Methods to reduce aggregation in preparations of anti-CD40 antibody-inactivated virus.

Abstract

The present invention relates to compositions of virus-inactivated antibody preparations having reduced levels of high molecular weight aggregates, methods for producing the same, and / or methods for minimizing the formation of high molecular weight aggregates in virus-inactivated antibody preparations, said virus-inactivated antibody preparations comprising an antibody or its antigen-binding portion, such as an anti-CD40 antibody or its antigen-binding portion.

Description

Related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 617,366, filed January 3, 2024, the entire contents of which are incorporated herein by reference.

[0002] sequence list This application contains a sequence list that has been electronically submitted in XML format, the entire contents of which are incorporated herein by reference. The XML file was created on December 27, 2024, named 132261_00202.xml, and is 17,440 bytes in size. Background of the Invention Antibodies, such as monoclonal antibodies, are an important class of therapeutic drugs in the pharmaceutical industry. Antibody therapeutics have been developed to treat many diseases, such as autoinflammatory and autoimmune diseases.

[0004] The production of proteins (such as monoclonal antibodies) for pharmaceutical applications typically involves upstream process technologies (such as cell culture) and downstream process technologies (such as protein purification). Antibodies are usually produced as recombinant proteins in mammalian cell cultures to ensure proper folding and post-translational modifications. Monoclonal antibodies derived from cell cultures require purification from host cell impurities, product-related impurities, and other process-related impurities for efficient utilization, for example, to improve safety and potency.

[0005] The purification process of monoclonal antibodies typically consists of primary recovery, capture (e.g., protein A chromatography), virus inactivation via low pH or detergent, purification (e.g., ion exchange chromatography), virus filtration, and ultrafiltration and percolation steps.

[0006] The virus inactivation step is dedicated exclusively to virus reduction to achieve an effective virus clearance strategy. Low-pH virus inactivation is typically performed after the protein A capture step. The pool is titrated to a sufficiently acidic pH to disrupt the viral envelope and held statically, then titrated to a pH where the protein is more stable and more suitable for the next chromatographic step.

[0007] The inventors have discovered that the low-pH virus inactivation step inadvertently induces protein aggregation of anti-CD40 monoclonal antibodies during this process. Establishing a robust preparation process that minimizes protein aggregation is crucial in the development of therapeutic antibodies. In clinical development, it is necessary to establish virus inactivation conditions that do not negatively impact product quality. Invention Overview The present invention is based at least on the identification of surprising and unexpected virus inactivation and neutralization conditions during the purification process of anti-CD40 antibodies or their antigen-binding moieties (e.g., KPL-404), which stabilize proteins and / or reduce the formation of high molecular weight aggregates in the preparation of virus-inactivated proteins.

[0009] A typical virus inactivation process used in the industry involves titrating a sample containing the protein of interest to a low pH to inactivate any enveloped viruses and viral components. The pH condition is chosen as a balance between a low enough pH to induce virus inactivation and a sufficiently high pH to avoid protein denaturation.

[0010] The inventors of this invention made a surprising and unexpected discovery: although pH ≤ 3.6 is the industry standard for virus inactivation processes, significant and unacceptable levels of protein aggregates were observed during the virus inactivation step for anti-CD40 antibodies (e.g., KPL-404) at this pH range. Therefore, new and unexpected conditions for virus inactivation were established during the purification process to ensure adequate virus inactivation while minimizing protein aggregation.

[0011] Therefore, in one aspect, the present invention provides a method for producing a virus-inactivated antibody preparation comprising an anti-CD40 antibody or its antigen-binding portion having reduced levels of high molecular weight aggregates, the method comprising, during a virus inactivation step, incubating a sample comprising an anti-CD40 antibody or its antigen-binding portion at a pH of about 3.6-3.9 to produce a virus-inactivated antibody preparation having reduced levels of high molecular weight aggregates.

[0012] In another aspect, the present invention provides a method for minimizing the formation of high molecular weight aggregates in antibody preparations containing anti-CD40 antibody or its antigen-binding moiety during a virus inactivation step, the method comprising incubating a sample containing anti-CD40 antibody or its antigen-binding moiety at a pH of about 3.6-3.9 during the virus inactivation step, thereby minimizing the formation of high molecular weight aggregates in the virus-inactivated antibody preparation.

[0013] In one aspect, the present invention provides a method for reducing the formation of high molecular weight aggregates in an antibody preparation containing an anti-CD40 antibody or its antigen-binding portion during a virus inactivation step, the method comprising incubating a sample containing an anti-CD40 antibody or its antigen-binding portion at a pH of about 3.6-3.9 during the virus inactivation step, thereby reducing the formation of high molecular weight aggregates in the virus-inactivated antibody preparation.

[0014] In another aspect, the present invention provides a method for maximizing the level of antibody monomers in a virus-inactivated antibody preparation containing an anti-CD40 antibody or its antigen-binding moiety thereof, the method comprising, during a virus inactivation step, incubating a sample containing an anti-CD40 antibody or its antigen-binding moiety at a pH of about 3.6-3.9, thereby maximizing the antibody monomers in the virus-inactivated antibody preparation.

[0015] In one aspect, the present invention provides a method for stabilizing a virus-inactivated antibody preparation comprising an anti-CD40 antibody or an antigen-binding moiety thereof, the method comprising, during a virus inactivation step, incubating a sample comprising an anti-CD40 antibody or an antigen-binding moiety thereof at a pH of about 3.6-3.9, thereby stabilizing the virus-inactivated antibody preparation.

[0016] In another aspect, the present invention provides a method for reducing the number and / or activity of viral particles in an antibody preparation containing an anti-CD40 antibody or its antigen-binding moiety thereof, the method comprising, during a virus inactivation step, incubating a sample containing an anti-CD40 antibody or its antigen-binding moiety at a pH of about 3.6-3.9, thereby reducing the number and / or activity level of viral particles in the antibody preparation.

[0017] In some implementations, the virus inactivation step includes a virus inactivation period and an optional static retention period.

[0018] In some embodiments, the anti-CD40 antibody or its antigen-binding portion includes a heavy chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 1, CDR2 having the amino acid sequence of SEQ ID NO: 2, and CDR3 having the amino acid sequence of SEQ ID NO: 3.

[0019] In some embodiments, the anti-CD40 antibody or its antigen-binding portion comprises a light chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0020] In some embodiments, the anti-CD40 antibody or its antigen-binding portion comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8.

[0021] In some embodiments, the anti-CD40 antibody or its antigen-binding portion comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and the light chain comprising the amino acid sequence of SEQ ID NO: 10.

[0022] In some embodiments, the anti-CD40 antibody or its antigen-binding portion is KPL-404.

[0023] In some implementations, during the virus inactivation step, the sample is incubated at a pH of about 3.6-3.9, about 3.6-3.8, about 3.7-3.9, about 3.6-3.7, about 3.7-3.8, or about 3.8-3.9.

[0024] In some implementations, the sample is incubated at a pH of about 3.6, about 3.7, about 3.8, or about 3.9 during the virus inactivation step.

[0025] In some implementations, the sample is incubated for approximately 15-360 minutes during the virus inactivation step.

[0026] In some embodiments, during the virus inactivation step, the sample is incubated for approximately 15-50 minutes, approximately 20-40 minutes, approximately 20-60 minutes, approximately 30-70 minutes, approximately 30-60 minutes, approximately 30-120 minutes, approximately 60-120 minutes, approximately 40-80 minutes, approximately 50-70 minutes, approximately 50-90 minutes, approximately 60-100 minutes, approximately 70-110 minutes, approximately 80-100 minutes, approximately 80-120 minutes, approximately 90-130 minutes, approximately 100-140 minutes, approximately 110- 130 minutes, approximately 110-150 minutes, approximately 120-160 minutes, approximately 130-170 minutes, approximately 140-180 minutes, approximately 150-190 minutes, approximately 160-200 minutes, approximately 170-190 minutes, approximately 170-210 minutes, approximately 180-220 minutes, approximately 200-240 minutes, approximately 220-260 minutes, approximately 230-250 minutes, approximately 240-280 minutes, approximately 280-320 minutes, approximately 290-310 minutes, or approximately 320-360 minutes.

[0027] In some embodiments, during the virus inactivation step, the sample is incubated at temperatures of approximately 13°C-37°C, approximately 15°C-37°C, approximately 15°C-30°C, approximately 13°C-25°C, or approximately 15°C-25°C.

[0028] In some embodiments, during the virus inactivation step, the sample is incubated at a pH of approximately 3.7-3.9 for approximately 30-120 minutes. In some embodiments, during the virus inactivation step, the sample is incubated at a pH of approximately 3.7-3.9 for approximately 60-120 minutes. In other embodiments, during the virus inactivation step, the sample is incubated at a temperature of approximately 15°C-25°C and a pH of approximately 3.7-3.9 for approximately 50-70 minutes.

[0029] In some embodiments, during the virus inactivation step, the sample is incubated at a pH of approximately 3.6-3.8 for approximately 30-120 minutes. In some embodiments, during the virus inactivation step, the sample is incubated at a pH of approximately 3.6-3.8 for approximately 60-120 minutes. In other embodiments, during the virus inactivation step, the sample is incubated at a temperature of approximately 15°C-25°C and a pH of approximately 3.6-3.8 for approximately 50-70 minutes.

[0030] In some embodiments, during the virus inactivation step, the sample is incubated at a temperature of about 15°C-25°C and a pH of about 3.6-3.8 for about 30-120 minutes. In some embodiments, during the virus inactivation step, the sample is incubated at a temperature of about 15°C-25°C and a pH of about 3.6-3.9 for about 60-120 minutes.

[0031] In some embodiments, during the virus inactivation step, the sample is incubated at a temperature of about 15°C-25°C and a pH of about 3.6-3.8 for about 30-120 minutes. In some embodiments, during the virus inactivation step, the sample is incubated at a temperature of about 15°C-25°C and a pH of about 3.6-3.8 for about 60-120 minutes.

[0032] In some implementations, during the virus inactivation step, the sample is incubated for about 30-120 minutes at a temperature of about 13°C-25°C and a pH of about 3.7-3.8.

[0033] In some embodiments, during the virus inactivation step, the sample is incubated at a pH of about 3.7 for about 60-120 minutes. In some embodiments, during the virus inactivation step, the sample is incubated at a pH of about 3.7 for about 120 minutes.

[0034] In some embodiments, during the virus inactivation step, the sample is incubated at a temperature of about 15°C-25°C and a pH of about 3.7 for about 60-120 minutes. In some embodiments, during the virus inactivation step, the sample is incubated at a temperature of about 15°C-25°C and a pH of about 3.7 for about 120 minutes. In some embodiments, during the virus inactivation step, the sample is incubated at a temperature of about 25°C and a pH of about 3.7 for about 120 minutes.

[0035] In some embodiments, during the virus inactivation step, the sample is incubated at a pH of about 3.8 for about 60-120 minutes. In some embodiments, during the virus inactivation step, the sample is incubated at a pH of about 3.8 for about 60 minutes. In other embodiments, during the virus inactivation step, the sample is incubated at a temperature of about 15°C-25°C and a pH of about 3.8 for about 60 minutes.

[0036] In some implementations, the sample is passed through a protein A column prior to the virus inactivation step.

[0037] In some embodiments, the virus-inactivated antibody preparation contains less than 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1% high molecular weight aggregates.

