Methods for producing antibody compositions

Abstract

Provided herein is a method for determining the product quality of an antibody composition, wherein the ADCC activity level of the antibody composition is a criterion on which the product quality of the antibody composition is based. In an exemplary embodiment, the method includes: (i) determining the total nonfucosylated (TAF) glycan content of a sample of the antibody composition; and (ii) determining that the product quality is acceptable and / or has achieved the ADCC activity level standard if the TAF glycan content determined in (i) is within a target range. Related methods for monitoring product quality and methods for manufacturing antibody compositions are further provided herein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62 / 906,709, filed September 26, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Incorporation by Reference of Electronically Submitted Materials The computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith is incorporated by reference in its entirety and is identified as follows: 26,660 byte ASCII (Text) file with the file name "A-2451-WO-PCT_SeqList_ST25.txt", created on September 24, 2020. [Background technology]

[0003] Glycosylation is one of the most common and important post-translational modifications because it is involved in many cellular functions, including protein folding, quality control, molecular transport and localization, and cell surface receptor interactions. Glycosylation influences the bioactivity, pharmacokinetics, immunogenicity, solubility, and in vivo clearance of therapeutic glycoproteins, thereby affecting the therapeutic efficacy of recombinant protein drugs. In particular, the Fc glycoform profile is an important product quality attribute for recombinant antibodies, as it directly influences the clinical efficacy and pharmacokinetics of the antibody.

[0004] Specific glycan structures associated with the conserved biantennary glycans in the Fc-CH2 domain can strongly influence antibody effector functions, such as interaction with FcγRs mediating antibody-dependent cellular cytotoxicity (ADCC) (see Reusch D, Tejada ML. Fc glycans of therapeutic antibodies as critical quality attributes. Glycobiology 2015;25:1325-34). For example, it has been shown that core fucose has a profound effect on FcγRIIIa binding affinity, resulting in substantial changes in ADCC activity (see Okazaki A, et al. Fucose depletion from human IgG1 oligosaccharide enhances binding enthalpy and association rate between IgG1 and FcγRIIIa. Journal of molecular biology 2004;336:1239-49; Ferrara C, et al. Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcγRIII and antibodies lacking core fucose. Proceedings of the National Academy of Sciences of the United States of America 2011;108:12669-74). High mannose has also been shown to modulate ADCC activity, albeit to a much smaller and less predictable extent than core fucose (Thomann M, et al. Fc-galactosylation modulates antibody-dependent cellular cytotoxicity of therapeutic antibodies. Molecular immunology 2016;73:69-75).

[0005] Various factors affect glycan structure and therefore the final glycosylation form (glycoform) of a protein (glycoprotein). For example, the cell line expressing the antibody, the cell culture medium, the feed medium composition, and the timing of feeding during cell culture can affect the production of protein glycoforms. While research groups have suggested many methods to influence the levels of specific glycoforms of antibodies, there remains a need in the biopharmaceutical industry for a simple and efficient method to predict the level of effector function that a particular antibody composition will exhibit based on the antibody composition's given glycoform profile. Furthermore, there is a need in the art for a method to determine the level of specific glycans (e.g., non-fucosylated glycans, high-mannose glycans) that will achieve a desired level of effector function. Summary of the Invention [Means for solving the problem]

