one perfiltration volume with a second perfiltration buffer, followed by perfiltration of 9 perfiltration volumes with a third perfiltration buffer.

[0291] In some embodiments, perfiltration includes perfiltration of two perfiltration volumes with a second perfiltration buffer, followed by perfiltration of 5 to 8 perfiltration volumes with a third perfiltration buffer. In some embodiments, perfiltration includes perfiltration of two perfiltration volumes with a second perfiltration buffer, followed by perfiltration of 5 perfiltration volumes with a third perfiltration buffer. In some embodiments, perfiltration includes perfiltration of two perfiltration volumes with a second perfiltration buffer, followed by perfiltration of 6 perfiltration volumes with a third perfiltration buffer. In some embodiments, perfiltration includes perfiltration of two perfiltration volumes with a second perfiltration buffer, followed by perfiltration of 7 perfiltration volumes with a third perfiltration buffer. In some embodiments, perfiltration includes perfiltration of two perfiltration volumes with a second perfiltration buffer, followed by perfiltration of 8 perfiltration volumes with a third perfiltration buffer.

[0292] In some embodiments, perfiltration includes perfiltration of three perfiltration volumes with a second perfiltration buffer, followed by perfiltration of four to seven perfiltration volumes with a third perfiltration buffer. In some embodiments, perfiltration includes perfiltration of three perfiltration volumes with a second perfiltration buffer, followed by perfiltration of four perfiltration volumes with a third perfiltration buffer. In some embodiments, perfiltration includes perfiltration of three perfiltration volumes with a second perfiltration buffer, followed by perfiltration of five perfiltration volumes with a third perfiltration buffer. In some embodiments, perfiltration includes perfiltration of three perfiltration volumes with a second perfiltration buffer, followed by perfiltration of six perfiltration volumes with a third perfiltration buffer. In some embodiments, perfiltration includes perfiltration of three perfiltration volumes with a second perfiltration buffer, followed by perfiltration of seven perfiltration volumes with a third perfiltration buffer.

[0293] In some embodiments, prior to percolation, the recombinant protein is present in the first concentrated composition at a concentration of at least 100% (e.g., at least 105%; at least 110%; at least 115%; at least 120%; at least 125%; at least 130%; at least 135%; at least 140%; 100%; 105%; 110%; 115%; 120%; 125%; 130%; 135%; 140%; 145%; 150%) of the concentration of the recombinant protein in the second composition, and optionally, the first concentrated composition is diluted with percolation buffer prior to percolation. In some embodiments, the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0294] In some embodiments, prior to percolation, the recombinant protein is present in the first concentrated composition at a concentration of at least 120% of the concentration of the recombinant protein in the second composition, and optionally, the first concentrated composition is diluted with percolation buffer prior to percolation. In some embodiments, the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0295] In some embodiments, prior to percolation, the recombinant protein is present in the first concentrated composition at a concentration of at least 140% of the concentration of the recombinant protein in the second composition, and optionally, the first concentrated composition is diluted with percolation buffer prior to percolation. In some embodiments, the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0296] In some embodiments, prior to percolation, the recombinant protein is present in the first concentrated composition at a concentration in the range of 100% to 150% of the concentration of the recombinant protein in the second composition (e.g., 110% to 140%; 120% to 140%; 105%; 110%; 115%; 120%; 125%; 130%; 135%; 140%; 145%; 150%), and optionally, the first concentrated composition is diluted with percolation buffer prior to percolation. In some embodiments, the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0297] This article provides a method for preparing formulations containing recombinant proteins, the method comprising: The first composition containing the recombinant protein is concentrated by ultrafiltration to obtain a first concentrated composition; and The first concentrated composition is percolated to obtain a second composition, wherein the recombinant protein is present in the first concentrated composition at a concentration of at least 120% of the concentration of the recombinant protein in the second composition.

[0298] This article also provides a method for preparing a formulation containing a recombinant protein, the method comprising: The first composition containing the recombinant protein is concentrated by ultrafiltration to obtain a first concentrated composition; and The first concentrated composition is percolated with a first percolation buffer and a second percolation buffer to obtain a second composition, wherein: The first percolation buffer is a concentrate of the second percolation buffer; and The recombinant protein is present in the first concentrated composition at a concentration of at least 120% of the concentration of the recombinant protein in the second composition.

[0299] In some embodiments, the first percolation buffer is at least a 2X (e.g., at least 2.5X; at least 3X; 2X; 2.25X; 2.5X; 3X; 3.25X; 3.5X; 3.75X; 4X; 4.25X; 4.5X; 4.75X; 5X; 2X to 3X; 2X to 4X; 2X to 5X) concentrate of the second percolation buffer. In some embodiments, the first percolation buffer is a 2X concentrate of the second percolation buffer. In some embodiments, the first percolation buffer is a 3X concentrate of the second percolation buffer. In some embodiments, the first percolation buffer is a 4X concentrate of the second percolation buffer. In some embodiments, the first percolation buffer is a 5X concentrate of the second percolation buffer.

[0300] In some embodiments, perfiltration includes perfiltration of 0.5 to 3 perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 6.5 to 9.5 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of 0.5 to 3 perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 6.5 to 9.5 perfiltration volumes with a second perfiltration buffer, wherein the total number of perfiltration volumes used during perfiltration is at most 10. In some embodiments, perfiltration includes perfiltration of 0.5 to 3 perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 6.5 to 9.5 perfiltration volumes with a second perfiltration buffer, wherein the total number of perfiltration volumes used during perfiltration is 10.

