Method for preparing highly concentrated PD-1 antibody solution by ultrafiltration / diafiltration (UF / DF)

Abstract

The present disclosure provides a method for preparing a highly concentrated antibody solution that binds to human programmed death receptor 1 (PD-1). In this process, a highly concentrated antibody solution can be produced by the ultrafiltration / diafiltration (UF / DF) unit operation described herein. The UF / DF preparation method mainly includes a first ultrafiltration concentration step, a buffer solution diafiltration step, and a second ultrafiltration concentration step. This process has a wider range of operating parameters compared to the preparation of low-concentration antibodies and maintains the stability and integrity of the antibodies.

Description

Technical Field

[0001] The present disclosure relates to a method for preparing a highly concentrated solution comprising an antibody or an antigen-binding fragment thereof that binds to human programmed death receptor 1 (PD-1). This methodology is intended to produce a highly concentrated antibody solution by ultrafiltration / diafiltration (UF / DF) as described herein. The high-concentration antibody solution is useful, for example, in subcutaneous administration. The UF / DF preparation method includes a first ultrafiltration concentration step, a buffer solution diafiltration step, and a second ultrafiltration concentration step. This process has a wide range of operating parameters and has demonstrated that the high-concentration solution prepared by this method retains the quality characteristics of the antibody found in low-concentration formulations.

Background Art

[0002] With the rapid development of antibody therapeutics, there is an increasing trend to focus on subcutaneous formulations rather than intravenous (IV) formulations and administrations in order to reduce clinical costs and improve patient compliance. In the case of the subcutaneous administration route of monoclonal antibody injections, the dosage is usually in the range of 50 mg to 800 mg, but the maximum subcutaneous volume is generally limited to about 2 ml, which enables the nominal volume to be delivered in a short time. Therefore, high-concentration protein preparations require additional processes to obtain protein concentrations of up to 100 mg / ml or higher without damaging the antibody itself. High-concentration monoclonal antibodies pose many challenges in the manufacturing process, process scaling, and ultimately patient administration. One of the most important challenges is high viscosity. Due to the properties of antibodies at high concentrations, therapeutic antibody formulations can form overly viscous solutions. In some cases, protein precipitation that blocks the membrane can occur during the ultrafiltration process, leading to either product loss or process failure. Another difficulty in concentrating high-concentration monoclonal antibody solutions by ultrafiltration is the possibility that the antibody aggregates to form clumps and / or precipitates after the concentration process is completed. Finally, if the final high-concentration protein solution is obtained by modifying the filtration process, the concentrated antibody formulation needs to have a viscosity suitable for use with disposable sterile syringes or prefilled needles for subcutaneous administration. In the manufacturing process, ultrafiltration / diafiltration (UF / DF) is typically the final process for obtaining antibody concentrations in the range of 10 - 60 mg / ml. However, the antibody dosage for intravenous injection is about 100 milligrams to about 1 gram. To achieve the same pharmacokinetics and efficacy with subcutaneous injection of the antibody solution, the ideal target antibody concentration in UF / DF can be 150 mg / ml or higher.

[0003] This high concentration causes technical challenges in the manufacturing process. First, highly concentrated antibody solutions may have high viscosities and exhibit different hydrodynamic behaviors in UF / DF. Due to the high pressure applied to the membrane, mass transfer is limited, and as a result, the flux passing through the membrane may decrease, which can lead to membrane fouling. Second, there is a large difference between the initial feed protein concentration and the protein concentration of the final solution, and a 40-fold concentration may be required in between. Especially in commercial-scale manufacturing, the volume change also becomes quite large. These factors play a role in the design of the UF / DF process and the selection of the skid. The UF / DF process setup should be able to handle high-flow and large-volume solutions, and in subsequent processing steps, it should be able to handle relatively low-flow and very small volumes (less than one-tenth or one-twentieth) for highly concentrated solutions. The range of pumps and sensors, the diameter of the tubes, the flow meters, and the dead volume cannot meet the process requirements of both the initial and later stages by using a conventional UF / DF process setup. Finally, the characteristics of the primary amino acid sequence of the antibody are one of the major determining factors in determining the solubility and / or stability properties of the antibody in various formulations. Highly concentrated antibody solutions can be modified to have low viscosity and high stability in the final formulation by screening for formulations and viscosity-reducing agents, as well as other stability tests. However, when the antibody concentration increases in the first ultrafiltration step, the antibody may not be able to maintain its structural stability in the feed buffer solution. Even if the antibody is stable in the feed buffer solution at a concentration of 150 mg / ml or higher, especially without adding viscosity-reducing chemicals such as salts, amino acids, sugars, polyols, and surfactants, the viscosity of the antibody solution is high, and the subsequent diafiltration process may take a very long time. The present disclosure provides a novel preparation method for producing highly concentrated antibody solutions by UF / DF steps. SUMMARY OF THE INVENTION

[0004] The present disclosure provides a method for preparing a highly concentrated anti-human PD-1 monoclonal antibody solution, preferably tislelizumab, for subcutaneous administration by UF / DF unit operations. The UF / DF process comprises the following steps: 1. Loading the feed material and buffer into the UF / DF system at the starting protein concentration after virus filtration; 2. In the UF1 step, ultrafiltrating the solution to obtain an intermediate concentration UF1 pool; 3. Diafiltrating the UF1 pool using the DF buffer to obtain a final drug substance formulation buffer, preferably His-HisHCl buffer, to obtain a DF pool; 4. Ultrafiltrating the DF pool to obtain a high-concentration protein solution as an over-concentrated pool at the required concentration; 5. Preparing a UF2 pool by combining the over-concentrated pool with a system flush or without a system flush, adding a surfactant and glycogen solution, diluting to a final high-concentration formulation solution, and achieving the target concentration of the final drug substance, including.

[0005] In some embodiments, the feed material is in 50 mM acetate buffer and the initial concentration varies from 3 g / L to 18 g / L. In some embodiments, the UF / DF membrane can be a Pellion3 Ultracel™ 30 kDa, D membrane, Sartocon Slice ECO Hydrosart™ 30 kDa membrane, or other 30 kDa or 50 kDa membrane. The membrane area can be adjusted according to the total amount of protein to be processed. In some embodiments, the membrane loading capacity is about 229.0 g / m 2 , 585.2 g / m 2 , 601.7 g / m 2 , 739.7 g / m 2 and preferably between 100 g / m 2 ~800 g / m 2 of.