[0006] The present disclosure provides a method for determining the product quality of an antibody composition, wherein the ADCC activity level of the antibody composition is a criterion on which the product quality of the antibody composition is based. In various aspects, the method determines the product quality with respect to the ADCC activity level criterion. In an exemplary embodiment, the method includes (i) determining the total nonfucosylated (TAF) glycan content of a sample of the antibody composition; and (ii) determining the product quality as acceptable and / or achieving the ADCC activity level criterion if the TAF glycan content determined in (i) is within a target range. In an exemplary aspect, the target range of the TAF glycan content is based on (1) a target range of the ADCC activity level of a reference antibody and (2) a first model correlating the ADCC activity level of the antibody composition with the TAF glycan content of the antibody composition. In an exemplary embodiment, the ADCC predicted by the first model is about 95% to about 105% of the ADCC predicted by the second model, where the second model correlates the ADCC activity level of an antibody composition with the HM glycan content of the antibody composition and the AF glycan content of the antibody composition. As used herein, the term "predicted" in connection with ADCC activity level refers to a calculated ADCC activity level, where the ADCC activity level is calculated by a model, e.g., the first model, the second model. Advantageously, the ADCC predicted by the first model is statistically significantly similar to the ADCC predicted by the second model. For example, the ADCC activity level predicted by the first model is about 95% to about 105% of the ADCC activity level predicted by the second model. The ADCC activity level predicted by the first model is optionally about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 101%, about 102%, about 103%, about 104%, or about 105% of the ADCC activity level predicted by the second model. The ADCC activity level predicted by the first model, in various examples, is about 100% of the ADCC predicted by the second model. In certain aspects, there is a one-to-one correspondence between the ADCC predicted by the first model and the ADCC predicted by the second model.In various examples, the first model and / or the second model are statistically significant. For example, the p-value of the first model is less than 0.0001, and / or the p-value of the second model is less than 0.0001. Optionally, each of the first model and the second model has a p-value less than 0.0001. In an exemplary embodiment, the ADCC activity level predicted by the first model is approximately 12Q x TAF%, where Q is the number of antibody binding sites on the antigen to which the antibody binds, and TAF% is the TAF glycan content of the antibody composition. In an exemplary case, the target range of TAF glycan content is m to n, where m is [ADCC. min / 12Q] (where ADCC min is the minimum of the target range of ADCC activity levels for the reference antibody), and n is [ADCC max ] / 12Q (where ADCC max is the maximum of the target range of ADCC activity levels for the reference antibody). In various examples, Q is 2. In various examples, the ADCC activity level predicted by the first model is about 24×TAF%. In various examples, the target range of TAF glycan content is m to n (where m is the maximum of the target range of ADCC activity levels for the reference antibody). min / 24], and n is [ADCC max ] / 24). In various examples, the ADCC activity level predicted by the second model is about 27×HM% + about 22×AF%, where AF% is the AF glycan content of the antibody composition and HM% is the HM glycan content of the antibody composition. In various examples, Q is 1. In various embodiments, the ADCC activity level predicted by the first model is about 12×TAF%. In various examples, the target range for TAF glycan content is m to n, where m is [ADCC min / 12], and n is [ADCC max] / 12. In various examples, the ADCC activity level predicted by the second model is about 14.8×HM% + about 12.8×AF%. Suitable alternative first and second models are described herein. In exemplary cases, the first model is any one of the models (e.g., equations) described herein that correlate ADCC with TAF glycan content, including, but not limited to, Equations 1, 3, 5, and 7, and Equation A. In exemplary cases, the second model is any one of the models (e.g., equations) described herein that correlate ADCC with HM glycan content and AF glycan content, including, but not limited to, Equations 2, 4, 6, and 8, and Equation B. For example, in various embodiments, the target range for TAF glycan content is m° to n°, where m° is [[ADCC min -y] / x] (where ADCC min is the minimum value of the target range of ADCC activity levels), and n° is [[ADCC max -y] / x] (where ADCC max is the maximum value of the target range for ADCC activity levels). Optionally, x is about 20.4 to about 27.7 and y is about -11.4 to about 16.7. Alternatively, x is about 9.7 to about 15.2 and y is about -15.6 to about 34.2. In various embodiments, the target range for TAF glycan content is m' to n', where m' is the maximum value of the target range for ADCC activity levels. min / x'] (where ADCC min is the minimum of the target range of ADCC activity levels), and n' is [ADCC max ] / x' (where ADCC max(where x' is the maximum of the target range of ADCC activity levels). Optionally, x' is about 24.1 to about 25.4. Alternatively, x' is about 13.0 to about 13.95. In various examples, the ADCC activity level of the antibody composition is about 13.5% ± 0.5% for every 1% of TAFs present in the antibody composition, where, optionally, the antibodies of the antibody composition bind to an antigen that comprises only one antibody binding site. In various embodiments, the ADCC activity level of the antibody composition is about 24.74% ± 0.625% for every 1% of TAFs present in the antibody composition, where, optionally, the antibodies of the antibody composition bind to an antigen that comprises only two antibody binding sites. In an exemplary embodiment, the ADCC activity level of the antibody composition is about 12% ± 1.5% × Q for every 1% of TAFs present in the antibody composition, where Q is the number of antibody binding sites present on the antigen. In an exemplary case, the reference antibody is infliximab. In exemplary embodiments, the reference antibody is rituximab. In exemplary embodiments, the method is a quality control (QC) assay. In exemplary embodiments, the method is an in-process QC assay. In various embodiments, the sample is a sample of in-process material. In various examples, the TAF glycan content is determined before or after collection. In exemplary cases, the TAF glycan content is determined after a chromatography step. Optionally, the chromatography step includes capture chromatography, intermediate chromatography, and / or polishing chromatography. In some embodiments, the TAF glycan content is determined after viral inactivation and neutralization, viral filtration, or buffer exchange. In various examples, the method is a lot release assay. In some embodiments, the sample is a sample of a manufacturing lot. In various embodiments, the method further includes selecting the antibody composition for downstream processing if the TAF glycan content determined in (i) is within a target range. If the TAF glycan content determined in (i) is not within the target range, in various embodiments, one or more conditions of the cell culture are modified to obtain an altered cell culture. In some embodiments, the method further comprises determining the TAF glycan content of a sample of the antibody composition obtained after modifying one or more conditions of the cell culture.In various embodiments, if the TAF glycan content determined in (i) is not within the target range, the method further includes (iii) modifying one or more conditions of the cell culture to obtain an altered cell culture, and (iv) determining the TAF glycan content of a sample of the antibody composition obtained from the altered cell culture. In an exemplary embodiment, if the TAF glycan content determined in (i) is not within the target range, the method further includes (iii) and (iv) until the TAF glycan content determined in (iv) is within the target range. In an exemplary case, an assay directly measuring the ADCC activity of the antibody composition is performed on the antibody composition only if the TAF glycan content determined in (i) is not within the target range, e.g., outside the target range. An assay directly measuring ADCC activity includes, for example, a cell-based assay that measures the release of a detectable agent upon lysis of antigen-expressing cells containing the detectable agent by effector cells that bind to an antibody that binds to both the antigen-expressing cells and the effector cells. In an exemplary case, an assay directly measuring the ADCC activity of the antibody composition is not performed on the antibody composition. In various embodiments, determining the TAF glycan content is the only step required to determine product quality based on the ADCC activity level. Without being bound by theory, the statistically significant correlation between the first model and the second model allows the TAF glycan content to indicate the ADCC activity level, eliminating the need for an assay to directly measure the ADCC activity level. Therefore, directly measuring the ADCC activity level of the antibody composition is not necessary, and therefore is not performed in various embodiments of the methods disclosed herein.