[0038] In some embodiments, the virus-inactivated antibody preparation contains less than 2% high molecular weight aggregates. In some embodiments, the virus-inactivated antibody preparation contains less than 1% high molecular weight aggregates.

[0039] In some implementations, the level of high molecular weight aggregates is determined by size exclusion chromatography.

[0040] In one aspect, the present invention provides a composition comprising an anti-CD40 antibody or its antigen-binding portion thereof, wherein the composition comprises a virus-inactivated eluent collected from an affinity chromatography column, wherein the eluent comprises less than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1% of high molecular weight aggregates.

[0041] In some embodiments, the eluent from virus inactivation contains less than 2% high molecular weight aggregates. In some embodiments, the eluent from virus inactivation contains less than 1% high molecular weight aggregates.

[0042] In some implementations, the affinity chromatography column includes a protein A chromatography column.

[0043] In some embodiments, the virus-inactivated eluent has undergone a virus inactivation step at a pH of about 3.6-3.9.

[0044] In some embodiments, the anti-CD40 antibody or its antigen-binding portion includes a heavy chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 1, CDR2 having the amino acid sequence of SEQ ID NO: 2, and CDR3 having the amino acid sequence of SEQ ID NO: 3.

[0045] In some embodiments, the anti-CD40 antibody or its antigen-binding portion comprises a light chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0046] In some embodiments, the anti-CD40 antibody or its antigen-binding portion comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8.

[0047] In some embodiments, the anti-CD40 antibody or its antigen-binding portion comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and the light chain comprising the amino acid sequence of SEQ ID NO: 10.

[0048] In some embodiments, the anti-CD40 antibody or its antigen-binding portion is KPL-404.

[0049] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising an anti-CD40 antibody and a pharmaceutically acceptable carrier, the method comprising, during a virus inactivation step, incubating a sample comprising an anti-CD40 antibody or an antigen-binding portion thereof at a pH of about 3.6-3.9, thereby producing a pharmaceutical composition comprising an anti-CD40 antibody and a pharmaceutically acceptable carrier.

[0050] In some embodiments, the antibody preparation comprises charged anti-CD40 antibodies, wherein the charged species comprises at least about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% of the major species.

[0051] In some embodiments, the pharmaceutical composition comprises charged anti-CD40 antibodies, wherein the charged species comprises at least about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% of the major species. Attached Figure Description

[0052] Figure 1 An overview of an exemplary purification process is described.

[0053] Figure 2 The acid titration curves of KPL-404 under prolonged virus inactivation were plotted.

[0054] Figure 3 The percentage of high molecular weight (HMW) protein aggregates in KPL-404 samples after prolonged virus inactivation retention (up to 300 minutes) at pH 3.5 and 3.6 was depicted. Invention Details This invention is based at least on the identification of surprising and unexpected viral inactivation and neutralization conditions during the purification process of proteins of interest (e.g., anti-CD40 antibodies or their antigen-binding moieties), conditions that stabilize proteins and / or minimize the formation of high molecular weight aggregates in the preparation of virally inactivated proteins. In particular, the inventors of this invention made the surprising and unexpected discovery that, although pH ≤ 3.6 is the industry standard for viral inactivation processes, significant and unacceptable levels of protein aggregates were observed during the viral inactivation step for anti-CD40 antibodies (e.g., KPL-404) at this pH range. Therefore, new and unexpected ranges for pH and / or holding time for viral inactivation were established to ensure that KPL-404 can be held for extended periods without compromising the quality of the final product.

[0056] The following description is presented to enable those skilled in the art to make and use various embodiments. The descriptions of specific methods, compositions, techniques, and applications are provided by way of example only. Various modifications to the examples described herein will be apparent to those skilled in the art, and the general principles described herein can be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Therefore, the various embodiments are not intended to be limited to the examples described and shown herein, but are consistent with the scope of the claims.

[0057] 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 application pertains. If any definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in a patent, application, publication, or other publication incorporated herein by reference, the definition set forth in this section shall prevail. The headings provided herein are for convenience only and do not limit this application in any way. The entire contents of all patents, applications, publications, and other publications mentioned herein are incorporated herein by reference.

[0058] I. Definition To facilitate understanding of this invention, certain terms are first defined. Furthermore, it should be noted that whenever a value or range of a parameter is listed, it means that values ​​and ranges within the listed values ​​are also intended to be part of this invention.

[0059] The articles “a” and “a kind” are used in this text to refer to one or more (i.e., at least one) grammatical objects of the article. For example, “an element” means one element or more elements, such as multiple elements.

[0060] The term “including” is used in this document to mean the phrase “including but not limited to” and is used interchangeably with the phrase “including but not limited to”.

[0061] The term “or” is used herein to mean the term “and / or” and is used interchangeably with the term “and / or” unless the context clearly indicates otherwise. For example, “semantic chain or antisemantic chain” is understood as “semantic chain or antisemantic chain or semantic chain and antisemantic chain”.

[0062] The term “about” is used herein to indicate a typical tolerance range in the art. For example, “about” can be understood as about 2 standard deviations from the mean. In some embodiments, about means ±3%. In some embodiments, about means ±2%. In some embodiments, about means ±1%. When about is present before a series of numbers or ranges, it should be understood that “about” may modify each number in that series or range.

[0063] The terms "peptide" and "protein" are used interchangeably to refer to polymers of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues can contain native or non-native amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and polymers of amino acid residues. Full-length proteins and fragments thereof are included in this definition. The term also includes post-expression modifications of peptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. Furthermore, for the purposes of this invention, "peptide" refers to a protein that includes modifications to its native sequence, such as deletions, additions, and substitutions (generally conserved in nature), provided the protein retains the desired activity. These modifications can be intentional, such as through site-directed mutagenesis, or can be accidental, such as through mutations in the host producing the protein or errors due to PCR amplification.

[0064] The term "antibody" refers to an immunoglobulin molecule composed of four polypeptide chains (two heavy (H) chains and two light (L) chains) linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as HCVR or VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains: CHI, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into highly denatured regions called complementarity-determining regions (CDRs) and more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0065] The term antibody also includes chimeric antibodies, humanized antibodies, and antibodies from various species such as mice, humans, cynomolgus monkeys, llamas, camels, etc. The term also includes multivalent antibodies such as bivalent or tetravalent antibodies. Multivalent antibodies include, for example, a single polypeptide chain containing multiple antigen-binding (CDR) domains, and two or more polypeptide chains, each containing one or more antigen-binding domains, which are associated with each other, for example, through hinge regions capable of forming disulfide bonds or any other covalent or non-covalent interactions.

[0066] The term "antigen-binding moiety" (or "antibody moiety") of an antibody includes fragments of an antibody, such as one or more antigen-binding domains, that retain the ability to specifically bind antigens (e.g., in the case of KPL-404, CD40). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding moiety" of an antibody include molecules comprising at least CDR1, CDR2, and CDR3 of a single-domain antibody (sdAb), wherein said molecule is capable of binding antigens. The term "antibody-binding moiety" also refers to molecules comprising at least heavy chains of CDR1, CDR2, and CDR3 and light chains of CDR1, CDR2, and CDR3, wherein said molecule is capable of binding antigens. The term antibody-binding fraction also includes fragments capable of binding antigens, such as (i) Fab fragments, monovalent fragments containing VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments containing two Fab fragments linked by disulfide bonds in a hinge region; (iii) Fab' fragments, which can be formed by reducing F(ab')2 fragments; (iv) Fc fragments, containing CH2 and CH3 regions held together by one or more disulfide bonds and non-covalent interactions, as well as a portion of the hinge region; (v) Fd fragments, containing VH and CH1 domains; (vi) Fv fragments, containing VL and VH domains in an antibody single arm; (vii) reduced IgG or half-IgG; and (viii) dAb fragments (Ward et al. (1989) Nature 341:544-546, the entire teachings of which are incorporated herein by reference), which contains the VH domain. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be joined via synthetic linkers using recombination methods, allowing them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called a single-stranded Fv (scFv); see, for example, Bird). et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883, the entire teachings of which are incorporated herein by reference. This type of single-chain antibody is also intended to be covered within the “antigen-binding portion” of the term antibody. Other forms of single-chain antibodies are also covered, such as biantibodies. Biantibodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thus forcing the domains to pair with complementary domains of another chain to form two antigen-binding sites (see, for example, Holliger, P., et al.(1993) Proc. Natl.Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123, the entire teachings of which are incorporated herein by reference. Furthermore, an antibody or its antigen-binding portion may be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include tetrameric scFv molecules (Kipriyanov, SM, using the streptavidin core region). et al. (1995) Human Antibodies and Hybridomas 6:93-101, the entire teachings of which are incorporated herein by reference) and the preparation of divalent and biotinylated scFv molecules using cysteine ​​residues, labeled peptides, and C-terminal multihistidine tags (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058, the entire teachings of which are incorporated herein by reference). Antibody moieties, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as digestion of whole antibodies with papain or pepsin, respectively. Furthermore, antibodies, antibody moieties, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques as described herein. In one aspect, the antigen-binding moiety is a whole domain or a pair of whole domains.

[0067] The term "human antibody" includes antibodies having variable and constant regions corresponding to the human immunoglobulin sequences described by Kabat et al. (see Kabat, et al.(1991) Sequences of proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIHP Publication No. 91-3242. The human antibodies of this invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo), such as in the CDR, and particularly in CDR3. Mutations may be introduced using a “selective mutagenesis method.” The human antibody may have at least one position where an amino acid residue can be substituted, such as an activity-enhancing amino acid residue not encoded by a human germline immunoglobulin sequence. The human antibody may have up to 20 positions where amino acid residues are substituted that are not part of a human germline immunoglobulin sequence. In other embodiments, up to ten, up to five, up to three, or up to two positions are substituted. In one embodiment, these substitutions are within the CDR region. However, as used herein, the term “human antibody” is intended to exclude antibodies in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human frame sequence.

[0068] The phrase "recombinant human antibody" includes human antibodies prepared, expressed, produced, or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into host cells, antibodies isolated from a recombinant combined human antibody library, and antibodies isolated from animals (e.g., mice) that are transgenic for human immunoglobulin genes (see, for example, Taylor, LD, et al. (1992) Nucl. Acids Res. 20:6287-6295, the entire teachings of which are incorporated herein by reference) or antibodies prepared, expressed, constructed, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. These recombinant human antibodies possess variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, EA, et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242. However, in some embodiments, these recombinant human antibodies are subjected to in vitro mutagenesis (or, when using animals that are transgenic for the human Ig sequence, in vivo somatic cell mutagenesis), so that the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that, although derived from and associated with human germline VH and VL sequences, may not be naturally present in the in vivo human antibody germline library. However, in some embodiments, these recombinant antibodies are the result of selective mutagenesis or reversion mutations, or both.

[0069] As used herein, "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to CD40 is substantially free of antibodies that specifically bind to antigens other than CD40). However, isolated antibodies that specifically bind to CD40 may be cross-reactive with other antigens (e.g., CD40 molecules from other species). Furthermore, isolated antibodies may be substantially free of other cellular material and / or chemicals. A suitable anti-CD40 antibody is KPL-404.