[0006] In some embodiments, the transmembrane pressure (TMP) of UF1 is in the range of 6 - 29 Psi, preferably about 14.5 Psi. The feed flow rate is 4 L / min / m 2 , 5 L / min / m 2 , or up to 6 L / min / m 2It can be. The protein concentration in the UF1 pool after the first ultrafiltration step can be 25 g / L, 50 g / L, or 75 g / L. In some embodiments, the UF1 pool concentration is 30 g / L, 70 g / L, or any value between 25 and 75 g / L. In some embodiments, in the UF1 step, the VCF is 3.41 to 10.23, preferably 25 or less.

[0007] In some embodiments, the TMP of the DF is in the range of 6 to 29 Psi, preferably about 14.5 Psi. The feed flow rate is 4 L / min / m 2 , 5 L / min / m 2 , or up to 6 L / min / m 2 It can be. The starting protein concentration of the UF1 pool in the DF step is 25 to 75 g / L, preferably 50 g / L. The volume exchange times of the DF step should be greater than 4, preferably 6 or more.

[0008] In some embodiments, the TMP of the UF2 is in the range of 6 to 29 Psi, preferably about 14.5 Psi. In some embodiments, the feed flow rate is about 0.5 L / min / m 2 , 1 L / min / m 2 , 2.5 L / min / m 2 , 5 L / min / m 2 or up to 6 L / min / m 2 It can be. The feed flow rate needs to be adjusted by keeping the TMP relatively constant at the target pressure. The adjustment can be done manually or automatically by proportional-integral-derivative (PID) settings.

[0009] In some embodiments, the over-concentration pool of the UF2 step can have a concentration of 60 g / L, 180 g / L, 200 g / L, 240 g / L, and any value from 50 g / L to 250 g / L at room temperature, and the solution viscosity can be up to 300 mPa·s. The UF2 pool prepared from the over-concentration pool can have a required concentration of 50 g / L to 243 g / L at room temperature by dilution with the DF buffer. The protein concentration of the UF2 pool for subcutaneous administration purposes requires a high concentration, preferably a concentration exceeding 150 g / L. In some embodiments, the UF2 pool has a concentration of 167 g / L, 174 g / L, 184 g / L, 204 g / L, and 243 g / L.

[0010] In some embodiments, the UF1 pool, DF pool, over-concentration pool, and UF2 pool are stable at room temperature for 1 hour and up to 5 hours. The protein quality data (SEC-HPLC and CE-SDS (NR)) is consistent even when maintained at room temperature for 5 hours in the very high concentration 243 g / L treatment during the UF / DF process from UF1 to UF2 pools. In some embodiments, the final highly concentrated drug substance produced by UF / DF for subcutaneous administration according to the present disclosure has quality data (SEC, CE-SDS (NR), and CZE) equivalent to that of the drug substance for intravenous injection.

[0011] In some embodiments, the UF / DF unit operation is processed at 30 °C using a buffer, and all intermediate product pools are maintained at 30 °C. The processing time at 30 °C may be approximately 1 / 4 shorter than the time at room temperature. The over-concentration pool and UF2 pool can achieve up to 250 g / L at 30 °C.

[0012] In some embodiments, the viscosity of the protein solution is 1.56 mPa·s in the feed solution, about 33.47 mPa·s in the UF1 pool and DF pool, and up to 292.4 mPa·s in the over-concentration pool and UF2 pool. The UF / DF process and system can handle solutions with a wide range of viscosities up to 300 mPa·s.

[0013] In some embodiments, the formulation buffer is selected from histidine, acetate, and a mixture of histidine and acetic acid. In some embodiments, the formulation buffer can be a histidine buffer. In some embodiments, the concentration of the histidine buffer is from about 10 mM to about 30 mM. In some embodiments, the concentration of the histidine buffer is about 20 mM of histidine. [Table 1]

[0014] In some embodiments, the PD-1 antibody is tislelizumab (BGB-A317, Table 2) or an antigen-binding fragment of tislelizumab.

[0015] In some embodiments, the subcutaneous antibody formulation has an antibody concentration between about 50 mg and 800 mg. In another embodiment, the subcutaneous antibody formulation has an antibody concentration of about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, or about 600 mg. [Brief Description of the Drawings]

[0016]

Figure 1A

Figure 1B

Figure 1C

Figure 1D

Figure 1E

Figure 1F

Figure 1G

Figure 2A

Figure 2B

Figure 3A

Figure 3B

Figure 4A

Figure 4B

Figure 4C

Figure 4D

Figure 4E

Figure 4F

Figure 5A

Figure 5B

Figure 5C

Mode for Carrying Out the Invention

[0017] Definitions Unless otherwise specifically defined elsewhere in this document, all other technical and scientific terms used in this specification have the meanings commonly understood by those skilled in the art.

[0018] As used in this specification, including in the appended claims, singular words such as "a", "an", and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0019] The term "or" is used to mean "and / or" and is used interchangeably unless the context clearly dictates otherwise.

[0020] Throughout this specification and the claims that follow, unless the context clearly dictates otherwise, the word "comprise" and variations such as "comprises" and "comprising" are to be understood to mean that the recited amino acid sequences, DNA sequences, steps, or groups thereof are included, but do not exclude any other amino acid sequences, DNA sequences, steps. As used in this specification, the term "comprising" can be replaced by the terms "containing", "including", or in some cases "having".

[0021] The term "antibody" as used herein is used in the broadest sense and specifically encompasses antibodies (including full-length monoclonal antibodies) and antibody fragments as long as they recognize an antigen, e.g., PD-1. Antibodies are usually monospecific but can also be called idiotypic, heterospecific, or multispecific. Antibody molecules bind to specific antigenic determinants or epitopes on an antigen by specific binding sites.