[0007] The present disclosure also provides a method for monitoring the product quality of an antibody composition, wherein the ADCC activity level of the antibody composition is a criterion on which the product quality of the antibody composition is based. In an exemplary embodiment, the method includes determining the product quality of the antibody composition using a first sample obtained at a first time point and a second sample taken at a second time point different from the first time point according to the method of the present disclosure. In various examples, each of the first sample and the second sample is a sample of in-process material. In various aspects, the first sample is a sample of in-process material, and the second sample is a sample of a manufacturing lot. Optionally, the first sample is a sample obtained before modifying one or more conditions of cell culture, and the second sample is a sample obtained after modifying one or more conditions of cell culture. In an exemplary case, the TAF glycan content is determined for each of the first sample and the second sample. The product quality of the antibody composition depends on whether the TAF glycan content is within a target range. In an exemplary embodiment, the target range of TAF glycan content is based on (1) a target range of ADCC activity levels of a reference antibody and (2) a first model correlating the ADCC activity level of an antibody composition with the TAF glycan content of the antibody composition. In an exemplary embodiment, the ADCC predicted by the first model is about 95% to about 105% of the ADCC predicted by a second model, where the second model correlates the ADCC activity level of an antibody composition with the HM glycan content of the antibody composition and the AF glycan content of the antibody composition.