[0301] In some embodiments, perfiltration includes perfiltration of 0.5 perfiltration volume with a first perfiltration buffer, followed by perfiltration of 6.5 to 9.5 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of 0.5 perfiltration volume with a first perfiltration buffer, followed by perfiltration of 6.5 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of 0.5 perfiltration volume with a first perfiltration buffer, followed by perfiltration of 7.5 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of 0.5 perfiltration volume with a first perfiltration buffer, followed by perfiltration of 8.5 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of 0.5 perfiltration volume with a first perfiltration buffer, followed by perfiltration of 9.5 perfiltration volumes with a second perfiltration buffer.

[0302] In some embodiments, perfiltration includes perfiltration of one perfiltration volume with a first perfiltration buffer, followed by perfiltration of 6 to 9 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of one perfiltration volume with a first perfiltration buffer, followed by perfiltration of 6 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of one perfiltration volume with a first perfiltration buffer, followed by perfiltration of 7 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of one perfiltration volume with a first perfiltration buffer, followed by perfiltration of 8 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of one perfiltration volume with a first perfiltration buffer, followed by perfiltration of 9 perfiltration volumes with a second perfiltration buffer.

[0303] In some embodiments, perfiltration includes perfiltration of two perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 5 to 8 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of two perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 5 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of two perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 6 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of two perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 7 perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of two perfiltration volumes with a first perfiltration buffer, followed by perfiltration of 8 perfiltration volumes with a second perfiltration buffer.

[0304] In some embodiments, perfiltration includes perfiltration of three perfiltration volumes with a first perfiltration buffer, followed by perfiltration of four to seven perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of three perfiltration volumes with a first perfiltration buffer, followed by perfiltration of four perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of three perfiltration volumes with a first perfiltration buffer, followed by perfiltration of five perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of three perfiltration volumes with a first perfiltration buffer, followed by perfiltration of six perfiltration volumes with a second perfiltration buffer. In some embodiments, perfiltration includes perfiltration of three perfiltration volumes with a first perfiltration buffer, followed by perfiltration of seven perfiltration volumes with a second perfiltration buffer.

[0305] In some embodiments, the first concentrated composition is diluted with a percolation buffer prior to percolation. In some embodiments, the first concentrated composition is diluted with a percolation buffer such that the concentration of recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0306] In some embodiments, the recombinant protein is present in the first concentrated composition at a concentration of at least 140% of the concentration of the recombinant protein in the second composition, and the first concentrated composition is optionally diluted with a percolation buffer prior to percolation. In some embodiments, the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0307] In some embodiments, the recombinant protein is present in the first concentrated composition at a concentration in the range of 120% to 150% of the concentration of the recombinant protein in the second composition (e.g., 120% to 140%; 120%; 125%; 130%; 135%; 140%; 145%; 150%), and the first concentrated composition is diluted with a percolation buffer prior to percolation. In some embodiments, the first concentrated composition may optionally be diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

[0308] In some embodiments, the recombinant protein is an antigen-binding protein. In some embodiments, the recombinant protein is an antibody or an antibody fragment. In some embodiments, the recombinant protein is an antibody. In some embodiments, the recombinant protein is an antibody fragment.

[0309] In some embodiments, the recombinant protein is an IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is an IgG1 antibody. In some embodiments, the recombinant protein is an IgG2 antibody. In some embodiments, the recombinant protein is an IgG4 antibody.

[0310] In some embodiments, the recombinant protein is a human antibody. In some embodiments, the recombinant protein is a human IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is a human IgG1 antibody. In some embodiments, the recombinant protein is a human IgG2 antibody. In some embodiments, the recombinant protein is a human IgG4 antibody.

[0311] In some embodiments, the recombinant protein is a fragment of an IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is a fragment of an IgG1 antibody. In some embodiments, the recombinant protein is a fragment of an IgG2 antibody. In some embodiments, the recombinant protein is a fragment of an IgG4 antibody.

[0312] In some embodiments, the recombinant protein is a fragment of a human antibody. In some embodiments, the recombinant protein is a fragment of a human IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is a fragment of a human IgG1 antibody. In some embodiments, the recombinant protein is a fragment of a human IgG2 antibody. In some embodiments, the recombinant protein is a fragment of a human IgG4 antibody.

[0313] In some embodiments, the recombinant protein is a fusion protein.

[0314] Purification process prior to UF / DF Compositions subjected to the enhanced UF / DF method disclosed herein may be derived from cell culture processes and may undergo one or more purification processes prior to UF / DF. In some embodiments, one or more purification processes (e.g., one or more unit operations described herein) may be performed sequentially with the enhanced UF / DF method described herein. In some embodiments, one or more purification processes (e.g., one or more unit operations described herein) may be linked to the enhanced UF / DF method described herein. For example, a virus filtering unit operation may be performed sequentially with or linked to the enhanced UF / DF method described herein.