[0022] As used herein, the terms "monoclonal antibody," "mAb," or "Mab" mean a substantially homogeneous population of antibodies, i.e., the antibody molecules within the population have the same amino acid sequence except for possible naturally occurring mutations that may be present in trace amounts. In contrast, conventional (polyclonal) antibody preparations typically contain a number of different antibodies having different amino acid sequences within the variable domains, particularly in the complementarity determining regions (CDRs) that are often specific for different epitopes. The modifier "monoclonal" indicates the antibody's characteristic of being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring the production of the antibody by any particular method. Monoclonal antibodies (mAbs) may be obtained by methods known to those of skill in the art. See, for example, Kohler G et al., Nature 1975 256:495-497; U.S.Pat.No.4,376,110; Ausubel FM et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1992; Harlow E et al., ANTIBODIES: A LABORATORY MANUAL, Cold spring Harbor Laboratory 1988; and Colligan JE et al., CURRENT PROTOCOLS IN IMMUNOLOGY 1993. The mAbs disclosed herein may be of any immunoglobulin class including IgG, IgM, IgD, IgE, IgA, and any of their subclasses. Hybridomas producing mAbs may be cultured in vitro or in vivo. In vivo production can yield high titers of mAbs. In in vivo production, cells from individual hybridomas are injected intraperitoneally into a mouse, e.g., a pristine-primed Balb / c mouse, to generate ascites containing high concentrations of the desired mAb. MAbs of isotype IgM or IgG may be purified from such ascites or culture supernatants using column chromatography methods well known to those of skill in the art.

[0023] Generally, the basic antibody structural unit contains a tetramer. Each tetramer contains a pair of two identical polypeptide chains, and each pair has one "light chain" (about 25 kDa) and one "heavy chain" (about 50 - 70 kDa). The amino-terminal portion of each chain contains a variable region of about 100 - 110 or more amino acids in length that is mainly involved in antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region that is mainly involved in effector functions. Typically, human light chains are classified into kappa light chains and lambda light chains. Furthermore, human heavy chains are typically classified as α, δ, ε, γ, or μ, and the antibody isotypes are defined as IgA, IgD, IgE, IgG, and IgM, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region consisting of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 10 or more amino acids.

[0024] The variable regions of each light chain / heavy chain (VL / VH) pair form the antibody binding site. Thus, generally, an intact antibody has two binding sites. Except for bifunctional antibodies or bispecific antibodies, the two binding sites are generally the same.

[0025] Typically, both the variable domains of the heavy and light chains contain three hypervariable regions, also known as "complementarity determining regions (CDRs)", which are located between relatively conserved framework regions (FRs). The CDRs are typically aligned by the framework regions and enable binding to a particular epitope. Generally, both the variable domains of the light and heavy chains, from the N-terminus to the C-terminus, sequentially contain FR-1 (or FR1), CDR-1 (or CDR1), FR-2 (FR2), CDR-2 (CDR2), FR-3 (or FR3), CDR-3 (CDR3), and FR-4 (or FR4). The amino acid assignments to each domain generally follow the following definitions: Sequences of Proteins of Immunological Interest, Kabat, et al., National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al, (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

[0026] The term "hypervariable region" means the amino acid residues of an antibody that are involved in antigen binding. The hypervariable regions include the amino acid residues of the "CDRs" (i.e., VL-CDR1, VL-CDR2, and VL-CDR3 of the light chain variable domain and VH-CDR1, VH-CDR2, and VH-CDR3 of the heavy chain variable domain). See the following: Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (defining the CDR regions of antibodies by sequence); see also the following: Chothia and Lesk (1987) J. Mol. Biol. 196:901-917 (defining the CDR regions of antibodies by structure). The term "framework" or "FR" residues means the variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0027] Unless otherwise specified, an "antibody fragment" or "antigen-binding fragment" means an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to an antigen to which the full-length antibody binds, e.g., a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., single-chain Fv (ScFv); nanobodies and multispecific antibodies formed from antibody fragments, but are not limited thereto.

[0028] An antibody that binds specifically to a particular target protein is also described as binding specifically to that particular target protein. This indicates that the antibody binds preferentially to its target compared to other proteins, but this specificity does not mean that absolute binding specificity is required. An antibody is considered "specific" for its intended target when its binding allows the presence of the target protein in a sample to be determined without producing undesirable results such as false positives. Antibodies or binding fragments thereof useful in the present disclosure bind to the target protein with an affinity at least 2-fold, preferably at least 10-fold, more preferably at least 20-fold, and most preferably at least 100-fold higher than the affinity for non-target proteins. The antibodies herein are said to bind specifically to a polypeptide containing a given amino acid sequence.

[0029] The term "human antibody" as used herein means an antibody that contains only human immunoglobulin protein sequences. A human antibody may contain mouse sugar chains when produced in a mouse, mouse cells, or a hybridoma derived from mouse cells. Similarly, a "mouse antibody" or a "rat antibody" means an antibody that contains only mouse or rat immunoglobulin protein sequences, respectively.

[0030] The term "humanized antibody" means a form of antibody that also contains the sequences of non-human (e.g., mouse) antibodies and human antibodies. Such antibodies contain a minimal sequence derived from non-human immunoglobulins. Generally, a humanized antibody contains substantially all of at least one, typically two, variable domains, and all or substantially all of the hypervariable loops of the variable domains correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the FR regions are the FRs of human immunoglobulin sequences. A humanized antibody optionally also contains at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin constant region (Fc). The prefixes "hum", "hu", "Hu", or "h" are added to the antibody clone name when it is necessary to distinguish the humanized antibody from the parental rodent antibody. The humanized form of a rodent antibody generally contains the same CDR sequences as the parental rodent antibody, but may contain certain amino acid substitutions to improve affinity, to improve the stability of the humanized antibody, or for other reasons.

[0031] Furthermore, the antibodies of the present application potentially have therapeutic use in the control of viral infections and other human diseases that are mechanistically involved in immune tolerance or "exhaustion". In the context of the present application, the term "exhaustion" refers to a process leading to the exhaustion of the ability of immune cells to respond to cancer or chronic viral infection.

[0032] The term "transmembrane pressure difference" or "TMP" is the pressure across the UF / DF membrane. TMP is calculated by the following equation (1):

Equation

[0033] "Ultrafiltration step 1" (UF1) means the first ultrafiltration step in the process, which is shown in Figure 1A.

[0034] The "diafiltration step" (DF) means any diafiltration step in the process, which is shown in Figure 1A.

[0035] The term "ultrafiltration step 2" (UF2) means the ultrafiltration step 2 in the process, which is shown in Figure 1A.

[0036] The abbreviation "VCF" means "volume concentration factor", which is the amount by which the volume of the feed stream has decreased from the initial volume calculated in equation (2).

Number

[0037] The term "WFI" means "water for injection".