[0008] The present disclosure provides methods for producing an antibody composition. In exemplary embodiments, the method includes determining the product quality of the antibody composition, wherein the product quality of the antibody composition is determined according to a method of the present disclosure. Optionally, the method includes (i) determining the TAF glycan content of a sample of the antibody composition, where the sample is a sample of in-process material. In various examples, the method includes determining the product quality of the antibody composition as acceptable and / or achieving a standard ADCC activity level if the TAF glycan content determined in (i) is within a target range as defined herein. In exemplary embodiments, the target range of the TAF glycan content is based on (1) a target range of the ADCC activity level of a reference antibody and (2) a first model correlating the ADCC activity level of the antibody composition with the TAF glycan content of the antibody composition. In exemplary embodiments, the ADCC predicted by the first model is about 95% to about 105% of the ADCC predicted by the second model, where the second model correlates the ADCC activity level of the antibody composition with the HM glycan content of the antibody composition and the AF glycan content of the antibody composition. In various embodiments, if the TAF glycan content determined in (i) is not within the target range, the method further includes (iii) modifying one or more conditions of the cell culture to obtain an modified cell culture, and (iv) determining the TAF glycan content of a sample of the antibody composition obtained from the modified cell culture, optionally repeating steps (iii) and (iv) until the TAF glycan content is within the target range. In various examples, the sample is a sample of a cell culture containing cells expressing an antibody of the antibody composition. In various examples, one or more conditions of the cell culture are modified to modify the TAF glycan content. In various embodiments, the TAF glycan content of the antibody composition is achieved by modifying the AF glycan content. In exemplary embodiments, one or more conditions of cell culture are modified to modify the AF glycan content of the antibody composition.In exemplary embodiments, one or more conditions mainly modify the AF glycan content.In various examples, one or more conditions modify the AF glycan content and do not modify the HM glycan content.In exemplary embodiments, the method includes achieving a TAF glycan content of the antibody composition by modifying the HM glycan content. Optionally, one or more conditions of the cell culture are modified to modify the HM glycan content of the antibody composition. In some examples, the one or more conditions modify primarily the HM glycan content. In some embodiments, the one or more conditions modify the HM glycan content but not the AF glycan content. In various examples, the method includes repeating the modification of the nonfucosylated (AF) glycan content and / or repeating the modification of the high mannose (HM) glycan content until the TAF glycan content is within a target range.