[0070] The terms “Kabat numbering,” “Kabat definition,” and “Kabat labeling” are used interchangeably herein. These terms, as generally accepted in the art, refer to a system for numbering amino acid residues that are more variable (i.e., highly variable) than other amino acid residues in the variable regions of the heavy and light chains of the antibody or its antigen-binding moiety. et al. (1971) Ann. NY Acad, Sci. 190:382-391 and Kabat, EA, et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, the entire teachings of which are incorporated herein by reference. For the heavy chain variable region, the range of the hypervariable region is amino acid positions 31 to 35 for CDR1, 50 to 65 for CDR2, and 95 to 102 for CDR3. For the light chain variable region, the range of the hypervariable region is amino acid positions 24 to 34 for CDR1, 50 to 56 for CDR2, and 89 to 97 for CDR3.

[0071] As used herein, the term "product" refers to a protein of interest that may be present in the background of a sample containing one or more variants and / or impurities, such as product-related substances, product aggregates, fragments, or charged species, such as acidic or basic species, and / or process-related impurities, such as host cell proteins. In some embodiments, the product, i.e., the protein of interest, is an antibody or an antigen-binding fragment thereof.

[0072] As used herein, the term "fragment" refers to any truncated protein species derived from a protein of interest due to the breaking of one or more bonds along the peptide backbone of the protein of interest or the dissociation by enzymatic and / or chemical modification. For example, antibody fragments include, but are not limited to, Fab, F(ab')2, Fab', Fc, Fv, scFv, Fd, dAb, haptens, or other compositions containing a portion of an antibody molecule.

[0073] As used herein, the terms “aggregate” or “high molecular weight aggregate” or “high molecular weight impurity” refer to the oligomerization of two or more individual protein molecules of interest, including but not limited to protein dimers, trimers, tetramers, oligomers and other high molecular weight types.

[0074] As used herein, the terms "charge variant" or "charged species" refer to the complete complement of products with different charges. In some embodiments, such variants may include product aggregates and / or product fragments to such extent that such aggregates and / or fragmentation produce products with charge changes, as seen in analytical techniques used for this purpose. In some embodiments, such variants refer to products having different modifications that cause charge heterogeneity. In monoclonal antibody preparation, charged variants, such as acidic or basic species, can be detected by charge-based separation techniques, such as isoelectric focusing (IEF) gel electrophoresis, capillary isoelectric focusing (cIEF) gel electrophoresis, cation exchange chromatography (CEX), and anion exchange chromatography (AEX).

[0075] As used herein, the term "acidic species" refers to a variant of a protein (e.g., an antibody or its antigen-binding moiety) characterized by a total acidic charge. When analyzing antibodies using IEF-based methods, an acidic species is a variant with a lower apparent pI. When analyzed by chromatographic methods, acidic and basic species are defined based on their retention times relative to the main peak. An acidic species is a variant that elutes earlier than the main peak from CEX or later than the main peak from AEX.

[0076] As used herein, the term "basic species" refers to a variant of a protein, such as an antibody or its antigen-binding moiety, characterized by a total basic charge. When analyzing antibodies using IEF-based methods, a basic species is a variant with a higher apparent p1. When analyzed by chromatographic methods, a basic species is a variant that elutes later than the main peak from CEX or earlier than the main peak from AEX.

[0077] As used herein, the term "major species" refers to the form of protein eluted as the main peak on a chromatogram (e.g., an antibody or its antigen-binding portion), i.e., the majority of species detected during the fractionation of charged variants of proteins.

[0078] As used herein, the term "process-related impurity" refers to an impurity present in a protein-containing composition but not derived from the protein itself. Process-related impurities include, but are not limited to, host cell proteins (HCPs), host cell nucleic acids (e.g., DNA or RNA), chromatographic materials, and culture medium components. Removal of process-related impurities (such as host cell proteins) from the resulting protein product (e.g., an antibody or its antigen-binding portion) is desirable so that the resulting protein product will provide therapeutic benefits with higher potency, greater efficacy, or better stability without undesirable effects.

[0079] As used in this article, “host cell protein” (HCP) refers to protein-related impurities that are non-target proteins derived from host cells.

[0080] As used herein, the term "CD40" refers to the well-known gene and protein, a member of the tumor necrosis factor (TNF) receptor superfamily. The encoded protein is a receptor on antigen-presenting cells of the immune system and is essential for mediating a wide range of immune and inflammatory responses, including T-cell-dependent immunoglobulin class switching, memory B-cell development, and germinal center formation. CD40 is also known as Bp50, TNFRSF5, tumor necrosis factor receptor superfamily member 5, B-cell surface antigen CD40, CD40 antigen, CDw40, or CD40L receptor. The CD40 antigen is displayed on the surface of various cell types, such as normal and neoplastic human B cells, dendritic cells, other antigen-presenting cells (APCs), endothelial cells, monocytes, CD8+ T cells, epithelial cells, some epithelial carcinomas, and many solid tumors, including lung, breast, ovarian, and colon cancers. Malignant B cells from several B-cell lineage tumors express high levels of CD40 and appear to depend on CD40 signaling for survival and proliferation.

[0081] The term "CD40" includes human CD40, the amino acid sequence of which can be found, for example, in GenBank accession numbers NP_001241.1 (SEQ ID NO:11) or NP_690593.1 (SEQ ID NO:12). The term "CD40" also includes cynomolgus monkey CD40, mouse CD40, and rat CD40. The term "CD40" includes wild-type, variant, or isotype of the CD40 protein or a fragment or domain thereof. In some embodiments, the CD40 protein may be coupled to a signal peptide sequence and / or a protein tag.

[0082] As used herein, the term "KPL-404" refers to a monoclonal antibody designed to inhibit the interaction between CD40 and CD154 (CD40 ligands), a well-known pathway that plays a key role in regulating B cell proliferation, T cell activation, and antibody production (see PCT Publication No. WO2017040932, the entire contents of which (including the sequences described therein) are incorporated herein by reference). KPL-404 comprises: a heavy chain containing the sequence shown in SEQ ID NO: 9 and a light chain containing the sequence shown in SEQ ID NO: 10. The variable region of the heavy chain of KPL-404 contains the sequence shown in SEQ ID NO: 7, and the variable region of the light chain of KPL-404 contains the sequence shown in SEQ ID NO: 8. The variable region of the heavy chain of KPL-404 contains CDR1 having the sequence shown in SEQ ID NO: 1, CDR2 having the sequence shown in SEQ ID NO: 2, and CDR3 having the sequence shown in SEQ ID NO: 3. The light chain variable region of KPL-404 includes CDR1 having the sequence shown in SEQ ID NO: 4, CDR2 having the sequence shown in SEQ ID NO: 5, and CDR3 having the sequence shown in SEQ ID NO: 6.

[0083] As used herein, the term "cell incubation" refers to methods for generating and maintaining a host cell population capable of producing recombinant proteins of interest, as well as methods and techniques for optimizing the production and collection of proteins of interest. For example, once an expression vector is incorporated into a suitable host, the host can be maintained under conditions suitable for expressing the relevant nucleotide coding sequence and for collecting and purifying the desired recombinant protein.

[0084] As used herein, the term "downstream process technology" refers to one or more techniques used after upstream process technologies to purify proteins of interest (e.g., antibodies). For example, downstream process technologies include purifying protein products using, for instance, affinity chromatography (including protein A affinity chromatography), ion exchange chromatography (such as anion or cation exchange chromatography), hydrophobic interaction chromatography, mixed-mode or multi-mode chromatography, or displacement chromatography.

[0085] As used herein, the term "virus inactivation" or "VI" refers to treating a sample containing one or more viruses in a manner that renders one or more viruses incapable of replication or inactive. Virus inactivation is intended to refer to a reduction in the number of viral particles in a particular sample, and a decrease in the activity (e.g., but not limited to infectivity or replication capacity) of the viral particles in that sample. Virus inactivation can be achieved by physical means (e.g., heat, ultraviolet light, ultrasonic vibration) or by chemical means (e.g., pH changes or the addition of chemicals). Virus inactivation is a process step commonly used in most protein purification processes, particularly in the purification of therapeutic proteins. It should be understood that the absence of detection of one or more viruses in a sample using standard assays known in the art and those described herein indicates complete inactivation of one or more viruses after sample treatment. In some embodiments, virus inactivation is achieved through low pH treatment.

[0086] As used herein, the term "virus inactivation step" includes (a) a virus inactivation period, which is the period immediately following the step of titrating a sample containing the protein to be purified with acid to a sufficiently acidic pH to disrupt the viral envelope, during which a large level of virus present is inactivated (e.g., about 15 minutes), and optionally, (b) a static holding period, which is the period following the virus inactivation period during which the sample undergoes static holding or extended static holding.

[0087] Following the virus inactivation step, the sample is neutralized with alkali to titrate it back to a pH that is more stable for proteins and more suitable for the next purification step. The neutralized material is then filtered for further processing.

[0088] The phrase “recombinant host cell” (or simply “host cell”) includes cells in which a recombinant expression vector has been introduced. It should be understood that this terminology is intended to refer not only to the specific subject cell but also to the offspring of such cells. Because certain modifications may occur in the next generation due to mutations or environmental influences, these offspring may actually differ from the parent cell, but are still included within the scope of the term “host cell” as used herein.

[0089] As used herein, the term “chromatography” refers to any type of technique that separates a product of interest (e.g., a therapeutic protein or antibody) from contaminants and / or protein aggregates in a formulation.

[0090] The term "affinity chromatography" refers to a protein separation technique in which a target protein (e.g., an Fc region containing a protein of interest or an antibody) specifically binds to a ligand (e.g., protein A), which is typically immobilized on a solid support (the ligand immobilized on the solid support is referred to herein as the "chromatographic matrix"). The target protein typically retains its specific binding affinity to the ligand during the chromatographic step, while other solutes and / or proteins in the mixture bind to the ligand in a less obvious or specific manner. The binding of the target protein to the immobilized ligand allows impurities (including contaminating proteins or protein impurities (e.g., HCP)) to pass through the chromatographic matrix, while the target protein maintains its specific binding to the immobilized ligand on the solid support material; however, some non-specific binding of contaminating proteins to the matrix is ​​typically observed. The chromatographic matrix is ​​typically washed once or multiple times with a suitable washing buffer to remove non-specifically bound proteins (e.g., HCP) and other impurities before eluting the bound proteins from the matrix. Subsequently, the specifically bound protein of interest is eluted from the matrix using a suitable elution buffer that facilitates the separation of the protein of interest from the matrix. In embodiments of the invention, one or more intermediate washing steps are eliminated from such a process without reducing the purity of the eluted target protein. In other words, in some embodiments of the invention, the protein of interest is allowed to bind to a chromatographic matrix containing protein A and subsequently elute without requiring one or more intermediate washing steps; however, the purity of the protein of interest in the protein A elution pool is unaffected. In other embodiments, the number of intermediate washing steps is reduced compared to processes that typically use a certain number of washing steps to achieve a certain level of purity of the protein of interest in the protein A elution pool. In various embodiments of the invention, despite the elimination or reduction of the number of intermediate washing steps, the level of host cell protein in the protein A elution pool is reduced.