[0038] "CIP" means "clean-in-place".

[0039] The term "NWP" is an abbreviation for "normalized water permeability". The NWP test is a method for evaluating the effectiveness of the membrane CIP process.

[0040] The term "permeate flux" is defined as the flux of the solution through the UF / DF membrane.

[0041] Anti-PD-1 antibody The present disclosure provides an anti-PD-1 antibody and a subcutaneous formulation thereof. For example, tislelizumab (BGB-A317) is an anti-PD-1 antibody disclosed in U.S. Patent No. 8,735,553, and its sequence is presented below.

Table 2-1

Table 2-2

[0042] The anti-PD-1 antibody may include, but is not limited to, tislelizumab, pembrolizumab, or nivolumab. Pembrolizumab (formerly MK-3475), disclosed in US8,354,509 and US8,900,587 by Merck, is a humanized IgG4-K immunoglobulin that targets the PD-1 receptor and inhibits the binding of the PD-1 receptor ligands PD-L1 and PD-L2. Pembrolizumab is approved for the indications of metastatic melanoma and metastatic non-small cell lung cancer (NSCLC), and is under clinical investigation for the treatment of head and neck squamous cell carcinoma (HNSCC) and refractory Hodgkin lymphoma (cHL). Nivolumab (disclosed by Bristol-Meyers Squibb) is a fully human IgG4-K monoclonal antibody. Nivolumab (clone 5C4) is disclosed in US Patent No. 8,008,449 and WO2006 / 121168. Nivolumab is approved for the treatment of melanoma, lung cancer, kidney cancer, and Hodgkin lymphoma.

[0043] Antibody production The anti-PD-1 antibody and its antigen-binding fragments can be produced by any means known in the art, including but not limited to recombinant expression of antibody tetramers, chemical synthesis, and enzymatic digestion, while full-length monoclonal antibodies can be obtained, for example, by hybridoma or recombinant production. Recombinant expression can be derived from any suitable host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0044] The present disclosure further provides polynucleotides encoding the antibodies described herein, for example, polynucleotides encoding heavy or light chain variable regions or segments comprising the complementarity determining regions described herein. In some embodiments, the polynucleotide encoding the heavy chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity to the polynucleotide encoding the polypeptide of SEQ ID NO: 7. In some embodiments, the polynucleotide encoding the light chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity to the polynucleotide encoding the polypeptide of SEQ ID NO: 8.

[0045] The polynucleotides of the present disclosure can encode the variable region sequences of anti-PD-1 antibodies. The polynucleotides of the present disclosure can also encode both the variable and constant regions of an antibody. Some of the polynucleotide sequences encode polypeptides comprising the variable regions of both the heavy and light chains of the exemplified tislelizumab antibody.

[0046] Also provided herein are expression vectors and host cells for producing tisrelizumab antibodies. The choice of expression vector depends on the host cell in which the vector is intended to be expressed. Typically, an expression vector contains a promoter and other regulatory sequences (e.g., enhancers) operably linked to a polynucleotide encoding a tisrelizumab antibody chain or antigen-binding fragment. In some embodiments, an inducible promoter is used to prevent expression of the inserted sequence except when under the control of inducing conditions. Examples of inducible promoters include, for example, the arabinose, lacZ, metallothionein promoter, or heat shock promoter. Cultures of the transformed organisms can be grown under non-inducing conditions without biasing the population for a coding sequence whose expression product is better tolerated by the host cell. In addition to the promoter, other regulatory elements may be required or desired for efficient expression of the tisrelizumab antibody or antigen-binding fragment. These elements typically include the ATG start codon and adjacent ribosome binding site or other sequences. Additionally, the efficiency of expression can be enhanced by including an enhancer appropriate for the cell line in use (see, e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

[0047] Host cells for harboring and expressing the tisrelizumab antibody chain may be prokaryotic cells or eukaryotic cells. E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present disclosure. Other microbial hosts suitable for use include bacilli such as Bacillus subtilis, and other Enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, expression vectors can also be prepared, which typically contain expression control sequences compatible with the host cell (e.g., origin of replication). In addition, any number of various well-known promoters exist, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system derived from phage lambda. The promoter typically optionally controls expression by an operator sequence and has a ribosome binding site sequence, etc. for initiating and completing transcription and translation. Other microorganisms such as yeast can also be used to express tisrelizumab. Insect cells combined with a baculovirus vector can also be used.

[0048] In other embodiments, mammalian host cells are used to express and produce tislelizumab. For example, they can be either a hybridoma cell line that expresses an endogenous immunoglobulin gene or a mammalian cell line that carries an exogenous expression vector. These include any normal lethal or normal or abnormal immortal animal or human cells. For example, a plurality of suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HEK293 cells, myeloma cell lines, transformed B cells, and hybridomas. The use of mammalian tissue cell cultures for expressing polypeptides is generally discussed, for example, in Winnacker, From Genes to Clones, VCH Publishers, NY, N.Y., 1987. Expression vectors for mammalian host cells can include expression control sequences such as an origin of replication, a promoter, and an enhancer (see, for example, Queen et al., Immunol. Rev. 89:49-68, 1986), and necessary processing information sites such as a ribosome binding site, an RNA splice site, a polyadenylation site, and a transcription terminator sequence. These expression vectors usually contain a promoter derived from a mammalian gene or a mammalian virus. Suitable promoters can be constitutive, cell-type specific, stage-specific, and / or modulatable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate early CMV promoter), the constitutive CMV promoter, and combinations of promoters-enhancers known in the art.

Example

[0049] Examples and descriptions of certain embodiments are not limiting of the disclosure as defined by the claims and can be used by way of illustration. As will be readily understood, numerous variations and combinations of the features described above can be utilized without departing from the disclosure as set forth in the claims. All such variations are intended to be included within the scope of the disclosure. All references cited are hereby incorporated by reference in their entirety.

[0050] Analysis method The section on this method shows an overview of the methods used in Examples 1 - 5 below.

[0051] SEC-HPLC The formation of soluble aggregates is analyzed by size exclusion chromatography (SEC) using a Waters HPLC system. Using an isocratic gradient method, the protein is separated based on molecular size on a TSKgel G3000 (TM) SWXL column maintained at 37 ± 5 °C. Molecular weight species are eluted and detected by UV absorption at 280 nm. The distribution of aggregates, monomers, and fragments is quantified by the peak areas of standards and samples.