[0315] Illustratively, in an example upstream process preceding the enhanced UF / DF method described herein, the expressed recombinant protein can be secreted into a culture medium from which it can be recovered and / or collected. Harvesting operations including acid precipitation can be combined with other harvesting strategies, including centrifugation, such as disc centrifugation, intermittent discharge centrifugation, or continuous solid discharge centrifugation; filtration, including, for example, tangential flow filtration, microfiltration, ultrafiltration, and depth filtration; precipitation / sedimentation methods, such as flocculation; and separation based on chromatographic media.

[0316] Additionally, post-harvest recovery techniques, such as protein A purification of immunoglobulins and immunoglobulin-like biological products, and chromatographic separation and purification steps can be employed prior to the UF / DF method described herein, including column and alternative mode chromatographic separations via ion exchange chromatography (IEX), including anion exchange chromatography (AEX) and / or cation exchange chromatography (CEX), hydrophobic interaction chromatography (HIC), mixed-mode or multi-mode chromatography (MM), hydroxyapatite chromatography (HA), reversed-phase chromatography, size exclusion chromatography (SEC), gel filtration, or any other known form of chromatographic separation of biological and / or biochemical substances.

[0317] In some embodiments, recombinant proteins recovered from host cells or cell culture media may be further purified or partially purified to remove cell culture medium components, host cell proteins or nucleic acids, or other process or product-related impurities by one or more unit operations before or after virus filtration. Those skilled in the art can select one or more appropriate unit operations for further purification of the recombinant protein based on the characteristics of the recombinant protein to be purified, the characteristics of the host cells expressing the recombinant protein, and the composition of the culture medium in which the host cells are grown. Illustratively, in some embodiments, the recombinant protein is purified from the harvest permeate by one or more of flocculation, precipitation, centrifugation, deep filtration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, mixed-mode anion exchange chromatography, hydrophobic interaction chromatography, or hydroxyapatite chromatography.

[0318] Capture unit operations may include capture chromatography using resins and / or membranes containing reagents that bind to the target recombinant protein, such as affinity chromatography, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography (HIC), solid-phase metal affinity chromatography (IMAC), etc. Such chromatographic materials are known in the art and are commercially available. For example, if the recombinant protein is an antibody or contains components derived from an antibody (e.g., an Fc domain), affinity chromatography using ligands such as protein A, protein G, protein A / G, or protein L can be used as a capture chromatographic unit operation to further purify the recombinant protein. In other embodiments, the target recombinant protein may contain a polyhistidine tag at its amino or carboxyl terminus, followed by purification using IMAC. The recombinant protein may be engineered to include other purification tags, such as FLAG® tags or c-myc epitopes, and subsequently purified by affinity chromatography using antibodies specific to such tags or epitopes.

[0319] Additional unit operations for inactivating, reducing, and / or eliminating viral contaminants may include filtration processes and / or conditioning of solution conditions. One method for achieving viral inactivation is incubation at a low pH (e.g., pH < 4). Following a low-pH viral inactivation operation, a neutralization unit operation may be performed to readjust the pH of the virus-inactivated solution to better suit the requirements of subsequent unit operations. Filtration, such as depth filtration with an optional viral pre-filter, may also be performed after a low-pH viral inactivation operation to remove any resulting turbidity or precipitation. Conditioning of temperature or chemical composition (e.g., using a detergent) may also be used to achieve viral inactivation.

[0320] Virus filters are polymer membranes with various membrane chemistry compositions and complex pore structures designed to trap viral particles. Virus filters are commercially available from multiple manufacturers (e.g., Millibo Sigma, Sartorius, Pall, and Asahi Kasei) and include large and small virus filters. Large virus filters are designed to trap viruses larger than 60 nm, while small virus filters are designed to trap viruses larger than 20 nm. Virus filters can be used in normal flow filtration (NFF) or tangential flow filtration (TFF) modes. In TFF or NFF modes, filtration is performed under conditions that trap viral contaminants (e.g., viruses with a diameter of 20 to 100 nm) on the membrane surface while allowing recombinant proteins to pass through the filter membrane.

[0321] Non-limiting examples of virus filters include those formed from regenerated cellulose (e.g., cuprammonium regenerated cellulose), polyethersulfone, polyarylsulfone, polysulfone, polyimide, polyamide, polyvinylidene fluoride (PVDF), etc. For example, non-limiting examples of virus filters include VIRESOLVE® membranes and RETROPORE™ membranes available from EMD Millipore, Bill Ricard, Massachusetts. These can be supplied in cartridge (NFF) form (such as the VIRESOLVE® NFP virus filter) or in boxes (for TFF) (such as the PELLICON® box), available from EMD Millipore, Bill Ricard, Massachusetts.

[0322] Another non-limiting example is Sartorius's Virosart® CPV small virus filter, which comprises a polyethersulfone membrane. Other PES membrane filters include, but are not limited to, Viresolve® NFR, Viresolve® Pro, Virosart® CPV, Virosart® HF, Virosart® HC, and Pegasus™ grade Prime. Other non-limiting example filters include the Planova™ BioEX filter and the Viresolve® NFP filter, both comprising a polyvinylidene fluoride (PVDF) membrane, and Asahi Kasei Corporation's 15N, 20N, and 35N filters, which comprise a cuprammonium regenerated cellulose membrane (e.g., a hollow fiber cuprammonium regenerated cellulose membrane). Examples of alternative virus filters are known in the art and include hydrophilic acrylate-modified PVDF membranes from Pall Corporation, such as Ultipor® VF grade DV20, Ultipor® VF grade DV50, and Pegasus™ grade PV4.