[0091] As used interchangeably herein, the terms “ion exchange” and “ion exchange chromatography” refer to a chromatographic process in which a solute or analyte of interest in a mixture interacts with a charged compound attached (e.g., by covalent attachment) to a solid-phase ion exchange material, such that the solute or analyte of interest interacts more or less nonspecifically with the charged compound compared to solute impurities or contaminants in the mixture. Contaminating solutes in the mixture elute from the column of the ion exchange material faster or slower than the solute of interest, or bind to or are excluded from the resin relative to the solute of interest. “Ion exchange chromatography” includes cation exchange chromatography, anion exchange chromatography, and mixed-mode ion exchange chromatography. For example, cation exchange chromatography may bind to a target molecule (e.g., a target protein containing an Fc region) and then elute (cation exchange binding and elution chromatography), or it may primarily bind impurities while the target molecule “flows” through the column (cation exchange flow-through chromatography). In the case of anion exchange chromatography, the solid-phase material may bind to a target molecule (e.g., a target protein containing an Fc region) and then elute, or it may primarily bind impurities as the target molecule “flows” through the column.

[0092] As used herein, the term "recombinant protein" refers to a protein resulting from the transcription and translation of a gene carried on a recombinant expression vector that has been introduced into a host cell. In some embodiments, the recombinant protein is an antibody, such as a chimeric antibody, a humanized antibody, or a fully human antibody. In some embodiments, the recombinant protein is an antibody selected from the following isotypes: IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA1, IgA2, IgD, or IgE. In some embodiments, the antibody molecule is a full-length antibody (e.g., IgG1 or IgG4 immunoglobulin), or alternatively, the antibody may be a fragment (e.g., an Fc fragment or a Fab fragment).

[0093] II. The method of the present invention The present invention is based at least on the identification of unexpected viral inactivation and neutralization conditions during the purification process of proteins of interest (e.g., anti-CD40 antibodies or those that stabilize antigen-binding moieties, such as KPL-404), which stabilize proteins and / or reduce the formation of high molecular weight aggregates in the preparation of virally inactivated proteins.

[0094] In a typical purification process, once the protein of interest is expressed in a cell culture, the cell culture medium undergoes a clarification step to remove impurities and particles, such as cells and cell debris. The clarified cell culture medium containing the protein of interest is then subjected to one or more chromatographic steps. To ensure the safety of the protein of interest, especially in the case of therapeutic candidates, it is necessary to inactivate any enveloped viruses that may be present in the sample containing the protein of interest during the purification process.

[0095] Virus inactivation is typically performed after a chromatographic step (e.g., affinity chromatography). For example, after a chromatographic step, an elution pool containing the protein of interest is collected and subjected to a virus inactivation step for a period of time to inactivate enveloped viruses that may be present in the elution pool. In some embodiments, the chromatographic step is an affinity chromatography step, such as protein A affinity chromatography. Virus inactivation can be achieved by physical means (e.g., heat, ultraviolet light, ultrasonic vibration) or by using chemical means (e.g., pH changes or the addition of chemicals). During purification, one or more of a variety of virus reduction / inactivation methods can be used. It should be understood that the failure to detect one or more viruses in a sample using standard assays known in the art and those described herein indicates complete inactivation of one or more viruses after sample treatment.

[0096] Large enveloped viruses (e.g., X-MuLV) have been reliably demonstrated to be inactivated in commercial purification processes via low-pH viral inactivation. In pH inactivation, the operator titrates the pooled product to a sufficiently acidic pH to disrupt the viral envelope and allow the product to remain statically held, before titrating it back to a pH where the protein is more stable and more suitable for the next chromatographic step. The pH condition is chosen as a balance between a low enough pH to induce viral inactivation and a sufficiently high pH to avoid protein denaturation.

[0097] The inventors of this invention made a surprising and unexpected discovery: although pH ≤ 3.6 is the industry standard for virus inactivation processes, significant and unacceptable levels of protein aggregates were observed during the virus inactivation step for anti-CD40 antibodies (e.g., KPL-404) within this pH range. Therefore, new and unexpected conditions for virus inactivation were established during the purification process of KPL-404 to ensure adequate virus inactivation while reducing and / or minimizing protein aggregation.

[0098] Therefore, the present invention provides a method for producing a virus-inactivated antibody preparation comprising an anti-CD40 antibody or its antigen-binding moiety having reduced levels of high molecular weight aggregates; a method for minimizing the formation of high molecular weight aggregates in an antibody preparation comprising an anti-CD40 antibody or its antigen-binding moiety; a method for reducing the formation of high molecular weight aggregates in an antibody preparation comprising an anti-CD40 antibody or its antigen-binding moiety; a method for maximizing the antibody monomer level in a virus-inactivated antibody preparation comprising an anti-CD40 antibody or its antigen-binding moiety; a method for stabilizing a virus-inactivated antibody preparation comprising an anti-CD40 antibody or its antigen-binding moiety; a method for reducing the number and / or activity of virus particles in an antibody preparation comprising an anti-CD40 antibody or its antigen-binding moiety; and a method for producing a pharmaceutical composition comprising an anti-CD40 antibody or its antigen-binding moiety and a pharmaceutically acceptable carrier. The method includes incubating a sample comprising an anti-CD40 antibody or its antigen-binding moiety at a pH of about 3.6-3.9 during a virus inactivation step.

[0099] In some embodiments, the virus inactivation step includes a virus inactivation period. In some embodiments, the virus inactivation step includes a static maintenance period. In some embodiments, the virus inactivation step includes both a virus inactivation period and a static maintenance period.

[0100] In some embodiments, the antibody or its antigen-binding portion includes a heavy chain variable region comprising CDR1 having the sequence shown in SEQ ID NO: 1, CDR2 having the sequence shown in SEQ ID NO: 2, and CDR3 having the sequence shown in SEQ ID NO: 3. In other embodiments, the antibody or its antigen-binding portion includes a light chain variable region comprising CDR1 having the sequence shown in SEQ ID NO: 4, CDR2 having the sequence shown in SEQ ID NO: 5, and CDR3 having the sequence shown in SEQ ID NO: 6.

[0101] In some embodiments, the antibody or its antigen-binding portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7 and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8.

[0102] In some embodiments, the antibody or its antigen-binding portion comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 9, and the light chain comprising the amino acid sequence shown in SEQ ID NO: 10.

[0103] In some embodiments, the anti-CD40 antibody or its antigen-binding portion is KPL-404 or its antigen-binding portion.

[0104] In some embodiments, the sample is passed through an affinity chromatography column, such as a protein A column, prior to the virus inactivation step. In some embodiments, during the virus inactivation step, such as during a static hold period or an extended static hold period, the sample is incubated at a pH of about 3.6-3.9, about 3.6-3.7, about 3.7-3.8, about 3.8-3.9, about 3.6-3.8, or about 3.7-3.9. In some embodiments, the sample has a pH of about 3.6-3.9, about 3.6-3.7, about 3.7-3.8, about 3.8-3.9, about 3.6-3.8, or about 3.7-3.9. In some embodiments, during the virus inactivation step, the sample is incubated at a pH of about 3.6, about 3.7, about 3.8, or about 3.9. In some embodiments, the sample has a pH of about 3.6, about 3.7, about 3.8, or about 3.9.

[0105] In some embodiments, the sample is incubated for approximately 10-360 minutes during the virus inactivation step. In some embodiments, the sample is incubated for approximately 30-120 minutes during the virus inactivation step, for example, approximately 30-60 minutes, approximately 60-90 minutes, approximately 60-120 minutes, or approximately 90-120 minutes. In some embodiments, the sample is incubated for approximately 60-120 minutes during the virus inactivation step. In some embodiments, during the virus inactivation step, the sample is incubated for approximately 10-50 minutes, approximately 20-40 minutes, approximately 20-60 minutes, approximately 30-70 minutes, approximately 40-80 minutes, approximately 50-70 minutes, approximately 50-90 minutes, approximately 60-100 minutes, approximately 70-110 minutes, approximately 80-100 minutes, approximately 80-120 minutes, approximately 60-120 minutes, approximately 90-130 minutes, approximately 100-140 minutes, approximately 110-130 minutes, or approximately 110 minutes. -150 minutes, approximately 120-160 minutes, approximately 130-170 minutes, approximately 140-180 minutes, approximately 150-190 minutes, approximately 160-200 minutes, approximately 170-190 minutes, approximately 170-210 minutes, approximately 180-220 minutes, approximately 200-240 minutes, approximately 220-260 minutes, approximately 230-250 minutes, approximately 240-280 minutes, approximately 280-320 minutes, approximately 290-310 minutes, or approximately 320-360 minutes.

[0106] In some implementations, the sample is incubated for at least about 15 minutes, at least about 30 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 120 minutes during the virus inactivation step. In some implementations, during the virus inactivation step, the sample is incubated for approximately 15 minutes, approximately 20 minutes, approximately 30 minutes, approximately 40 minutes, approximately 50 minutes, approximately 60 minutes, approximately 70 minutes, approximately 80 minutes, approximately 90 minutes, approximately 100 minutes, approximately 110 minutes, approximately 120 minutes, approximately 130 minutes, approximately 140 minutes, approximately 150 minutes, approximately 160 minutes, approximately 170 minutes, approximately 180 minutes, approximately 190 minutes, approximately 200 minutes, approximately 210 minutes, approximately 220 minutes, approximately 230 minutes, approximately 240 minutes, approximately 250 minutes, approximately 260 minutes, approximately 270 minutes, approximately 280 minutes, approximately 290 minutes, approximately 300 minutes, approximately 310 minutes, approximately 320 minutes, approximately 330 minutes, approximately 340 minutes, approximately 350 minutes, or approximately 360 minutes.

[0107] In some embodiments, during the virus inactivation step, the sample is incubated at temperatures of approximately 4°C-37°C, approximately 10°C-37°C, approximately 10°C-20°C, approximately 15°C-30°C, approximately 15°C-25°C, approximately 15°C-37°C, or approximately 13°C-25°C.

[0108] In some embodiments, during the virus inactivation step, the sample is incubated at a pH of about 3.6-3.9 for about 30-120 minutes. In some embodiments, during the virus inactivation step, the sample is incubated at a pH of about 3.6-3.9 for about 60-120 minutes.

[0109] In some embodiments, during the virus inactivation step, the sample is incubated at a pH of approximately 3.7-3.9 for approximately 30-120 minutes. In some embodiments, during the virus inactivation step, the sample is incubated at a pH of approximately 3.7-3.9 for approximately 60-120 minutes. In other embodiments, during the virus inactivation step, the sample is incubated at a temperature of approximately 15°C-25°C and a pH of approximately 3.7-3.9 for approximately 50-70 minutes.

[0110] In some embodiments, during the virus inactivation step, the sample is incubated at a pH of about 3.6-3.8 for about 30-120 minutes. In some embodiments, during the virus inactivation step, the sample is incubated at a pH of about 3.6-3.8 for about 60-120 minutes.

[0111] In some embodiments, during the virus inactivation step, the sample is incubated at a temperature of about 15°C-25°C and a pH of about 3.6-3.9 for about 30-120 minutes. In some embodiments, during the virus inactivation step, the sample is incubated at a temperature of about 15°C-25°C and a pH of about 3.7-3.9 for about 30-120 minutes. In other embodiments, during the virus inactivation step, the sample is incubated at a temperature of about 15°C-25°C and a pH of about 3.6-3.8 for about 30-120 minutes.

[0112] In some embodiments, during the virus inactivation step, the sample is incubated at a temperature of about 15°C-25°C and a pH of about 3.6-3.9 for about 60-120 minutes. In some embodiments, during the virus inactivation step, the sample is incubated at a temperature of about 15°C-25°C and a pH of about 3.7-3.9 for about 60-120 minutes. In other embodiments, during the virus inactivation step, the sample is incubated at a temperature of about 15°C-25°C and a pH of about 3.6-3.8 for about 60-120 minutes.