[0052] CZE Capillary zone electrophoresis (CZE), also known as free solution capillary electrophoresis, is used with a PA800 Plus (TM) (Beckman) to determine the charge heterogeneity of the sample. The sample is separated based on the electrophoretic mobility resulting from differences in the charge and hydrodynamic radius of the analytes within a capillary filled with a buffer solution containing capric acid. The sample is analyzed in its native state when an external electric field is applied, resulting in a specific peak pattern showing the various charge variants (acidic, basic, and major charge variant) of the antibody. The sample is injected by pressure and the migrated protein is detected by UV absorbance at 214 nm.

[0053] CE-SDS(NR) The purity of the sample is determined by capillary gel electrophoresis (CE) using a PA800 Plus (trademark) (Beckman). The sample is denatured with sodium dodecyl sulfate (SDS) and separated based on size within a capillary filled with a gel that functions as a sieving medium. For non-reducing (NR) samples, the alkylating agent N-ethylmaleimide (NEM) is added to avoid any fragmentation caused by the sample preparation and to confirm that the major IgG peak remains intact. The sample is electrokinetically injected and the migrated proteins are detected using a UV detector at a UV absorbance of 200 nm. The reportable value for non-reducing samples is the time-corrected area percent (TCA)% of the major IgG peak.

[0054] Protein concentration The protein concentration is determined at UV 280 nm.

[0055] Viscosity The viscosity of the antibody formulation is measured using a chip-based microVISC (trademark) instrument (Rheosense), and the pressure difference correlates with the kinematic viscosity of the solution. The sample size is approximately 70 - 100 μL. An aliquot is loaded into a 400 μL microVISC (trademark) disposable pipette and connected to the chip. The measurement is performed three times at a shear rate of 500 S -1 at a temperature of approximately 25 °C.

[0056] Osmolality The osmolality of the antibody solution or buffer solution is measured using an OSMOMAT 3000 (trademark) osmometer (Gonotec). 50 μl of each sample is loaded and tested twice to obtain an average osmolality value.

[0057] Example 1: Definition of parameters for the UF1 / DF / UF2 process of a high-concentration PD-1 antibody solution To define the parameters of UF / DF for a high-concentration PD-1 antibody solution, a laboratory-scale UF / DF system and process were designed for scale-up and future large-scale GMP production. The unit operations include multiple steps such as pre-treatment of the membrane before use (WFI flush, integrity test, CIP, NWP test), equilibration, ultrafiltration 1, diafiltration, ultrafiltration 2, system flush and recovery, UF / DF pool, and post-treatment of the membrane after use, which are shown in the process flow chart (Figure 1A). The UF / DF system consists of a selected UF / DF membrane and membrane housing skid, three pressure gauges for the flow paths of the feed solution, retentate, and permeate, valves for TMP and flow rate adjustment at the outlets of the retentate and permeate, one pump for feed solution loading, one pump for buffer replenishment, and three containers for the feed solution / retentate solution, buffer solution, and permeate solution, as shown in the diagram of Figure 1B. The fluid path of this system was well designed to minimize the dead volume of the system in order to reduce the dilution effect by system flushing.

[0058] In the first step of determining the process parameters of ultrafiltration 1 (UF1), after the virus filtration step, tislelizumab was prepared and purified as the UF / DF process feed solution. The antibody was dispersed in the process feed solution of 50 mM, pH 5.36 acetate buffer at an antibody concentration of 3, 8, 13, or 18 g / L and filtered through a 0.2 μm Corning™ filtration system. To evaluate the TMP-flux relationship, Pellion3 Ultracel™ 30 kDa, D membrane area 0.11 m 2 was used in the laboratory-scale UF / DF process design and tests. The recommended feed flow rate of the Pellion3™ membrane in this example was 4 - 6 L / min / m 2 . The permeate flux was monitored during all treatment conditions.

[0059] Figure 1C shows the feed flow rate of 5 L / min / m in the UF1 step 2It shows the influence of TMP and feed concentration on the permeate flux in this case. As TMP increased, the permeate flux increased accordingly. As the process continued, a high-concentration antibody protein layer was formed on the membrane surface, and the permeate flux reached the optimal point. Further concentration led to a decrease in the flux. For the 3 g / L feed solution, the optimal TMP was about 15 - 21 Psi. For higher feed concentrations of 13 or 18 g / L, the high-concentration layer was formed much earlier than under the 3 g / L feed condition, and the optimal TMP was about 6 - 18 Psi. For different feed solutions in the UF1 step, the TMP was controlled at 6 - 21 Psi.

[0060] Figure 1D shows the influence of TMP and feed flow rate on the permeate flux when using an 8 g / L feed solution in the UF1 step. At different feed flow rates of 4, 5, and 6 L / min / m 2 for all cases, the optimal permeate flux can be achieved by controlling the TMP at 15 - 18 Psi. At a lower TMP controlled at 9 - 15 Psi, the permeate flux did not decrease significantly, which is acceptable in a high-concentration UF / DF process. The feed flow rate of 4 - 6 L / min / m 2 was the best operating range for the UF1 step.

[0061] Figure 1E shows the UF1 intermediate pool protein concentration range during the UF1 step using the value of permeate flux * VCF. The initial concentration of antibody protein in the feed solution was 7.33 g / L. The higher the value of permeate flux * VCF, the better the ultrafiltration effect in the UF1 step and the diafiltration effect in the subsequent DF step. The TMP was controlled at 15 Psi and the feed flow rate was 5 L / min / m 2When controlled, the value of the permeate flux *VCF maintained near the maximum in the antibody protein range of 25 g / L to 75 g / L and decreased sharply at about 80 g / L due to the Donnan effect. The Donnan effect, also known as the Gibbs-Donnan effect or Donnan's law, explains the behavior of charged particles (such as proteins) that cannot be evenly distributed across both sides of the membrane. Since the UF1 intermediate pool protein concentration range: 25 g / L to 75 g / L, is wide, it brought about the robustness of the process in the subsequent DF step and UF2 step. The corresponding VCF was 3.41 (25 g / L / 7.33 g / L) to 10.23 (75 g / L / 7.33 g / L), preferably 25 (75 g / L / 3 g / L) or less. The concentration ratio of the UF2 step was 2 to 3, and a high antibody concentration solution of 50 g / L to 75 g / L could be achieved from the lowest 25 g / L UF1 intermediate pool. The concentration ratio was as low as about 3.2 (about 240 g / L / 75 g / L), and a very high concentration solution of about 240 g / L could be achieved in the UF2 step from the initial 75 g / L UF1 intermediate pool.