[0323] Virus pre-filters, commercially available from multiple sources (e.g., Millibo Sigma, Sartorius, Pall, Asahi Kasei Corporation), remove protein aggregates and other process impurities that may otherwise contaminate the virus filter. Common virus pre-filters utilize a variety of membrane chemistry compositions, such as PES membrane surfaces chemically modified via cross-linked polymeric sulfonic acid cation exchange (Viresolve® Shield, Millibo Sigma) and PES membrane surfaces chemically modified via cross-linked mixed-mode (Viresolve® Shield-H, Millibo Sigma). Additionally, depth filters (Viresolve® pre-filters, Millibo Sigma), pleated sheet membranes (Pegasus™ Protect, Pall; Pegasus™ grade UL6, Pall), and three-layer flat sheet membranes (Virosart® Max, Sartorius) composed of diatomaceous earth, cellulose fibers, and binders containing cationic imine groups can be used as pre-filters.

[0324] Virus prefilters typically function at least in part through size exclusion, for example, size exclusion cutoff values ​​between about 75 nm and about 0.2 µm (e.g., 75 nm (Planova 75N, Asahi Kasei Corporation); 0.1 µm (Virosart® Max, Sartorius Corporation; Viresolve® prefilter, Milli-Bose-Sigma Corporation); 0.2 µm (Viresolve® Shield, Milli-Bose-Sigma Corporation; Viresolve® Shield-H, Milli-Bose-Sigma Corporation; Pegasus™ Protect, Pall Corporation)).

[0325] Purification unit operations can utilize various chromatographic methods to purify target proteins and remove contaminants and impurities. Resins and / or membranes that can be used in purification chromatographic unit operations contain reagents that can be used in either "flow-through mode" (where the target protein is contained in the eluent and contaminants and impurities are bound to the chromatographic medium) or "bind-elute mode" (where the target protein is bound to the chromatographic medium and elutes after contaminants and impurities flow through or are washed away from the chromatographic medium). Examples of such purification chromatographic methods include, but are not limited to, ion exchange chromatography (IEX), such as anion exchange chromatography (AEX) and cation exchange chromatography (CEX); hydrophobic interaction chromatography (HIC); mixed-mode or multi-mode chromatography (MM), hydroxyapatite chromatography (HA); reversed-phase chromatography; and size exclusion chromatography (e.g., gel filtration).

[0326] Recombinant protein Compositions comprising any type of recombinant protein (including proteins containing a single polypeptide chain or multiple polypeptide chains) can be formulated according to the methods disclosed herein. Such recombinant proteins include, but are not limited to, secretory proteins, non-secretory proteins, intracellular proteins, or membrane-bound proteins. Illustratively, recombinant proteins may include, but are not limited to, cytokines, growth factors, hormones, mutant proteins, fusion proteins, antibodies, antibody fragments, peptide bodies, T-cell conjugating molecules, and multispecific antigen-binding proteins. In some embodiments, the recombinant protein is a fusion protein.

[0327] In other embodiments, the recombinant protein is an antigen-binding protein. Antigen-binding proteins include, but are not limited to, antibodies, peptides, antibody derivatives, antibody analogs, fusion proteins (including, for example, single-chain variable fragments (scFv), double-chain (bivalent) scFv, and IgG scFv (see, for example, Orcutt et al., 2010, Protein Eng Des Sel [Protein Engineering Design and Selection] 23:221-228)), heterologous IgG molecules (see, for example, Liu et al., 2015, J Biol Chem [Journal of Biochemistry] 290:7535-7562), mutant proteins, and XmAbs. ® (Xencor, Inc., Monova, California). Other antigen-binding proteins include, but are not limited to, bispecific T-cell conjugates (BiTE). ® Bispecific T-cell conjugates with extended durations (e.g., extended half-life) (e.g., HLE BiTE molecules, HeteroIg BITE molecules, etc.), chimeric antigen receptors (CAR, CAR T), and T-cell receptors (TCRs).