[0113] In some embodiments, during the virus inactivation step, the sample is incubated at a pH of about 3.7 for about 30-120 minutes. In some embodiments, during the virus inactivation step, the sample is incubated at a pH of about 3.7 for about 60-120 minutes.

[0114] In some embodiments, during the virus inactivation step, the sample is incubated at a temperature of about 15°C-25°C and a pH of about 3.7 for about 30-120 minutes. In some embodiments, during the virus inactivation step, the sample is incubated at a temperature of about 15°C-25°C and a pH of about 3.7 for about 60-120 minutes. In some embodiments, during the virus inactivation step, the sample is incubated at a pH of about 3.7 for about 120 minutes.

[0115] In some embodiments, the sample is incubated at a temperature of about 25°C and a pH of about 3.7 for about 30-120 minutes during the virus inactivation step. In some embodiments, the sample is incubated at a temperature of about 25°C and a pH of about 3.7 for about 120 minutes during the virus inactivation step.

[0116] In some implementations, during the virus inactivation step, the sample is incubated for about 30-120 minutes at a temperature of about 13°C-25°C and a pH of about 3.7-3.8.

[0117] In some implementations, during the virus inactivation step, the sample is incubated for about 30-120 minutes at a temperature of about 15°C-25°C and a pH of about 3.7-3.8.

[0118] In some implementations, during the virus inactivation step, the sample is incubated for about 60-120 minutes at a temperature of about 15°C-25°C and a pH of about 3.7-3.8.

[0119] In other embodiments, during the virus inactivation step, the sample is incubated for about 60 minutes at a temperature of about 15°C-25°C and a pH of about 3.8.

[0120] The virus-inactivated antibody preparation has reduced levels of high molecular weight aggregates. In some embodiments, the virus-inactivated antibody preparation contains less than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1% of high molecular weight aggregates, and within one or more of the foregoing ranges.

[0121] In some embodiments, the virus-inactivated antibody preparation contains less than about 10% high molecular weight aggregates. In some embodiments, the virus-inactivated antibody preparation contains less than about 5% high molecular weight aggregates. In some embodiments, the virus-inactivated antibody preparation contains less than about 2% high molecular weight aggregates. In some embodiments, the virus-inactivated antibody preparation contains less than about 1% high molecular weight aggregates. In some embodiments, the virus-inactivated antibody preparation contains less than about 0.5% high molecular weight aggregates.

[0122] In some embodiments, the virus-inactivated antibody preparation comprises about 0.1-10%, about 0.1-9%, about 0.1-8%, about 0.1-7%, about 0.1-6%, about 0.1-5%, about 0.1-4%, about 0.1%-3%, about 0.1%-2%, or about 0.1-1% of high molecular weight aggregates, and the ranges within one or more of the foregoing. In some embodiments, the virus-inactivated antibody preparation comprises about 0.1-2% of high molecular weight aggregates. In some embodiments, the virus-inactivated antibody preparation comprises about 0.1-1% of high molecular weight aggregates.

[0123] The levels of high molecular weight aggregates can be analyzed using any method known in the art. In some embodiments, size exclusion chromatography (SEC) is used to measure the levels of aggregates. Any other technique, such as mass spectrometry, can also be used to determine size variants.

[0124] In one embodiment, the virus-inactivated antibody preparation comprises charged antibodies, which contain at least about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or 51% of the major species. In other embodiments, the virus-inactivated antibody preparation comprises charged antibodies, which contain about 40-55%, 40-54%, 40-53%, 40-52%, 40-51%, about 40-50%, about 40-49%, about 40-48%, about 40-47%, about 40-46%, about 40-45%, about 46-51%, or about 40-44% of the major species.

[0125] The level of charged species can be analyzed using any method known in the art. In some embodiments, the level of charged species is measured by charge-based separation techniques, such as isoelectric focusing (IEF) gel electrophoresis methods, such as capillary IEF (cIEF) and imaging cIEF (cIEF). Any other techniques, such as cation exchange chromatography (CEX) and anion exchange chromatography (AEX), can also be used to determine charge variants.

[0126] In some embodiments, the virus-inactivated antibody preparation may undergo one or more additional purification steps, such as anion-exchange chromatography, cation-exchange chromatography, or mixed-mode column chromatography. Further purification procedures may also include virus filtration and ultrafiltration / distillation.

[0127] Following the purification chromatographic step, the eluent pool can be subjected to a nanofiltration step. In one embodiment, the nanofiltration step is performed via one or more nanofilters or virus filters. The filter can be any filter known in the art suitable for this purpose, and can include, for example, EMDMillipore Viresolve VPro, Viresolve NFP, Viresolve NFR, or Planova 15N, 20N, and 35N virus removal filters from Asashi Kasei Pharma. In some embodiments, the nanofiltration filter has an average pore size between about 15 nm and about 200 nm. Those skilled in the art will understand that the choice of the type and number of filters will depend on the volume of the sample being processed and the desired filtration performance.

[0128] Ultrafiltration and percolation steps may also be included to further concentrate and formulate proteins of interest, such as CD-40 antibodies or their antigen-binding moieties, such as KPL-404. Ultrafiltration and percolation can be performed after nanofiltration to obtain targeted drug concentrations and buffer conditions prior to formulation. Ultrafiltration is generally considered to refer to filtration using a filter with a molecular weight cutoff of approximately 10 kDa. By using a filter with such a small pore size, the sample volume can be reduced by permeating the sample buffer through the filter membrane pores, while proteins (e.g., antibodies) remain above the membrane surface. Percolation is a method that uses a membrane filter to remove and exchange salts, sugars, and non-aqueous solvents, separate free species from bound species, remove low molecular weight species, and / or cause rapid changes in ionic and / or pH environments. Microsolutes are most effectively removed by adding solvent to the solution to be percolated at a rate approximately equal to the percolate flow rate. This washes away microspecies from the solution at a constant volume, effectively purifying the retained proteins of interest. In some embodiments of the invention, optionally prior to further chromatographic or other purification steps, a percolation step is employed to exchange the various buffer solutions used in conjunction with the invention and to remove impurities from the protein formulation. Those skilled in the art can select suitable membrane filter devices for UF / DF operations. Examples of membrane cartridges suitable for the invention include, but are not limited to, Sartorius Vivaspin, Pellicon 2, or Pellicon 3 cartridges with 10 kD, 30 kD, or 50 kD membranes from EMD Millipore; Kvick 10 kD, 30 kD, or 50 kD cartridges from GE Healthcare; and Centramate or Centrasette 10 kD, 30 kD, or 50 kD cartridges from Pall Corporation. After the percolation step, the protein concentration of the solution can be adjusted to the desired final concentration using a percolation buffer.

[0129] III. The compositions of the present invention This invention includes, in one aspect, a composition comprising a virus-inactivated eluent collected from an affinity chromatography column (e.g., a protein A column), comprising a protein having reduced levels of high molecular weight aggregates. In one embodiment, the protein is an anti-CD40 antibody or its antigen-binding portion. In another embodiment, the anti-CD40 antibody is KPL-404.

[0130] In some embodiments, the composition comprises an eluent that has been subjected to affinity chromatography to inactivate the virus, wherein the eluent comprises less than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%, and within one or more of the foregoing ranges.

[0131] In some embodiments, the eluent from virus inactivation contains less than about 10% high molecular weight aggregates. In some embodiments, the eluent from virus inactivation contains less than about 5% high molecular weight aggregates. In some embodiments, the eluent from virus inactivation contains less than about 2% high molecular weight aggregates. In some embodiments, the eluent from virus inactivation contains less than about 1% high molecular weight aggregates. In some embodiments, the eluent from virus inactivation contains less than about 0.5% high molecular weight aggregates.

[0132] In some embodiments, the virus-inactivated eluent contains about 0.1-10%, about 0.1-9%, about 0.1-8%, about 0.1-7%, about 0.1-6%, about 0.1-5%, about 0.1-4%, about 0.1%-3%, about 0.1%-2%, or about 0.1-1% of high molecular weight aggregates, and within one or more of the foregoing ranges. In some embodiments, the virus-inactivated eluent contains about 0.1-2% of high molecular weight aggregates.

[0133] In some embodiments, the eluent, having passed through an affinity chromatography column (e.g., a protein A column), has additionally undergone a virus inactivation step and has, for example, a pH of about 3.6-3.7, about 3.7-3.8, about 3.8-3.9, about 3.6-3.8, about 3.6-3.9, or about 3.7-3.9 during a static hold period or an extended static hold period. In some embodiments, during the virus inactivation step, the eluent has a pH of about 3.6-3.7, about 3.7-3.8, about 3.8-3.9, about 3.6-3.8, about 3.6-3.9, or about 3.7-3.9. In some embodiments, during the virus inactivation step, the eluent, having passed through an affinity chromatography column, has a pH of about 3.6, about 3.7, about 3.8, or about 3.9. In some embodiments, the eluent has a pH of about 3.6, about 3.7, about 3.8, or about 3.9.

[0134] In some embodiments, the eluent during the virus inactivation step has a temperature of about 4°C-37°C, about 10°C-37°C, about 10°C-20°C, about 15°C-30°C, about 15°C-25°C, about 15°C-37°C, or about 13°C-25°C.

[0135] In some embodiments, the eluent has a pH of about 3.6-3.9 and a temperature of about 15°C-25°C. In some embodiments, the eluent has a pH of about 3.6-3.8 and a temperature of about 15°C-25°C. In some embodiments, the eluent has a pH of about 3.7-3.9 and a temperature of about 15°C-25°C. In some embodiments, the eluent has a pH of about 3.7-3.9 and a temperature of about 13°C-25°C.

[0136] In some embodiments, the eluent has a pH of about 3.7 and a temperature of about 15°C to 25°C. In some embodiments, the eluent has a pH of about 3.8 and a temperature of about 15°C to 25°C.

[0137] In some embodiments, the proteins in the virus-inactivated eluent collected from an affinity chromatography column (e.g., a protein A column) contain antibodies or their antigen-binding moieties, having reduced levels of high molecular weight aggregates. For example, the antibody or its antigen-binding moieties could be an anti-CD40 antibody or its antigen-binding moieties, such as KPL-404 or its antigen-binding moieties.

[0138] In some embodiments, the antibody or its antigen-binding portion includes a heavy chain variable region comprising CDR1 having the sequence shown in SEQ ID NO: 1, CDR2 having the sequence shown in SEQ ID NO: 2, and CDR3 having the sequence shown in SEQ ID NO: 3. In other embodiments, the antibody or its antigen-binding portion includes a light chain variable region comprising CDR1 having the sequence shown in SEQ ID NO: 4, CDR2 having the sequence shown in SEQ ID NO: 5, and CDR3 having the sequence shown in SEQ ID NO: 6.

[0139] In some embodiments, the antibody or its antigen-binding portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7 and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8.

[0140] In some embodiments, the antibody or its antigen-binding portion comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 9, and the light chain comprising the amino acid sequence shown in SEQ ID NO: 10.

[0141] In some embodiments, antibodies or antigen-binding portions thereof that can be used in the compositions of this disclosure can be generated by a variety of techniques, including immunizing animals with the antigen of interest followed by conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique of Kohler and Milstein (1975) Nature 256:495. Somatic cell hybridization procedures can be used. In principle, other techniques for generating monoclonal antibodies can also be employed, including viral or oncogenic transformation of B lymphocytes.