[0062] Figure 1F shows curves regarding the changes in pH and conductivity of the permeate flow due to the diafiltration volume exchange at different UF1 intermediate pool concentrations in the diafiltration (DF) step. After 4 volume exchanges, the diafiltration TMP controlled at 15 Psi, and the feed flow rate 5 L / min / m 2 in which the pH and conductivity curves became flat and the values became the same as those of the DF buffer (Table 2), indicating that when the number of volume exchanges is greater than 4, the DF step can be considered completed.

Table 3

[0063] The influence of TMP and feed flow rate on the permeate flux is important to monitor using a 50 g / L diafiltration pool solution in the UF2 step. The permeate flux curve was maintained flat in the TMP range of 6 - 22 Psi for all feed flow rate conditions. This demonstrated that a high - concentration protein layer had already formed on the membrane surface at the initial diafiltration pool concentration of 50 g / L, and there was no optimal TMP for the permeate flux in the UF2 step (Figure 1G). The permeate flux was only proportional to the feed flow rate in the UF2 step. During UF2, as the process progressed, the solution concentration and viscosity increased, which further increased the TMP. Therefore, by adjusting the feed flow rate from the initial target flow rate, for example, 5 L / min / m 2 to a lower value, 1 L / min / m 2 , the TMP can be controlled in UF2 to a specific target value, for example, 15 Psi, or within a range such as 6 - 29 Psi.

[0064] Example 2: UF / DF unit operations for producing a 167 g / L protein pool Tislelizumab was prepared and purified after virus filtration as the UF / DF process feed solution as described in Example 1. The antibody was dispersed at a concentration of 7.97 g / L in a 50 mM, pH 5.37 acetate buffer. For the UF / DF process, a laboratory - scale UF / DF system was used, and Pellion3 Ultracel™ 30 kDa, membrane A with an area of 0.11 m 2 was used. The loading capacity of membrane A was 229.0 g / m 2 . The unit operations included pre - treatment of the membrane (WFI flush, integrity test, CIP, NWP test), equilibration, ultrafiltration 1, diafiltration, ultrafiltration 2, system flush and recovery, UF / DF pool, and post - treatment of the membrane, as shown in the process flow chart (Figure 1A).

[0065] In the UF1 step, the TMP was controlled at approximately 14.5 Psi and the feed flow rate was controlled at 218 LMH. The antibody protein concentration was concentrated up to 48.5 g / L and the viscosity was 1.58 mPa.s. The same TMP and feed flow rate were controlled in the following DF step. After the DF buffer (20 mM His-His HCl, 70 mM NaCl, pH 6.04) was exchanged 6 times, the antibody protein concentration in the DF pool solution was 46.73 g / L and the pH was 5.99. As the protein concentration increased, the TMP was controlled at approximately 14.5 Psi by gradually adjusting the feed flow rate to be lower, down to 109 LMH, and the DF pool solution was further processed in the UF2 step. The over-concentrated pool achieved a protein concentration of 191 g / L with a viscosity of 33.47 mPa.s. After the entire UF / DF system was flushed and recycled with a certain amount of DF buffer, the antibody protein concentration in the final UF2 pool was 167 g / L in a buffer of 20 mM His-His HCl, 70 mM NaCl, pH 6.1.

[0066] Figure 2A shows the process chart of the 167 g / L UF / DF unit operation. The protein concentration increased in both the UF1 and UF2 steps. In the UF2 step, since the antibody protein concentration increased significantly in the high range (>100 g / L), it was necessary to maintain the TMP at the target value of 14.5 Psi and manually decrease the feed flow rate. Figure 2B shows the weight molar osmotic concentration and viscosity curves accompanying the change in the antibody protein concentration in the UF1 / DF / UF2 steps. When the antibody protein concentration exceeded 100 g / L in the UF2 step, the weight molar osmotic concentration and viscosity increased exponentially. This was consistent with the observed decrease in the permeate flux in the UF2 step in Figure 2A. This example also shows that the UF / DF system and the currently designed process were suitable for producing the final antibody protein solution with a viscosity of approximately 33.47 mPa.s.

[0067] Example 3: UF / DF unit operation for producing a 174 g / L protein pool Tisrelizumab was prepared and purified as a UF / DF process feed solution after virus filtration as described in Example 1. The antibody was dispersed in a 50 mM, pH 5.27 acetate buffer at a concentration of 8.27 g / L. For the UF / DF process, a laboratory-scale UF / DF system was used with Membrane B: three 0.14 m 2 Sartocon Slice ECO Hydrosart™ 30 kDa membranes with an area of (total area 0.42 m 2 ). The loading capacity of Membrane B was 739.7 g / m 2 . Unit operations included pre-treatment of the membrane (WFI flush, integrity test, CIP, NWP test), equilibration, ultrafiltration 1, diafiltration, ultrafiltration 2, system flush and recovery, UF / DF pool, post-treatment of the membrane, etc., which are shown in the process flow chart (Figure 1A).

[0068] In the UF1 step, the TMP was controlled at approximately 14.5 Psi and the feed flow rate was controlled at 338.57 LMH. The protein concentration was concentrated to 34.47 g / L in the UF1 pool. The same TMP and feed flow rate were controlled in the following DF step. After 8 volume exchanges of the DF buffer (20 mM His-His HCl, 70 mM NaCl, pH 6.01), the protein concentration of the DF pool solution was 34.64 g / L and the pH was 5.98. As the antibody protein concentration increased, the TMP was controlled at approximately 14.5 Psi (29 Psi or less) by gradually adjusting the feed flow rate to be even lower, down to 38.57 LMH, and the DF pool solution was further processed in the UF2 step. The over-concentration pool achieved an antibody protein concentration of 190.34 g / L. After flushing and recycling the entire UF / DF system with a certain amount of DF buffer, the antibody protein concentration of the final UF2 pool was 173.98 g / L in a buffer of 20 mM His-His HCl, 70 mM NaCl, pH 6.0. The quality data (SEC, CE-SDS (NR), and CZE) shown in Figure 3A demonstrated that the final high-protein-concentration solution (174 g / L) prepared by this UF / DF process was comparable to the current 10 g / L intravenous injection solution, indicating that the integrity of the tislelizumab antibody was maintained during the concentration process.