[0328] In some embodiments, the antigen-binding protein binds to one or more of the following (alone or in any combination): CD proteins, including but not limited to CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171, and CD174; HER receptor family proteins, including, for example, HER2, HER3, HER4, and EGF receptors, EGFRvIII, cell adhesion molecules (e.g., LFA-1, Mol, p150,95, VLA-4, ICAM-1, VCAM, and αv / β3 integrin); growth factors, including but not limited to, for example, vascular endothelial growth factor (“VEGF”); VEGFR2, growth hormone, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, growth hormone-releasing factor, parathyroid hormone, and Miller duct inhibitors. Inflammatory proteins of human macrophages (MIP-1-α), erythropoietin (EPO), nerve growth factors (such as NGF-β), platelet-derived growth factor (PDGF), fibroblast growth factors (including, for example, aFGF and bFGF), epidermal growth factor (EGF), Cripto, transforming growth factor (TGF) (especially including TGF-α and TGF-β (including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5)), insulin-like growth factor-I and insulin-like growth factor-II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I) and bone-inducing factor, insulin and insulin-related proteins (including but not limited to insulin, insulin A chain, insulin B chain, proinsulin, and insulin-like growth factor binding proteins); (coagulation proteins and coagulation-related proteins, especially factor VIII, tissue factor, van Wilbond). Willebrand factor, protein C, α-1-antitrypsin, plasminogen activators (such as urokinase and tissue plasminogen activator (“t-PA”)), bombazine, thrombin, thrombopoietin and thrombopoietin receptor, colony-stimulating factor (CSF) (especially including M-CSF, GM-CSF and G-CSF), other blood and serum proteins (including but not limited to albumin, IgE and blood group antigens), receptors and receptor-related proteins (including, for example, flk2 / flt3 receptors, obesity (OB) receptors, growth hormone receptors and T-cell receptors); neurotrophic factors, including but not limited to bone-derived neurotrophic factor (BDNF) and neurotrophin-3, neurotrophin-4, neurotrophin-5 or neurotrophin-6 (NT-3, NT-4, NT-5 or NT-6).Relaxin A chain, relaxin B chain and pro-relaxin, interferons (including, for example, interferon α, interferon β and interferon γ), interleukins (ILs) (e.g. IL-1 to IL-10, IL-12, IL-15, IL-17, IL-23, IL-12 / IL-23, IL-2Ra, IL-1-R1, IL-6 receptor, IL-4 receptor and / or IL-13 receptor, IL-13RA2 or IL-17 receptor, IL-1RAP); viral antigens, including but not limited to AIDS envelope virus antigens, lipoproteins, calcitonin, glucagon, atrial natriuretic peptide, pulmonary surfactant. Agents, tumor necrosis factor-α and tumor necrosis factor-β, enkephalin, BCMA, IgKappa, ROR-1, ERBB2, mesothelin, RANTES (activated and regulated normal T cell expression and secretion factors), mouse gonadotropin-related peptide, DNase, FR-α, inhibin and activin, integrin, protein A or D, rheumatoid factor, immunotoxin, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane protein, decay accelerator factor (DAF), AIDS envelope, transport protein, homing receptor, MIC (MIC-a, MIC-B), ULBP 1-6, EPCAM, addressin, regulatory protein, immunoadhesin, antigen-binding protein, growth hormone, CTGF, CTLA4, eotaxin-1, MUC1, CEA, c-MET, Claudin-18, GPC-3, EPHA2, FPA, LMP1, MG7, NY-ESO-1, PSCA, ganglioside GD2, ganglioside GM2, BAFF, OPGL (RANKL), myostatin, Dickkopf-1 (DKK-1), Ang2, NGF, IGF-1 receptor, hepatocyte growth factor (HGF), TRAIL-R2, c-Kit, B7RP-1, PSMA, NKG2D-1, programmed cell death protein 1 and ligand, PD1 and PDL1, mannose receptor / hCGβ, hepatitis C virus, mesothelin dsFv [PE38] conjugate, Legionella pneumophila (lly), IFN γ, interferon-gamma inducible protein 10 (IP10), IFNAR, TALL-1, thymic stromal lymphopoietin (TSLP), proprotein convertase subtilisin / Kexin type 9 (PCSK9), stem cell factor, Flt-3, calcitonin gene-related peptide (CGRP), OX40L, α4β7, platelet-specific (platelet glycoprotein IIb / IIIb (PAC-1)), transforming growth factor β (TFGβ), zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, platelet-derived growth factor receptor α (PDGFRα), sclerostin, and any bioactive fragments or variants of the foregoing.

[0329] In other embodiments, the recombinant protein is an antibody. In some embodiments, the antibody is a human antibody.

[0330] In some embodiments, the antibody is selected from abrilumab, brazikumab, brodalumab, crizanlizumab, denosumab, eculizumab, erenumab, evolocumab, fremanezumab, meplazumab, nemolizumab, and ontamalimab. The antibody is selected from panitumumab, prezalumab, ravulizumab, rilotumumab, romosozumab, satralizumab, tafolecimab, tanezumab, tezepelumab, tremelimumab, utomilumab, and volagidemab. In some embodiments, the antibody is selected from dinosumab, errenumab, ivosumab, panitumumab, romosozumab, and tezepelumab. In some embodiments, the antibody is dinosumab. In some embodiments, the antibody is errenumab. In some embodiments, the antibody is ivosumab. In some embodiments, the antibody is panitumumab. In some embodiments, the antibody is romosozumab. In some embodiments, the antibody is terzrolumab.

[0331] In some embodiments, the antibody is an IgG1, IgG2, or IgG4 antibody. In some embodiments, the antibody is a human IgG1, IgG2, or IgG4 antibody.

[0332] In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is a human IgG1 antibody.

[0333] In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is a human IgG2 antibody.

[0334] In some embodiments, the antibody is an IgG4 antibody. In some embodiments, the antibody is a human IgG4 antibody.

[0335] Example To provide a fuller understanding of this disclosure, the following examples are provided. It should be understood that these examples are for illustrative purposes only and should not be construed as limiting this disclosure in any way.

[0336] Example 1. Risk-based modeling approach using 2X percolation buffer concentrate Risk-based modeling was used to assess the process impacts of enhancing the ultrafiltration / percolation (UF / DF) unit operation used for preparing monoclonal antibodies. Specifically, process modeling was used to assess the risks associated with using percolation buffer concentrates at different stages of the UF / DF process as follows: batch processing at a concentration 1X higher than the target percolation concentration and online dilution with water during UF / DF operations to achieve the target 1X concentration. Higher concentration factors result in lower buffer batch volumes and associated resource usage. Figure 1 However, during process steps such as percolation and post-percolation product recovery, over-dilution or under-dilution of the percolation buffer can occur due to pump errors. Figure 2 This could affect key quality attributes (CQA, such as protein concentration, osmolar concentration, and / or pH), so process modeling examined the impact of the relative dilution accuracy of the 2X percolation buffer concentrate on the product pool CQA during different steps of the UF / DF process.