[0009] In an exemplary embodiment, a method for producing an antibody composition includes (i) determining the TAF glycan content of a sample of the antibody composition; and (ii) selecting an antibody composition for downstream processing based on the TAF glycan content determined in (i). In various aspects, the sample is taken from a cell culture containing cells expressing an antibody of the antibody composition. In various examples, the method further includes modifying the TAF glycan content of the antibody composition and determining the modified TAF glycan content. Optionally, one or more conditions of the cell culture are modified to modify the TAF glycan content. In an exemplary aspect, the method includes repeating the modification until the TAF glycan content falls within a target range. In an exemplary case, the target range is based on a target range of ADCC activity levels of the antibody. Without being bound by theory, the TAF glycan content correlates with the ADCC activity level of an antibody composition; therefore, the ADCC activity level of an antibody composition can be predicted based on the TAF glycan content of the antibody composition. The ADCC activity level of an antibody composition can be a criterion worth considering when determining whether the antibody composition should be selected for downstream processing. Thus, in various embodiments, the method includes: (i) determining the TAF glycan content of a sample of the antibody composition; (ii) determining the ADCC activity level of the antibody composition based on the TAF glycan content determined in (i); and, optionally, (iii) selecting the antibody composition for downstream processing if the ADCC level of the antibody composition determined in (ii) is within a target range of ADCC activity levels. In various embodiments, the target range of ADCC activity levels is known for the antibodies of the antibody composition. In various embodiments, the antibodies of the antibody composition are biosimilars of a reference antibody. In various examples, the target range of TAF glycan content is based on or determined (e.g., calculated) based on a known target range of ADCC activity levels. Thus, in an exemplary embodiment, the method includes: (i) determining the TAF glycan content of a sample of an antibody composition; and (ii) selecting the antibody composition for downstream processing if the TAF glycan content determined in (i) is within a target range.When the method further comprises modifying the TAF glycan content of the antibody composition, the method, in various examples, comprises modifying the nonfucosylated (AF) glycan content to modify the TAF glycan content. Optionally, one or more conditions of the cell culture are modified to modify the AF glycan content of the antibody composition, which then modifies the TAF glycan content. Alternatively, or in addition, when the method comprises modifying the TAF glycan content of the antibody composition, the method, in various examples, comprises modifying the high mannose (HM) glycan content to modify the TAF glycan content. Optionally, one or more conditions of the cell culture are modified to modify the HM glycan content of the antibody composition, which then modifies the TAF glycan content. In exemplary embodiments, one or more conditions primarily modify the AF glycan content. In exemplary cases, one or more conditions primarily modify the HM glycan content. In exemplary embodiments, one or more conditions modify the AF glycan content but not the HM glycan content. In exemplary cases, the one or more conditions modify HM glycan content but do not modify AF glycan content. The method optionally includes repeating the modification of nonfucosylated (AF) glycan content and / or repeating the modification of high mannose (HM) glycan content until the TAF glycan content is within the target range. In exemplary embodiments, the antibody of the antibody composition is an IgG, optionally an IgG1. In various embodiments, the target range for TAF glycan content is m to n, where m is [[ADCC. min -y] / x] (where ADCC min is the minimum of the target range of ADCC activity levels), and n is [[ADCC max -y] / x] (where ADCC max (where x is the maximum of the target range for ADCC activity levels). Optionally, x is about 20.4 to about 27.7 and y is about -11.4 to about 16.7. Alternatively, x is about 9.7 to about 15.2 and y is about -15.6 to about 34.2. In various embodiments, the target range for TAF glycan content is m' to n', where m' is the maximum of the target range for ADCC activity levels. min / x'] (where ADCC min is the minimum of the target range of ADCC activity levels), and n' is [ADCCmax ] / x' (where ADCC max (where x' is the maximum of the target range of ADCC activity levels). Optionally, x' is about 24.1 to about 25.4. Alternatively, x' is about 13.0 to about 13.95. In various examples, the ADCC activity level of the antibody composition is about 13.5%±0.5% for every 1% of TAFs present in the antibody composition, and optionally, the antibodies of the antibody composition bind to an antigen that comprises only one antibody binding site. In various embodiments, the ADCC activity level of the antibody composition is about 24.74%±0.625% for every 1% of TAFs present in the antibody composition, and optionally, the antibodies of the antibody composition bind to an antigen that comprises only two antibody binding sites. In an exemplary embodiment, the ADCC activity level of the antibody composition is about 12%±1.5%×Q for every 1% of TAFs present in the antibody composition, where Q is the number of antibody binding sites present on the antigen. In an exemplary case, Q is 1, and optionally, the antibody is infliximab or a biosimilar thereof. Optionally, Q is 2, and optionally the antibody is rituximab or a biosimilar thereof.

[0010] In an exemplary embodiment, the method of producing an antibody composition includes (i) determining the % total nonfucosylated (TAF) glycans of the antibody composition, (ii) determining the % total nonfucosylated (TAF) glycans of the antibody composition based on Equation A: Y=2.6+24.1×X [Equation A] where Y is the ADCC % and X is the TAF glycan % determined in step (i). calculating the antibody-dependent cellular cytotoxicity (ADCC) % of the antibody composition based on the TAF % using the following formula: and (iii) if Y is within the target ADCC % range, selecting the antibody composition for one or more downstream processing steps.