[0069] Figure 3B shows the process chart of the 174 g / L UF / DF unit operation. The antibody protein concentration increased in both the UF1 and UF2 steps. Due to the larger membrane area and the UF / DF skid (shown in Figure 1B), the TMP and feed flow rate were stably controlled at 14.5 Psi and 338.57 LMH during the UF1 and DF steps. In UF2, as the protein concentration increased, the TMP increased. Therefore, the TMP was controlled by reducing the feed flow rate to approximately 38.57 LMH.

[0070] Example 4: Evaluation of the maximum antibody protein concentration and antibody stability in the final UF / DF step To investigate the antibody protein concentration range and stability in the UF2 step, a new set of UF / DF experiments was designed by continuing the UF2 step until the solution viscosity reached approximately 300 mPa.s. Tislelizumab was prepared and purified as the UF / DF process feed solution after virus filtration as described above. The antibody was dispersed in 50 mM acetate buffer, pH 5.36, at a concentration of 8.15 g / L. For the UF / DF process, a laboratory-scale UF / DF system was used with membrane A: Pellion3 Ultracel™ 30 kDa with an area of 0.11 m 2 and membrane D. The loading capacity of membrane A was 601.67 g / m 2 . Unit operations included pre-treatment of the membrane (WFI flush, integrity test, CIP, NWP test), equilibration, ultrafiltration 1, diafiltration, ultrafiltration 2, system flush and recovery, UF / DF pool, and post-treatment of the membrane as shown in the process flowchart (Figure 1A). First, the solution was concentrated to 50 g / L in UF1 at a viscosity of 1.56 mPa.s. Next, the solution was diafiltered with a DF buffer (20 mM His-His HCl, 70 mM NaCl, pH 6.04) with 6 volume exchanges to obtain the initial test material for UF2.

[0071] Figure 4A shows the process flowchart of the concentrated antibody until the antibody reaches a concentration of 243 g / L. TMP and feed flow rate were maintained constant and well-controlled at 15 ± 0.5 Psi and 300 LMH in the UF1 and DF steps. As shown in Figure 4B, in the UF2 step, as the protein concentration increased, the osmolality and viscosity increased significantly. When the protein concentration exceeded 150 g / L, it was necessary to adjust the TMP and feed flow rate simultaneously to maintain the permeate flux passing through the membrane while keeping the TMP below 29 Psi. During UF2, three in-process samples were taken at protein concentrations of 62, 184, and 204 g / L for quality analysis and stability testing. As the concentration approached the final concentration of 243 g / L, the viscosity reached approximately 300 mPa.s, and both the feed flow rate and permeate flux approached zero, indicating that the limit of the UF2 step had been reached.

[0072] Figures 4C and 4D show the quality characteristics (SEC and CE-SDS (NR)) of the UF1 pool, DF pool, and four concentrated antibody pool samples from the above process at room temperature. Even for the 243 g / L sample, there was no difference in quality among these samples. Figures 4E and 4F show the comparison of the quality characteristics (SEC and CE-SDS (NR)) of the UF1 pool, DF pool, and maximum concentrated antibody pool samples during the 5-hour stability test. The results showed that all inter-process samples were stable for 5 hours, demonstrating the robustness of this UF / DF process even for very high-concentration antibody processes. Since the over-concentrated pool can achieve 243 g / L due to the trade-off between various system flush strategies and yield, the protein concentration range of the final UF2 pool can flexibly cover from 50 g / L (the lower limit of the UF1 pool of 25 g / L using a 2-fold concentration factor in UF2) to 243 g / L (the maximum concentrated pool without system flush accepting a relatively low yield).

[0073] Example 5: UF / DF Unit Operations for Very High Protein Concentrations at Higher Operating Temperatures As the solution temperature increases, it is known that the solution viscosity decreases. Theoretically, operating UF / DF at a higher temperature, such as 30 °C, may show better process performance, such as more uniform TMP and better flux control, or be able to achieve higher concentrations than the process at room temperature or lower temperatures. To evaluate the effect of temperature on this UF / DF process, the anti-IL-6R mAb was prepared and purified as the UF / DF process feed solution after virus filtration as described in Example 1 above. The antibody was dispersed in a 50 mM, pH 5.27 acetate buffer at a concentration of 8.08 g / L. For the UF / DF process, a laboratory-scale UF / DF system was used with membrane A: 0.11 m 2 area of Pellion3 Ultracel™ 30 kDa, and membrane D. The loading capacity of membrane A was 585.2 g / m 2It was as follows. Unit operations included membrane pre-treatment before use (WFI flush, integrity test, CIP, NWP test), equilibration, ultrafiltration 1, diafiltration, ultrafiltration 2, system flush and recovery, UF / DF pool, post-treatment after membrane use, etc., as shown in the process flow chart (Figure 1A). The feed solution and buffer solution were stored in individual containers and maintained at 30 °C under control by a water bath.

[0074] In the UF1 step, the TMP was controlled at about 15 Psi and the feed flow rate was controlled at 300 LMH. The antibody was concentrated to 48.77 g / L and the viscosity was 1.56 mPa·s. The same TMP and feed flow rate were controlled in the following DF step. After 6 volume exchanges of the DF buffer (20 mM His-His HCl, 70 mM NaCl, pH 5.99), the antibody protein concentration in the DF pool solution was 49.15 g / L and the pH was 5.99. As the antibody protein concentration increased, the feed flow rate was gradually adjusted to be lower, down to 120 LMH, so as to control the TMP at about 15 Psi (29 Psi or less), and the DF pool solution was further processed in the UF2 step. The concentrated antibody pool achieved a protein concentration of 248.54 g / L with a viscosity of 292.4 mPa·s in a buffer of 20 mM His-His HCl, 70 mM NaCl, pH 6.06.