[0337] The risk-based modeling approach used in this study takes into account the Donnan effect, a phenomenon of differential excipient distribution caused by the retention of the target protein on the membrane and the equal thermodynamic activities of the permeate and the retentate. A first-principles-based model for predicting the impact of the Donnan effect on UF / DF operations of different products and buffer types has been previously described (Ladwig et al., “Mechanistic model of pH and excipient concentration during ultrafiltration and diafiltration processes of therapeutic antibodies.” Biotechnology Progress, Sep 2020;36(5):e2993. doi: 10.1002 / btpr.2993. 2020 April 7 electronic version), and is adopted here.

[0338] The model was first used to evaluate the relationship between the excipient's percolation buffer concentration, the final UF / DF pool concentration of the excipient, and the final UF / DF pool osmolality. While the osmolality could be estimated using first-principles calculations based on predicted solute concentrations, experimental data were used in this model. Next, different scenarios for the dilution accuracy of the 2X percolation buffer concentrate were simulated by modeling the percolation buffer concentration (the model input) at the same level of variability, assuming pump dilution accuracy of 90%, 95%, 105%, or 110% of the target value. Batch variability of the percolation buffer was not considered.

[0339] The changes in final UF / DF pool osmolality were evaluated under the following four conditions: (1) the entire process (including the recovery phase) used a diluted 2X percolation buffer concentrate; (2) the entire process (excluding the recovery phase) used a diluted 2X percolation buffer concentrate; (3) 6 out of 7 percolation volumes used a diluted 2X percolation buffer concentrate and the remainder (the last 1 percolation volume of DF and the recovery phase) used 1X buffer (undiluted); and (4) 6.5 out of 7 percolation volumes used a diluted 2X percolation buffer concentrate and the remainder (the last 0.5 percolation volume of DF and the recovery phase) used 1X buffer (undiluted). For each simulation, the predicted pool additive concentration was used to predict the pool osmolality.

[0340] Figure 3 The relative changes in predicted osmolality for four UF / DF process conditions are shown compared to a conventional process where the entire process from equilibration to recovery uses 1X buffer (corresponding to 100% dilution accuracy). Figure 3 As shown, a large variability in the accuracy of buffer dilution by ±10% leads to a large variability in the predicted osmolality. Furthermore, limiting the use of the concentrated percolation buffer to the equilibration and percolation phases reduces the variability in the predicted osmolality, and further limiting the use of the concentrated buffer to only a portion of the percolation phase further reduces the variability.

[0341] Figure 4 The simulation shows the process capability (Ppk) of the osmotic pressure molar concentration relative to the product specification limit under the assumption that the pump variability is uniformly distributed in the worst case.

[0342] The equation for calculating Ppk is shown in Equation 1, where USL is the upper bound of the variable of interest and LSL is the lower bound of the variable of interest. It focuses on the average value of variables, and That is the standard deviation. For this analysis, It is the average osmolar concentration of the active pharmaceutical ingredient measured experimentally, and Calculate using Equation 2, which uses the maximum value. y and minimum value x The standard deviation of a uniform distribution is shown. A uniform distribution is the worst-case assumption for this type of analysis because it assigns an equal probability to a 5% error as to other values ​​within that range (e.g., 1%, 2%, etc.). This can be achieved with systematic errors in pump output throughout the process, but it represents the worst-case scenario compared to the more likely operation of the pump during periods of higher accuracy. (1) (2) Assuming ±5% pump error and a separate 1X buffer for the recovery phase (condition (2)) is determined to be the desired operating strategy, this level of accuracy is achievable on large-scale UF / DF skids, where the DF process does not include changes in dilution strategy (i.e., the final percolation volume changes from 2X to 1X), and the predicted osmolality exhibits Ppk (>1.3). Osmolality is used as a performance metric in this analysis because other parameters (i.e., pH) are less sensitive to inappropriate dilution of typical percolation buffers.

[0343] Example 2. A risk-based modeling approach for evaluating over-concentration in ultrafiltration and accelerated buffer exchange using buffer concentrates during perfiltration. The process impact of enhanced ultrafiltration / percolation (UF / DF) unit operation was evaluated using a risk-based modeling approach similar to that described in Example 1, as follows: an increased ultrafiltration concentration target was used, followed by dilution with percolation buffer at the start of percolation. The aim of this approach was to reduce percolation volume requirements by removing more excipients during the ultrafiltration process and potentially increase the percolation rate in the early stages of percolation. The use of a 2X percolation buffer concentrate at the start of the percolation process to accelerate buffer exchange was also evaluated.

[0344] Specifically, simulations were conducted to evaluate the following three “over-concentration” scenarios: (1) Concentration to Y (2) Concentrate to approximately 1.3 g / L. Y g / L, and (3) concentrated to approximately 1.6 g / L. Y g / L, of which Y g / L is the expected concentration after percolation. This initial over-concentration helps reduce the total auxiliary material level before percolation, resulting in a more efficient process. Figure 5 The simulated concentrations of excipients under each of these conditions are shown, wherein the UF pool was diluted with percolation buffer prior to the start of percolation. Y Concentration in g / L.