[0142] An exemplary animal system for preparing hybridomas is the mouse system. Hybridoma generation is a well-established procedure. Immunoimmunization protocols and techniques for isolating immunogenic spleen cells for fusion are known in the art. Fusion couples (e.g., mouse myeloma cells) and fusion procedures are also known.

[0143] The antibodies used in the compositions of this invention may be human antibodies, chimeric antibodies, or humanized antibodies.

[0144] In one non-limiting embodiment, the antibody used in the compositions of the present invention is a human monoclonal antibody. Such a human monoclonal antibody can be generated using transgenic or transchromosomal mice carrying a portion of the human immune system rather than the mouse system. These transgenic and transchromosomal mice include mice referred to herein as HuMAb Mouse® (Medarex, Inc.), KM Mouse® (Medarex, Inc.), and XenoMouse® (Amgen). The antibody or its antigen-binding portion used in the compositions of the present invention can also be generated using the method described in U.S. Patent No. 6,090,382, the entire contents of which are expressly incorporated herein by reference.

[0145] Furthermore, alternative transchromosomal animal systems expressing human immunoglobulin genes are available in the art and can be used to generate the antibodies disclosed herein. For example, mice carrying human heavy chain transchromosomal and human light chain transchromosomal chromosomes, referred to as "TC mice," can be used; such mice are described in Tomizuka. et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727. Furthermore, bovine transchromosomals carrying human heavy and light chains (e.g., Kuroiwa) have been described in the art. et al. (2002) Nature Biotechnology 20:889-894 and PCT application number WO 2002 / 092812), and can be used to generate antibodies of the present disclosure.

[0146] Recombinant human antibodies to be used in the compositions of the present invention can be isolated by screening recombinant antibody libraries (e.g., scFv phage display libraries) prepared using human VL and VH cDNAs derived from mRNA derived from human lymphocytes. Methods for preparing and screening such libraries are known in the art. In addition to commercially available kits for generating phage display libraries (e.g., Pharmacia Recombinant Phage Antibody System, catalog number 27-9400-01; and Stratagene SurfZAP), other methods are available. TM In addition to the phage display kit (catalog number 240612, all of which is taught herein), examples of methods and reagents particularly suitable for generating and screening antibody display libraries can be found, for example, in Ladner. et al. US Patent No. 5,223,409; Kang et al. PCT Publication No. WO 92 / 18619; Dower et al. PCT Publication No. WO 91 / 17271; Winter et al. PCT Publication No. WO 92 / 20791; Markland et al. PCT Publication No. WO 92 / 15679; Breitling et al. PCT Publication No. WO 93 / 01288; McCafferty et al. PCT Publication No. WO 92 / 01047; Garrard et al. PCT Publication No. WO 92 / 09690; Fuchs et al. (1991)Bio / Technology 9:1370-1372; Hay et al. (1992) Hum Antibody Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; McCafferty et al. , Nature (1990) 348:552-554; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J MolBiol 226:889-896;Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992)PNAS 89:3576-3580; Garrard et al. (1991) Bio / Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982; its entire teachings are incorporated into this paper.

[0147] Human monoclonal antibodies used in the compositions of this invention can also be prepared using SCID mice in which human immune cells have been reconstituted, enabling the generation of a human antibody response after immunization. Such mice are described, for example, in U.S. Patent Nos. 5,476,996 and 5,698,767 by Wilson et al.

[0148] In some embodiments, the human antibody used in the compositions of the present invention is an anti-CD40 antibody and its antibody portion, an anti-CD40-associated antibody and its antibody portion, and a human antibody and its antibody portion having properties equivalent to those of the anti-CD40 antibody, such as high affinity for binding CD40 with low dissociation kinetics and high neutralization capacity. In one embodiment, the anti-CD40 antibody to be used in the compositions of the present invention binds to the same epitope on CD40 as KPL-404. In another embodiment, the anti-CD40 antibody to be used in the compositions of the present invention competitively inhibits the binding of KPL-404 to CD40 under physiological conditions. In one embodiment, the compositions of the present invention comprise KPL-404 or its antigen-binding portion.

[0149] Antibodies or their antigen-binding portions in the compositions used in this invention can be modified, wherein the constant regions of the antibody are modified to reduce the biological effector function mediated by at least one constant region relative to the unmodified antibody. To modify the antibodies of this invention to exhibit reduced binding to the Fc receptor, the immunoglobulin constant region can be mutated in specific regions essential for Fc receptor (FcR) interaction (see, for example, Canfield and Morrison (1991) J. Exp. Med. 173:1483-1491; and Lund). et al. (1991) J. of Immunol. 147:2657-2662, all of which are incorporated herein by reference. Decreased FcR binding capacity of antibodies can also reduce the functions of other effectors that depend on FcR interactions, such as opsonization, phagocytosis, and antigen-dependent cytotoxicity.

[0150] In another aspect, the present invention provides compositions comprising antibody preparations, such as anti-CD40 antibodies or their antigen-binding moieties, such as KPL-404. The methods of the present invention for preparing anti-CD40 antibodies or their antigen-binding moieties involve samples containing anti-CD40 antibodies or their antigen-binding moieties having undergone a virus inactivation step, which stabilizes the antibody and / or reduces the formation of high molecular weight aggregates in the virus-inactivated antibody preparation.

[0151] In some implementations, during the virus inactivation step, the sample containing the anti-CD40 antibody or its antigen-binding portion has been incubated at a pH of about 3.6-3.9.

[0152] In some embodiments, compositions comprising anti-CD40 antibodies or their antigen-binding portions have reduced levels of high molecular weight aggregates.

[0153] In some embodiments, the composition comprising an anti-CD40 antibody or its antigen-binding portion comprises less than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1% of a high molecular weight aggregate, and within one or more of the foregoing ranges.

[0154] In some embodiments, the composition comprising an anti-CD40 antibody or its antigen-binding portion contains less than about 10% high molecular weight aggregates. In some embodiments, the composition comprising an anti-CD40 antibody or its antigen-binding portion contains less than about 5% high molecular weight aggregates. In some embodiments, the composition comprising an anti-CD40 antibody or its antigen-binding portion contains less than about 2% high molecular weight aggregates. In some embodiments, the composition comprising an anti-CD40 antibody or its antigen-binding portion contains less than about 1% high molecular weight aggregates. In some embodiments, the composition comprising an anti-CD40 antibody or its antigen-binding portion contains less than about 0.5% high molecular weight aggregates.

[0155] In some embodiments, the composition comprising an anti-CD40 antibody or an antigen-binding fragment thereof comprises about 0.1-10%, about 0.1-9%, about 0.1-8%, about 0.1-7%, about 0.1-6%, about 0.1-5%, about 0.1-4%, about 0.1%-3%, about 0.1%-2%, or about 0.1-1% of high molecular weight aggregates, and within one or more of the foregoing ranges. In some embodiments, the composition comprising an anti-CD40 antibody or an antigen-binding fragment thereof comprises about 0.1-2% of high molecular weight aggregates. In some embodiments, the composition comprising an anti-CD40 antibody or an antigen-binding fragment thereof comprises about 0.1-1% of high molecular weight aggregates.

[0156] In some embodiments, the anti-CD40 antibody or its antigen-binding portion includes a heavy chain variable region comprising CDR1 having the sequence shown in SEQ ID NO: 1, CDR2 having the sequence shown in SEQ ID NO: 2, and CDR3 having the sequence shown in SEQ ID NO: 3. In other embodiments, the anti-CD40 antibody or its antigen-binding portion includes a light chain variable region comprising CDR1 having the sequence shown in SEQ ID NO: 4, CDR2 having the sequence shown in SEQ ID NO: 5, and CDR3 having the sequence shown in SEQ ID NO: 6.

[0157] In some embodiments, the anti-CD40 antibody or its antigen-binding portion comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8.

[0158] In some embodiments, the anti-CD40 antibody or its antigen-binding portion comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 9, and the light chain comprising the amino acid sequence shown in SEQ ID NO: 10.

[0159] In some embodiments, the anti-CD40 antibody or its antigen-binding portion comprises KPL-404 or its antigen-binding portion.

[0160] IV. Treatment methods using the compositions of the present invention The compositions of the present invention, comprising antibody preparations having undergone a virus inactivation step at a pH of about 3.6-3.9, can be used to treat any condition in a subject, wherein the therapeutic protein contained in the compositions is suitable for treating said condition. In some embodiments, the antibody preparations have undergone a virus inactivation step at a pH of about 3.6-3.7, about 3.7-3.8, about 3.8-3.9, about 3.6-3.8, or about 3.7-3.9. In some embodiments, the antibody preparations have undergone a virus inactivation step at a pH of about 3.6, about 3.7, about 3.8, or about 3.9. In one embodiment, the antibody is an anti-CD40 antibody or its antigen-binding portion. In another embodiment, the anti-CD40 antibody is KPL-404.

[0161] "Symptom" refers to any condition that would benefit from treatment with a protein. This includes both chronic and acute conditions or diseases, including those pathological conditions that predispose a subject to the symptom discussed. In the case of an anti-CD40 antibody or its antigen-binding moiety such as KPL-404, a therapeutically effective amount of the composition can be administered to treat CD40-related conditions.

[0162] As used herein, the term "CD40-related disease or condition" means a disease or other condition in which the presence of CD40 in a subject suffering from the condition has been shown or is suspected to be a cause of the pathophysiology of the condition or a factor contributing to its exacerbation. Therefore, a CD40-related condition is a condition in which inhibiting CD40 activity is expected to alleviate symptoms and / or promote disease progression. The compositions of the present invention can be used to treat any CD40-related disease or condition known in the art, including but not limited to autoimmune diseases, immune disorders, inflammatory conditions, and cancer.

[0163] As used herein, the term "subject" means including living organisms. Examples of subjects include mammals such as humans, dogs, cattle, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In a specific embodiment of the invention, the subject is a human.

[0164] As used in this article, the terms "to treat" or "treatment" refer to both therapeutic treatment and preventative or preventative measures. Those who require treatment include those who already have a condition, as well as those whose condition needs to be prevented.

[0165] The composition can be administered by a variety of methods known in the art. Exemplary routes / modes of administration include intravenous, intramuscular, intranasal, oral, topical, or subcutaneous delivery. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired outcome.

[0166] Dosing regimens can be adjusted to provide the optimal desired response (e.g., therapeutic or preventative response). For example, a single bolus can be administered, several separate doses can be administered over time, or the dose can be reduced or increased proportionally as indicated by the urgency of the treatment situation. In some embodiments, it is particularly advantageous to formulate the parenteral composition in dose units for ease of administration and dose uniformity. As used herein, dose units refer to physically discrete units suitable as a unit dose for use in a mammalian subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the desired drug carrier. The specifications of the dose unit form of the present invention are determined by and directly depend on: (a) the unique characteristics of the active compound and the specific therapeutic or preventative effect to be achieved, and (b) the inherent limitations in the development of such active compounds in the art for treating a specific disease or condition in an individual.

[0167] It should be noted that the dosing regimen for therapeutic antibodies administered to subjects can vary depending on the characteristics of the specific antibody (e.g., binding affinity and pharmacokinetic and pharmacodynamic properties) and the type and severity of the condition to be alleviated. It should also be understood that, for any given subject, the specific dosage regimen should be adjusted over time based on individual needs, the desired outcome, and the professional judgment of the person administering or supervising the administration of the composition.