[0011] The present disclosure also provides a method of producing an antibody composition, the method comprising: (i) determining the % high mannose glycans and % non-fucosylated glycans of the antibody composition; (ii) determining the % high mannose glycans and % non-fucosylated glycans of the antibody composition based on Equation B: Y = (0.24 + 27 × HM + 22.1 × AF) [Equation B] where Y is ADCC %, HM is high mannose glycan % determined in step (i), and AF is non-fucosylated glycan % determined in step (i). calculating the % antibody-dependent cellular cytotoxicity (ADCC) of the antibody composition based on the % high mannose glycans and the % non-fucosylated glycans using the and (iii) if Y is within the target ADCC % range, selecting the antibody composition for one or more downstream processing steps.

[0012] The present disclosure further provides a method for producing an antibody composition having a target ADCC %. In an exemplary embodiment, the method comprises: (i) calculating a % ADCC of an antibody composition according to Equation A: Y=2.6+24.1×X [Equation A] where Y is the target ADCC % and X is the target TAF glycan %. Calculate the target total nonfucosylated (TAF) glycan % for the target ADCC % using: and (ii) maintaining the glycosylation-competent cells in cell culture to produce an antibody composition having a target TAF glycan %, X.

[0013] The present disclosure further provides a method for producing an antibody composition having a target ADCC %, the method comprising: (i) determining a target ADCC percentage based on Equation B: Y = (0.24 + 27 × HM + 22.1 × AF) [Equation B] where Y is the target ADCC %, HM is the target high mannose glycan %, and AF is the target non-fucosylated glycan %. calculating the target nonfucosylated glycan % and the target high mannose glycan % for the target ADCC % using the and (ii) maintaining the glycosylation-competent cells in cell culture to produce an antibody composition having a target % high mannose glycans and a target % nonfucosylated glycans.

[0014] In exemplary embodiments of the disclosed methods, the target ADCC% is within a target ADCC% range. Optionally, the target ADCC% range is from about 40 to about 170. In various embodiments, the target ADCC% range is from about 44 to about 165. In various examples, the target ADCC% range is from about 60 to about 130. In exemplary embodiments, the target ADCC% range is Y±20, e.g., Y±17 or Y±18.

[0015] Additionally provided are methods for producing an antibody composition having an ADCC%, Y, of between about 40 and about 170, the methods comprising (i) determining the % total nonfucosylated (TAF) glycans, X, of the antibody composition, and (ii) selecting the antibody composition for one or more downstream processing steps if X is equal to (Y-2.6) / 24.1. In exemplary embodiments, X is between about 1.55% and about 6.95%. In various embodiments, Y is between about 44% and about 165%, and optionally, X is between about 1.72% and about 6.74%.

[0016] The present disclosure provides a method of producing an antibody composition having an ADCC%, Y, comprising: (i) determining a total nonfucosylated (TAF) glycan %, X, of the antibody composition; and (ii) selecting the antibody composition for one or more downstream processing steps if X is equal to (Y-2.6) / 24.1, where, optionally, X is greater than or equal to about X-0.4 and less than or equal to about X+0.4, and the ADCC% is greater than or equal to about Y-17 and greater than or equal to about Y+17. Also provided is a method of producing an antibody composition having an ADCC%, the method comprising: (i) determining the nonfucosylated glycan % and the high mannose glycan % of the antibody composition; and (ii) determining a total nonfucosylated (TAF) glycan % of the antibody composition, where AF and HM satisfy Equation B: Y = (0.24 + 27 × HM + 22.1 × AF) [Equation B] where Y is ADCC %, HM is high mannose glycan % determined in step (i), and AF is non-fucosylated glycan % determined in step (i). selecting the antibody composition for one or more downstream process steps if it is related to Y according to: In exemplary embodiments, Y is between about 40 and about 175, optionally between about 41 and about 171, AF is between about 1 and about 4, and HM is between about 40 and about 175. Optionally, Y is between about 30 and about 185, optionally between about 32 and about 180, HM is between about 1 and about 4, and AF is between about 30 and about 185. In exemplary cases, the ADCC % of the antibody composition is within a range defined by Y. Optionally, the ADCC % of the antibody composition is within a range of Y ± 18. In exemplary embodiments, AF is between about 1 and about 4. Optionally, the % high mannose glycans is within a range defined by HM, optionally within a range of HM ± 1. In various examples, HM is between about 1 and about 4. Optionally, the % nonfucosylated glycans is a value within a range defined by AF, optionally the range is AF±1.