[0345] In addition, the effect of using 2X percolation buffer concentrate to accelerate buffer exchange before switching to using 1X percolation buffer was examined. The percolation process was (1) 0.5 percolation volumes (DV); (2) 1 percolation volume; (3) 2 percolation volumes; and (4) 3 percolation volumes. Figure 6 The simulated concentrations of excipients for each of these scenarios are shown.

[0346] Next, using the first-principles modeling approach previously described elsewhere (Ladwig et al., “Mechanistic model of pH and excipient concentration during ultrafiltration and diafiltration processes of therapeutic antibodies.” Biotechnology Progress, Sep 2020;36(5):e2993. doi: 10.1002 / btpr.2993. April 7, 2020, e.g.), different scenarios of diluting the UF pool with DF buffer or accelerating buffer exchange were simulated. For each simulation, the predicted UF / DF pool excipient concentration was used to predict the UF / DF pool osmolar concentration, similar to the simulation in Example 1. Table 1 shows the comparison of six scenarios at a fixed target excipient concentration, which was selected based on the equilibrium excipient concentration for complete buffer exchange (i.e., approximately 10 DV). Note that 5% of the target was used as a metric for measuring buffer exchange efficiency.

[0347] Table 1. Comparison of conditions Example 3. Properties of diluted 2X buffer concentrate The study aimed to verify that the target percolation (DF) buffer properties could be obtained by using a 2X buffer concentrate formulation followed by a 1:1 dilution with water.

[0348] To achieve a target composition of 1X DF buffer with 272 mM proline, 10 mM acetate, and pH 4.1, a 2X buffer concentrate formulation was created by doubling all buffer components (excipient-grade glacial acetic acid and L-proline), resulting in a target composition of 544 mM proline, 20 mM acetate, and pH 4.1. This 2X formulation was batch-processed and tested, followed by 1:1 dilution with deionized water to obtain a 1X buffer, which was then tested. Triple batches were prepared and tested. Buffer testing consisted of pH and conductivity measurements performed at 20°C–22°C using an Orion Star Series A benchtop instrument (Thermo Fisher Scientific), and osmolality measurements performed using an OsmoTECH XT osmoremeter (Advanced Instruments).

[0349] Table 2 shows the average results (standard deviation in parentheses; N = 3) for the diluted 2X buffer, as well as the target properties for the 1X buffer. All results are within the expected range, demonstrating the feasibility and robustness of the strategy involving a 1:1 dilution of the 2X concentrate formulation (based on the 2X component amount compared to the pure buffer formulation) in achieving the target buffer properties.

[0350] Table 2. Properties of diluted 2X buffer concentrate All documents or portions thereof cited in this application, including but not limited to patents, patent applications, articles, books, and monographs, are expressly incorporated herein by reference. Unless the context clearly indicates otherwise, the content described in the embodiments of this disclosure may be combined with one or more other embodiments of this disclosure.

[0351] The subject matter disclosed is not intended to be limited in scope to the specific embodiments described herein, which are rather intended as non-limiting illustrations of various aspects of this disclosure. Functionally equivalent methods and components are within the scope of this disclosure. In fact, various modifications to the disclosed subject matter, in addition to those shown and described herein, will be apparent to those skilled in the art based on the foregoing description and drawings. Such modifications are intended to fall within the scope of the disclosed subject matter.

[0352] The description of various embodiments and / or examples of the disclosed subject matter is presented for illustrative purposes and is not intended to be exhaustive or limiting in any way. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles of the embodiments, their practical application, or technical improvements to existing technologies in the market, and / or to enable others skilled in the art to understand the disclosed subject matter.

Claims

1. A method for preparing a formulation comprising a recombinant protein, the method comprising: The ultrafiltration / percolation (UF / DF) membrane filter is equilibrated in a first percolation buffer to obtain a balanced UF / DF membrane filter; Using a balanced UF / DF membrane filter, the first composition containing recombinant proteins was concentrated by ultrafiltration to obtain a first concentrated composition; as well as The first concentrated composition is percolated with the first percolation buffer to obtain the second composition. The first percolation buffer is prepared by diluting a percolation buffer concentrate.

2. The method as described in claim 1, wherein, The percolation involves percolating the first concentrated composition with 6 to 10 percolation volumes of the first percolation buffer.

3. The method as claimed in claim 1 or claim 2, wherein, The method further includes: Optionally, the second composition may be concentrated to obtain a second concentrated composition; The balanced UF / DF membrane filter was rinsed with a second percolation buffer to obtain the recovered composition, wherein the second percolation buffer was not prepared by diluting a percolation buffer concentrate; and The recycled composition is combined with the second composition or the second concentrated composition to obtain a third composition.

4. The method of claim 3, wherein, The first percolation buffer and the second percolation buffer have essentially the same composition.

5. The method as claimed in claim 3 or claim 4, wherein, Concentrating the second composition involves ultrafiltration using a balanced UF / DF membrane filter.