[0168] VII. Pharmaceutical preparations The present invention also provides preparations and formulations comprising the compositions of the present invention. It should be understood that compositions comprising antibody preparations having undergone a virus inactivation step at a pH of about 3.6-3.9 can be formulated or prepared as described below. In one embodiment, the antibody is an anti-CD40 antibody or its antigen-binding portion. In another embodiment, the anti-CD40 antibody is KPL-404.

[0169] In some embodiments, the compositions of the present invention may be formulated as pharmaceutical (therapeutic) compositions with pharmaceutically acceptable carriers and may be administered by a variety of methods known in the art. As those skilled in the art will understand, the formulation and route and / or mode of administration will vary depending on the physical and pharmacological properties of the therapeutic antibody and the desired results.

[0170] The term "pharmaceutically acceptable carrier" refers to one or more non-toxic materials that do not interfere with the effectiveness of the bioactivity of the active ingredient.

[0171] The compositions of the present invention are present in a form acceptable for therapeutic use. In one embodiment, the formulation of the composition of the present invention is a liquid formulation. In another embodiment, the formulation of the composition of the present invention is a lyophilized formulation. In a further embodiment, the formulation of the composition of the present invention is a reconstituted liquid formulation. In one embodiment, the formulation of the composition of the present invention is a stable liquid formulation. In one embodiment, the liquid formulation of the composition of the present invention is an aqueous formulation. In another embodiment, the liquid formulation is non-aqueous. In a specific embodiment, the liquid formulation of the composition of the present invention is an aqueous formulation, wherein the aqueous carrier is distilled water.

[0172] The compositions of the present invention can be formulated for specific routes of administration, such as oral, nasal, pulmonary, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. The formulations can be conveniently present in unit dosage forms and can be prepared by any method known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the specific administration modality. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally the amount of the composition that produces the therapeutic effect. For example, in some embodiments, antibodies (including antibody fragments) are formulated for intravenous administration. In some other embodiments, antibodies (including antibody fragments) are formulated for local delivery to the cardiovascular system, such as via catheters, stents, sutures, intramyocardial delivery, intrapericardial delivery, or intraendocardial delivery. In one specific embodiment, the composition comprises an anti-CD40 antibody such as KPL-404 and is formulated for subcutaneous administration.

[0173] Formulations of the compositions of the present invention suitable for topical or transdermal application include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalers. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservatives, buffers, or propellants (US Patent Nos. 7,378,110; 7,258,873; 7,135,180; 7,923,029 and US Publication No. 20040042972).

[0174] As used in this article, the phrases “parenteral administration” and “of which” mean administration other than intravenous and local administration, usually by injection, and including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-bursal, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0175] The actual dosage level of the active ingredient in the pharmaceutical composition of the compositions of the present invention can be modified to obtain an amount of active ingredient that is non-toxic to the patient and effectively achieves the desired therapeutic response for a particular patient, composition, and administration method. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health status, and medical history of the patient to be treated, and similar factors well known in the medical field.

[0176] The present invention is further illustrated by the following embodiments, which should not be construed as limiting in any way.

[0177] Example Example 1: Downstream processes for generating anti-CD40 antibodies KPL-404 is a monoclonal antibody that binds to CD40 and blocks CD154-mediated B cell activation. KPL-404 is derived from a stable CHO cell line and consists of two light chains and two heavy chains. Downstream preparation processes include protein A capture, low pH maintenance to neutralize the virus, ion exchange chromatography, nanofiltration and ultrafiltration / percolation (UFDF), followed by final formulation, filtration, and filling. Figure 1 ).

[0178] This example describes the response of KPL-404 to changes in process parameters, particularly pH during the virus inactivation step, to investigate its impact on product quality.

[0179] Run 1 Studies were conducted to establish a risk profile of product quality variations due to unexpected pH changes during virus inactivation (VI) maintenance. During the initial set of robustness studies, the first extended virus inactivation (VI) maintenance experiment was performed at pH 3.27 and spanned a total of 360 minutes. To ensure accurate establishment of the point of failure (if any) within this timeframe, samples of the merged product were extracted at regular intervals between 0 and 360 minutes, individually neutralized to pH 5.0, filtered, and retained. If testing yielded unacceptable product quality (PQ) data for the merged product at 360 minutes, these intermediate merged products could be tested to determine when degradation occurred. Five time points were taken, starting at 120 minutes. Samples were analyzed by size exclusion chromatography (SEC), imaging capillary isoelectric focusing (iCIEF), and non-reducing capillary electrophoresis with sodium dodecyl sulfate (NR-CSDS).

[0180] The extended holding parameters and acid titration curves are presented in Table 1 and... Figure 2 The analytical data from the extended retention, the VI retention for the major substance generation (MG) treatment merged and the reference VI merged for comparison are presented in Table 2.

[0181] Table 1: Extended Holding Parameters

[0182] Table 2: VI retention data

[0183] When thawed samples were diluted with SEC Mobile Phase A buffer (0.1 M sodium phosphate, 0.1 M sodium sulfate, pH 6.8) to prepare for SEC analysis, immediate and widespread protein precipitation was observed, which remained stable proportionally to the monomers over the following five hours. Because this precipitation made the samples unsuitable for SEC as a whole, they were spun down, and the supernatant (i.e., aggregated proteins) was analyzed instead. This supernatant consisted primarily of HMW species. An immediate and stable transition to basic species was also observed in prolonged hold VI pools (Table 2).

[0184] Data from this maintenance experiment confirmed that when the maintenance pH was below 3.6, the product aggregates reached unacceptably high levels, and this worsened over time. Notably, it was surprising that a maintenance pH of 3.5 produced unacceptably high levels of aggregates, as this pH falls within the range typically used for virus inactivation of protein A elutions, indicating that KPL-404 aggregates particularly readily at and below this pH, and that a new pH range that does not jeopardize product quality needs to be defined.

[0185] Run 2 Perform another run using the experimental design described below: 1) The pH of Experiment VI was 3.5, and the pH of the control group was 3.6. pH 3.6 is the upper limit of the range in Process 2.

[0186] 2) The control will consist of half of the ProA eluent instead of its small sample, thus ensuring that the volume is large enough so that its sample can be properly neutralized before analysis.

[0187] 3) The QC program allows samples to thaw and remain undisturbed for a period of time before dilution and testing, as this may prevent excessive aggregation.

[0188] The ProA eluent was filtered through a 0.2 μm filter and then adjusted to the target pH of 3.5 with 0.5 M acetic acid. Control and initial ProA (pH 4.15) eluent samples were also collected. The control sample was adjusted to pH 3.6. All VI pools were held in PETG containers and titrated by pipette under constant mixing conditions, maintaining a static state at ambient temperature. Every hour after the start of the holding period, 10 mL of sample was taken from the experimental group at pH 3.5, neutralized, and filtered. Simultaneously, samples were taken from the control group. The parameters for the VI operation are listed in Table 3.

[0189] Table 3: Operating parameters, run 2

[0190] The data from Run 2 are presented in Table 4-6. The percentage of high molecular weight (HMW) aggregates is displayed. Figure 3 According to SEC data, the aggregation of KPL-404 is evident. It begins rapidly within 60 minutes after acid addition, and there are indications that it may begin to plateauing around the 240-minute mark.

[0191] IcIEF and NR-CSDS were also measured in the control and experimental groups to determine fragmentation or charge distribution changes up to 300 minutes. The results are shown in Tables 4-6.

[0192] Table 4. SEC Results

[0193] Table 5. icIEF Results

[0194] Table 6. NR-CE-SDS Results

[0195] In summary, this work within the broader KPL-404 robustness study group includes extended virus inactivation retention experiments and assessments of the lower limits of the pH range (e.g., pH 3.3, 3.4, 3.5, and 3.6) to understand the risks to product quality. While pH ≤ 3.6 is generally the industry standard for virus inactivation processes, unacceptable levels of aggregation for KPL-404 were observed within this pH range. Therefore, new ranges of pH and / or retention times for virus inactivation have been developed to ensure that KPL-404 can be maintained at extended VI retention periods without compromising product quality.

[0196] Example 2: Evaluation of the corrected pH range for virus inactivation during KPL-404 purification As demonstrated in Example 1, the industry-standard pH range for virus inactivation processes is unacceptable for KPL-404 purification because significant levels of aggregates were observed within this range. To reduce protein aggregation during the virus inactivation step while minimizing the risk of reduced viral clearance, a modified pH range of pH 3.6–3.8 was proposed. Specifically, in this example, pH 3.85, at the upper limit of the proposed VI pH range, was selected and evaluated. Table 7 shows the SEC results for KPL-404 after VI maintenance at pH 3.8, confirming that VI maintenance at pH 3.8 does not impair the quality of the KPL-404 product.

[0197] Table 7. SEC Results

[0198] KPL-404 is produced from a stable CHO cell line. To ensure the safety of CHO-derived products, their purification process needs to be evaluated by adding model viruses to demonstrate the removal or inactivation of several logs of viral infectivity. These studies can provide some assurance that the purified product is free of foreign factors, including viral contaminants that may be introduced by the starting materials (or materials used during preparation). In this example, the KPL-404 purification process was evaluated for its ability to inactivate heterophilic murine leukemia virus (X-MuLV).

[0199] The following table summarizes the characteristics of the virus:

[0200] Heterophilic murine leukemia virus (pNFS Th1 strain) is an 80-130 nm, enveloped, RNA-containing retrovirus. X-MuLV is used as a model for retroviral particles frequently found in CHO cell lines. This virus was initially characterized by identifying its species host range [positive growth test on Mv1Lu cells (mink lung) and negative growth test on NIH3T3 cells (mouse)]. The X-MuLV stock solution used in this study was positive for X-MuLV identity testing and free of potential bovine (BAV, BPV, BRSV, BTV, BVDV, IBR, and Reo-3) and swine (PAV, PPV, and TGE) viral contaminants. The strength (titer) of the X-MuLV Ultra 2 stock virus (lot number 03Jun20) used in this study was determined by plaque assay using PG4 indicator cells. The approximate titer of the X-MuLV stock solution is typically 5 × 10⁻⁶. 6 Up to 3×10 7 Between PFU / ml.

[0201] For virus inactivation studies, all times were ±1 minute. All temperatures were monitored and recorded. All samples were aseptically processed to avoid introducing contamination. All stock virus solutions were sonicated and filtered prior to incorporation.

[0202] Low pH treated samples Using a low pH buffer (0.5M acetic acid), adjust a portion of the Low pH Starting Material to pH 3.80–3.90 (target pH 3.85). Add the material to a target of 7.0–8.0 log10 total PFU with an appropriate stock virus solution (Ultra 2X-MuLV), and readjust to pH 3.80–3.90 (target pH 3.85) using Low pH Buffer, 0.5M acetic acid (if necessary). Incubate the added material at 14.0 ± 1.0 °C for 120 minutes. Remove the samples at 5, 15, 30, 60, 90, and 120 minutes after incubation, adjust to pH 6.5–7.5 using Low pH Buffer, 0.5M Tris Base, and filter (0.45 μm). Test each sample for infectivity immediately. Divide the remaining material into multiple aliquots, rapidly freeze, and store at or below -60 °C for later use. For "Low pH-T" 60分钟 "The samples were subjected to additional batch testing to improve the sensitivity of the test."