[0075] Figures 5A, 5B, and 5C show the process charts of the TMP curve, feed flow rate curve, antibody protein concentration curve, and permeate flow rate curve in the UF1, DF, and UF2 steps; the process chart of the pH curve and conductivity curve in the DF step; and the process charts of the protein concentration curve, osmotic concentration curve, and viscosity curve in each of the UF1, DF, and UF2 steps. The trends of each curve in all steps were the same as those in Example 2, Example 3, and Example 4. Since the viscosity at 30 °C was lower, the permeate flux was slightly higher than that of the process operated at room temperature in the previous example under the same TMP. The permeate flux in the UF1 / DF step was 22.9 LMH at 30 °C and 16.8 LMH at room temperature. The treatment time of UF1 at 30 °C was 72.2 minutes compared to 97 minutes at room temperature. The time for 6 volume exchanges of the target during the DF step at 30 °C was also significantly shorter: 210 minutes (30 °C) compared to 285 minutes (RT).

[0076] As reflected in the process chart, the total operation time to achieve the required target protein concentration was shorter at 30 °C, especially in the UF2 step. For the case of a target antibody concentration of 200 g / L in the UF2 step, the average permeate flux was 10.06 LMH and it took only 54.79 minutes at 30 °C. In contrast, when the temperature was lowered to room temperature, the average permeate flux was 7.03 LMH and it took 71.8 minutes. Also, relatively high concentrations can be achieved. Therefore, when a very high concentration protein solution is required, such as up to 250 g / L, for example, it can be produced by the process shown in this example by controlling the operation temperature at 30 °C.

Claims

Claim 1 An ultrafiltration (UF) / diafiltration (DF) process for a highly concentrated PD-1 antibody solution, comprising the following steps: A. Ultrafiltering the antibody in the process feed material (Ultrafiltration 1 (UF1)) to obtain a UF1 pool protein with an intermediate antibody concentration; B. Diafiltering the UF1 pool protein from step A using a diafiltration (DF) buffer to obtain a final drug substance formulation buffer and a DF pool; C. Ultrafiltering the DF pool from step B to obtain a high-concentration antibody protein solution (over-concentrated pool) with a desired concentration; D. Adjusting the over-concentrated pool, with or without system flush, to a final drug substance target concentration to prepare a UF2 pool, and then further diluting the UF2 pool to a final high-concentration formulation solution. Claim 2 The process according to claim 1, wherein the PD-1 antibody or its antigen-binding fragment comprises (a) CDR (complementary determining region) 1 of SEQ ID NO: 1; (b) CDR2 of SEQ ID NO: 2; (c) CDR3 of SEQ ID NO: 3; (d) CDR1 of SEQ ID NO: 4; (e) CDR2 of SEQ ID NO: 5; and (f) CDR3 of SEQ ID NO: 6, and a light chain variable region. Claim 3 The process according to claim 1, wherein the PD-1 antibody or its antigen-binding fragment comprises SEQ ID NO: 7 and SEQ ID NO:

8. Claim 4 The process according to claim 1, wherein the feed material in step A comprises a buffer, and the buffer is selected from the group consisting of histidine, acetate, citrate, succinate, phosphate, a mixture of histidine and acetic acid, and a mixture of histidine and citric acid. Claim 5 The process according to claim 4, wherein the feed material in step A comprises a buffer, and the buffer is histidine, a mixture of histidine and acetic acid, or a mixture of histidine and citric acid. Claim 6 The process according to claim 1, wherein the concentration of the PD-1 antibody solution is 3 g / L to 18 g / L. Claim 7 The process according to claim 1, wherein steps A to C comprise a 30 kDa or 50 kDa membrane. Claim 8 Membrane load capacity: 100 g / m ２ Up to 800 g / m ２ 8. The process of claim 7, wherein Claim 9 The process according to claim 1, wherein step A further comprises a transmembrane pressure (TMP) in the range of 6 to 29 Psi. Claim 10 The process according to claim 9, wherein the TMP is about 14.5 Psi.

11. Step A further includes a feed flow rate of up to 6 L / min / m ２ The process according to claim 1, further comprising

12. The process according to claim 1, wherein in step A, the UF1 pool protein concentration is in the range of 25 to 75 g / L.

13. The process according to claim 1, wherein step A results in a volume concentration factor (VCF) in the range of 2 to 25.

14. The process according to claim 1, wherein step B further comprises a TMP in the range of 6 to 29 Psi.

15. The process according to claim 14, wherein the TMP is about 14.5 Psi.

16. Step B is further included with a feed flow rate of up to 6 L / min / m ２ The process according to claim 1, further comprising a feed flow rate of up to 6 L / min / m.

17. The process according to claim 1, wherein in step B, the UF1 pool protein has a protein concentration between 25 and 75 g / L.

18. The process according to claim 17, wherein the UF1 pool protein has a concentration of about 50 g / L.

19. The process according to claim 1, wherein in step B, the volume exchange of the diafiltration buffer is 4 to 8.

20. The process according to claim 19, wherein the volume exchange of the diafiltration (DF) buffer is 6.

21. The process according to claim 1, wherein in step C, it further comprises a TMP in the range of 6 to 29 Psi.

22. The process according to claim 21, wherein the TMP is about 14.5 Psi.

23. Step C further includes a feed flow rate of up to 6 L / min / m ２ The process according to claim 1, further comprising.

24. The process according to claim 23, wherein the feed flux is adjusted by maintaining the TMP at a target pressure of about 14.5 Psi.

25. The process according to claim 24, wherein the feed flow rate adjustment can be adjusted manually or automatically.

26. The process according to claim 1, wherein in step C, the DF pool has a protein concentration between 25 and 75 g / L.

27. The process according to claim 26, wherein the protein concentration is about 50 g / L.

28. The process according to claim 1, wherein in step D, the over-concentration pool has a protein concentration of 60 g / L to 250 g / L.

29. The process according to claim 1, wherein the UF2 pool in step D is prepared by diluting the over-concentration pool to a concentration of 60 g / L to 250 g / L.

30. The process according to claim 29, wherein the UF2 pool is prepared by diluting the over-concentration pool to 167 g / L.

31. The process according to claim 30, wherein the UF2 pool in step D is buffered with histidine.

32. The process according to claim 31, wherein the concentration of histidine is 15 mM to 25 mM.

33. The process according to claim 32, wherein the buffer solution contains a 20 mM histidine buffer with a pH between 5.5 and 6.

0.

34. The process according to claim 1, wherein the temperature in all of the process steps is carried out between 22 °C and 30 °C.

35. The process according to claim 1, wherein the viscosity of the antibody solution during any step of the process can be from 0.8 mPa·s to 300 mPa·s.

Images