6. A method for preparing a formulation comprising a recombinant protein, the method comprising: The ultrafiltration / percolation (UF / DF) membrane filter is equilibrated in a first percolation buffer to obtain a balanced UF / DF membrane filter; Using a balanced UF / DF membrane filter, the first composition containing recombinant proteins was concentrated by ultrafiltration to obtain a first concentrated composition; as well as The first concentrated composition is percolated with the first percolation buffer and the second percolation buffer to obtain the second composition. in: The first percolation buffer was prepared by diluting a percolation buffer concentrate; This second percolation buffer was not prepared by diluting a percolation buffer concentrate; and The first percolation buffer and the second percolation buffer have essentially the same composition.

7. The method of claim 6, wherein, The percolation includes: Percolation is performed using the first percolation buffer for 6.5 percolation volumes, followed by percolation using the second percolation buffer for 0.5 percolation volumes; or Six percolation volumes were percolated with the first percolation buffer, followed by one percolation volume percolated with the second percolation buffer.

8. The method of claim 6 or claim 7, wherein, The method further includes: Optionally, the second composition may be concentrated to obtain a second concentrated composition; The balanced UF / DF membrane filter was rinsed with a third percolation buffer to obtain the recovered composition, wherein the third percolation buffer was not prepared by diluting a percolation buffer concentrate; and The recycled composition is combined with the second composition or the second concentrated composition to obtain a third composition.

9. The method of claim 8, wherein, The first percolation buffer, the second percolation buffer, and the third percolation buffer have essentially the same composition.

10. The method according to any one of claims 6 to 9, wherein, Concentrating the second composition involves ultrafiltration using a balanced UF / DF membrane filter.

11. The method according to any one of claims 1 to 10, wherein, The first percolation buffer was prepared by diluting the percolation buffer concentrate with water.

12. The method according to any one of claims 1 to 11, wherein, The first percolation buffer was prepared by diluting the percolation buffer concentrate at the desired dilution ratio of 1:

1.

13. The method according to any one of claims 1 to 12, wherein, The dilution was performed using an online dilution system.

14. The method according to any one of claims 1 to 13, wherein, The dilution was performed continuously using an online dilution system.

15. The method of claim 13 or claim 14, wherein, The pumps in this online dilution system have relative dilution accuracy in the range of 90% to 110% of the expected dilution ratio.

16. A method for preparing a formulation comprising a recombinant protein, the method comprising: The ultrafiltration / percolation (UF / DF) membrane filter is equilibrated in a first percolation buffer to obtain a balanced UF / DF membrane filter; Using a balanced UF / DF membrane filter, the first composition containing recombinant proteins was concentrated by ultrafiltration to obtain a first concentrated composition; as well as The first concentrated composition is percolated with a second percolation buffer and a third percolation buffer to obtain a second composition, wherein the second percolation buffer is a concentrate of the third percolation buffer.

17. The method of claim 16, wherein, The second percolation buffer is at least a 2X concentrate of the third percolation buffer.

18. The method of claim 16 or claim 17, wherein, The second percolation buffer is a 2X concentrate of the third percolation buffer.

19. The method according to any one of claims 16 to 18, wherein, The first and third percolation buffers have essentially the same composition.

20. The method according to any one of claims 16 to 19, wherein, The percolation includes: Percolation is performed with the second percolation buffer for 0.5 percolation volumes, followed by percolation with the third percolation buffer for 6.5 to 9.5 percolation volumes; or Percolate one percolation volume using the second percolation buffer, then percolate 6 to 9 percolation volumes using the third percolation buffer; or Percolate two percolation volumes using the second percolation buffer, then percolate 5 to 8 percolation volumes using the third percolation buffer; or Three percolation volumes were percolated with the second percolation buffer, followed by four to seven percolation volumes with the third percolation buffer.

21. The method according to any one of claims 1 to 20, wherein, The recombinant protein is present in the first concentrated composition at a concentration of at least 100% of the concentration of the recombinant protein in the second composition, and is optionally diluted with a percolation buffer prior to percolation, wherein the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

22. The method according to any one of claims 1 to 21, wherein, The recombinant protein is present in the first concentrated composition at a concentration of at least 120% of the concentration of the recombinant protein in the second composition, and is optionally diluted with a percolation buffer prior to percolation, wherein the first concentrated composition is diluted such that the concentration of the recombinant protein in the diluted composition is substantially the same as the concentration in the second composition.

23. A method for preparing a formulation comprising a recombinant protein, the method comprising: The first composition containing recombinant protein is concentrated by ultrafiltration to obtain a first concentrated composition; as well as The first concentrated composition is percolated with a first percolation buffer and a second percolation buffer to obtain a second composition, wherein: The first percolation buffer is a concentrate of the second percolation buffer; and The recombinant protein is present in the first concentrated composition at a concentration of at least 120% of the concentration of the recombinant protein in the second composition.

24. The method of claim 23, wherein, The first percolation buffer is at least a 2X concentrate of the second percolation buffer.

25. The method of claim 23 or claim 24, wherein, The percolation includes: Percolation is performed with the first percolation buffer for 0.5 percolation volumes, followed by percolation with the second percolation buffer for 6.5 to 9.5 percolation volumes; or Percolate one percolation volume using the first percolation buffer, then percolate 6 to 9 percolation volumes using the second percolation buffer; or Percolate two percolation volumes using the first percolation buffer, then percolate 5 to 8 percolation volumes using the second percolation buffer; or Three percolation volumes were percolated with the first percolation buffer, followed by four to seven percolation volumes with the second percolation buffer.

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