[0203] neutral control sample Using Low pH Buffer, 0.5M Tris Base, adjust the pH of another sample of Low pH Starting Material to pH 6.5–7.5, adding the same %v / v ratio as the stock virus solution used for adding the treated sample with 7.0–8.0 log10 total PFU, and confirm the pH to 6.5–7.5. Immediately remove the aliquot of the Low pH Starting Material, confirm the pH to 6.5–7.5, and filter (0.45 μm). Test the infectivity of one sample immediately. Divide the remaining material into multiple aliquots, rapidly freeze, and store at or below -60°C for later use. This serves as the “Low pH-T0” sample.

[0204] The remaining admixture was incubated at 14.0 ± 1.0 °C for 120 minutes. After incubation, the sample was removed, confirmed to be pH 6.5–7.5, and filtered (0.45 μm). One sample was immediately tested for infectivity. The remaining material was aliquoted into multiple equal aliquots, rapidly frozen, and stored at or below -60 °C for later use. This served as the “Low pH-treated control” sample.

[0205] This process is performed in duplicate. The following samples are generated and tested for the presence of X-MuLV using an infectiousness test:

[0206] Control group in virus removal studies Reserve virus control Aliquots of each sonicated and filtered stock virus solution used for incorporation were tested in an appropriate plaque assay. This determined the initial titer of the stock virus solution used for incorporation.

[0207] Quantitative analysis of infectious viruses by plaque assay At the start of the test, one copy of each test and control sample was diluted in serum-free medium to the endpoint (10⁻⁶). 0 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 and 10 -9 (As needed). Each appropriate dilution shall be determined by the standard viral titration procedure described below for each virus.

[0208] X-MuLV: Infectious viral particles were determined for each appropriate dilution in multiple wells and / or incubation dishes using X-MuLV plaque assays on PG4-indicating cells.

[0209] In each plaque assay, the following controls were tested: For each virus, aliquots of stock virus solutions, sonicated and filtered as assay controls, were used to generate an effectiveness standard for each assay. Serum-free culture medium was used as a negative control for each assay.

[0210] Validity The study was considered valid because the “stock virus control” defined above showed a value greater than 1 × 10⁻⁶. 6 Infective unit: viral titer per ml. Additionally, the negative control contained no virus (i.e., no viral plaques were observed).

[0211] Process Results Table 8 summarizes the viral log for samples that have been kept at pH 3.85 for 60 minutes using VI. 10 The reduction values ​​and 95% confidence limits are shown. The viral titers observed in samples held at pH 3.6 for 60 minutes are presented in Table 9, demonstrating that the worst-case viral clearance conditions produce acceptable viral clearance capabilities for the adjusted pH range (pH 3.6–3.8). Viral titers were determined by multiplying the “Average PFU” by the “Dilution” and dividing by the plate volume.

[0212] Table 8 - Log10 The final pH was lowered to 3.85.

[0213] Table 9. Summary of pH reduction by Log10 to 3.65

Claims

1. A method for producing a virus-inactivated antibody preparation comprising an anti-CD40 antibody or its antigen-binding moiety having reduced levels of high molecular weight aggregates, the method comprising, during a virus inactivation step, incubating a sample comprising an anti-CD40 antibody or its antigen-binding moiety at a pH of about 3.6-3.9 to produce a virus-inactivated antibody preparation having reduced levels of high molecular weight aggregates.

2. A method for minimizing the formation of high molecular weight aggregates in an antibody preparation containing an anti-CD40 antibody or its antigen-binding moiety during a virus inactivation step, the method comprising incubating a sample containing an anti-CD40 antibody or its antigen-binding moiety at a pH of about 3.6-3.9 during the virus inactivation step, thereby minimizing the formation of high molecular weight aggregates in the virus-inactivated antibody preparation.

3. A method for reducing the formation of high molecular weight aggregates in an antibody preparation containing an anti-CD40 antibody or its antigen-binding moiety during a virus inactivation step, the method comprising incubating a sample containing an anti-CD40 antibody or its antigen-binding moiety at a pH of about 3.6-3.9 during the virus inactivation step, thereby reducing the formation of high molecular weight aggregates in the virus-inactivated antibody preparation.

4. A method for maximizing the level of antibody monomers in a virus-inactivated antibody preparation containing an anti-CD40 antibody or its antigen-binding moiety thereof, the method comprising, during a virus inactivation step, incubating a sample containing an anti-CD40 antibody or its antigen-binding moiety at a pH of about 3.6-3.9, thereby maximizing the level of antibody monomers in the virus-inactivated antibody preparation.

5. A method for stabilizing a virus-inactivated antibody preparation comprising an anti-CD40 antibody or its antigen-binding moiety thereof, the method comprising, during a virus inactivation step, incubating a sample comprising an anti-CD40 antibody or its antigen-binding moiety thereof at a pH of about 3.6-3.9, thereby stabilizing the virus-inactivated antibody preparation.

6. A method for reducing the number and / or activity of viral particles in an antibody preparation containing an anti-CD40 antibody or its antigen-binding moiety thereof, the method comprising, during a virus inactivation step, incubating the sample containing the anti-CD40 antibody or its antigen-binding moiety at a pH of about 3.6-3.9, thereby reducing the number and / or activity level of viral particles in the antibody preparation.

7. The method of any one of claims 1-6, wherein the virus inactivation step includes a virus inactivation period and an optional static retention period.

8. The method of any one of claims 1-7, wherein the anti-CD40 antibody or its antigen-binding portion comprises a heavy chain variable region, said heavy chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 1, CDR2 having the amino acid sequence of SEQ ID NO: 2, and CDR3 having the amino acid sequence of SEQ ID NO:

3.

9. The method of any one of claims 1-8, wherein the anti-CD40 antibody or its antigen-binding portion comprises a light chain variable region, said light chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO:

6.

10. The method of any one of claims 1-9, wherein the anti-CD40 antibody or its antigen-binding portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:

8.

11. The method of any one of claims 1-10, wherein the anti-CD40 antibody or its antigen-binding portion comprises a heavy chain and a light chain, said heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and said light chain comprising the amino acid sequence of SEQ ID NO:

10.

12. The method of any one of claims 1-11, wherein the anti-CD40 antibody or its antigen-binding portion is KPL-404.

13. The method of any one of claims 1-12, wherein during the virus inactivation step, the sample is incubated at a pH of about 3.6-3.9, about 3.6-3.8, about 3.7-3.9, about 3.6-3.7, about 3.7-3.8 or about 3.8-3.

9.

14. The method of any one of claims 1-13, wherein during the virus inactivation step, the sample is incubated at a pH of about 3.6, about 3.7, about 3.8 or about 3.

9.

15. The method of any one of claims 1-14, wherein the sample is incubated for about 15-360 minutes during the virus inactivation step.

16. The method of any one of claims 1-15, wherein during the virus inactivation step, the sample is incubated for about 15-50 minutes, about 20-40 minutes, about 20-60 minutes, about 30-70 minutes, about 30-60 minutes, about 30-120 minutes, about 60-120 minutes, about 40-80 minutes, about 50-70 minutes, about 50-90 minutes, about 60-100 minutes, about 70-110 minutes, about 80-100 minutes, about 80-120 minutes, about 90-130 minutes, or about 100-140 minutes. Approximately 110-130 minutes, approximately 110-150 minutes, approximately 120-160 minutes, approximately 130-170 minutes, approximately 140-180 minutes, approximately 150-190 minutes, approximately 160-200 minutes, approximately 170-190 minutes, approximately 170-210 minutes, approximately 180-220 minutes, approximately 200-240 minutes, approximately 220-260 minutes, approximately 230-250 minutes, approximately 240-280 minutes, approximately 280-320 minutes, approximately 290-310 minutes, or approximately 320-360 minutes.

17. The method of any one of claims 1-16, wherein during the virus inactivation step, the sample is incubated at a temperature of about 13°C-37°C, about 15°C-37°C, about 15°C-30°C, about 13°C-25°C, or about 15°C-25°C.

18. The method of any one of claims 1-17, wherein during the virus inactivation step, the sample is incubated at a pH of about 3.6-3.8 for about 30-120 minutes.

19. The method of any one of claims 1-18, wherein during the virus inactivation step, the sample is incubated for about 30-120 minutes at a temperature of about 13°C-25°C and a pH of about 3.7-3.

8.

20. The method of any one of claims 1-19, wherein during the virus inactivation step, the sample is incubated at a pH of about 3.7 for about 60-120 minutes.

21. The method of any one of claims 1-20, wherein during the virus inactivation step, the sample is incubated for about 120 minutes at a temperature of about 25°C and a pH of about 3.

7.

22. The method of any one of claims 1-21, wherein the sample is passed through a protein A column prior to the virus inactivation step.

23. The method of any one of claims 1-22, wherein the virus-inactivated antibody preparation comprises less than 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1% high molecular weight aggregates.

24. The method of any one of claims 1-23, wherein the virus-inactivated antibody preparation contains less than 2% high molecular weight aggregates.

25. The method of any one of claims 1-24, wherein the level of high molecular weight aggregates is determined by size exclusion chromatography.

26. A composition comprising an anti-CD40 antibody or its antigen-binding moiety, wherein the composition comprises a virus-inactivated eluent collected from an affinity chromatography column, wherein the eluent comprises less than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1% of high molecular weight aggregates.

27. The composition of claim 26, wherein the virus-inactivated eluent contains less than 2% high molecular weight aggregates.

28. The composition of claim 26 or 27, wherein the affinity chromatography column comprises a protein A chromatography column.

29. The composition of any one of claims 26-28, wherein the virus-inactivated eluent has undergone a virus inactivation step at a pH of about 3.6-3.

9.

30. The composition of any one of claims 26-29, wherein the anti-CD40 antibody or its antigen-binding portion comprises a heavy chain variable region, said heavy chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 1, CDR2 having the amino acid sequence of SEQ ID NO: 2, and CDR3 having the amino acid sequence of SEQ ID NO:

3.

31. The composition of any one of claims 26-30, wherein the anti-CD40 antibody or its antigen-binding portion comprises a light chain variable region, said light chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO:

6.

32. The composition according to any one of claims 26-31, wherein the anti-CD40 antibody or its antigen-binding portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:

8.

33. The composition according to any one of claims 26-32, wherein the anti-CD40 antibody or its antigen-binding portion comprises a heavy chain and a light chain, said heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and said light chain comprising the amino acid sequence of SEQ ID NO:

10.

34. The composition of any one of claims 26-33, wherein the anti-CD40 antibody or its antigen-binding portion is KPL-404.

35. A method for producing a pharmaceutical composition comprising an anti-CD40 antibody and a pharmaceutically acceptable carrier, the method comprising, during a virus inactivation step, incubating a sample comprising an anti-CD40 antibody or its antigen-binding moiety at a pH of about 3.6-3.9, thereby producing a pharmaceutical composition comprising an anti-CD40 antibody and a pharmaceutically acceptable carrier.

36. The method of any one of claims 1-34, wherein the antibody preparation comprises charged anti-CD40 antibodies, wherein the charged species comprises at least about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% or 50% of a major species.

37. The method of claim 35, wherein the pharmaceutical composition comprises a charged anti-CD40 antibody, wherein the charged species comprises at least about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% of a major species.

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