[0017] In exemplary embodiments, the disclosed methods for producing an antibody composition include modifying the total nonfucosylated (TAF) glycan content of an antibody composition produced by cells in a cell culture. In various examples, one or more conditions of the cell culture are modified to modify the TAF glycan content. In various aspects, the method includes determining the modified TAF glycan content. Optionally, the modification is repeated until the determined TAF glycan content is within a target TAF range. Without being bound by theory, TAF glycan content can be modified by changing the nonfucosylated (AF) glycan content or the high mannose (HM) content, or a combination thereof, as each affects TAF glycan content. Thus, these methods advantageously allow for achieving a target range of TAF glycan content in multiple ways. For example, one or more conditions of the cell culture are modified to modify the AF glycan content in order to modify the TAF glycan content. Alternatively, one or more conditions of the cell culture are modified to modify the HM glycan content in order to modify the TAF glycan content. In various examples, one or more conditions of the cell culture are modified to modify the AF glycan content and the HM glycan content in order to modify the TAF glycan content. Thus, the present disclosure further provides a method for modifying the total nonfucosylated (TAF) glycan content of an antibody composition produced by cells of a cell culture. In exemplary embodiments, the method comprises modifying the AF glycan content. In exemplary embodiments, the method comprises modifying the HM glycan content. In various aspects, the method includes (i) determining the nonfucosylated (AF) glycan content and the high mannose (HM) glycan content of a sample of the antibody composition; (ii) determining a target range for the AF glycan content based on a target range of ADCC activity levels of antibodies of the antibody composition, assuming that the HM glycan content is constant; and (iii) selecting the antibody composition for downstream processing if the AF glycan content is within the target range of AF glycan content.In various examples, the method includes (i) determining the nonfucosylated (AF) glycan content and high mannose (HM) glycan content of a sample of an antibody composition; (ii) determining a target range of HM glycan content based on a target range of ADCC activity levels of antibodies in the antibody composition, assuming the AF glycan content is constant; and (iii) selecting the antibody composition for downstream processing if the HM glycan content is within the target range of HM glycan content. In various examples, the method includes (i) determining the AF glycan content and HM glycan content of a sample of the antibody composition, and (ii) determining a target range of AF glycan content based on the HM glycan content determined in (i), and (iii) modifying the AF glycan content until the AF glycan content is within the target range of AF glycan content, wherein the HM glycan content is not modified. Alternatively, the method includes (i) determining the AF glycan content and the HM glycan content of a sample of the antibody composition, and (ii) determining a target range of the HM glycan content based on the AF glycan content determined in (i), and (iii) modifying the HM glycan content until the HM glycan content is within the target range of HM glycan content, wherein the AF glycan content is not modified. In an exemplary embodiment, a model correlating the ADCC activity level of an antibody composition with the TAF glycan content of the antibody composition predicts substantially the same ADCC activity level as the level predicted by a model correlating ADCC with HM and AF glycan content.

[0018] In various embodiments of the disclosed methods, the % TAF glycan is determined by calculating the sum of the % high mannose glycan and the % nonfucosylated glycan. In various examples, the % high mannose glycan and the % nonfucosylated glycan are determined by hydrophilic interaction chromatography. Optionally, the % high mannose glycan and the % nonfucosylated glycan are determined by the method described in Example 1. In various embodiments, the % ADCC is determined by a quantitative cell-based assay that measures the ability of antibodies of the antibody composition to mediate cytotoxicity in a dose-dependent manner in cells that express the antibody's antigen and engage the Fc-γRIIIA receptor on effector cells via the antibody's Fc domain. In various examples, the % ADCC is determined by the assay described in Example 2. In exemplary embodiments, the determining step is performed after the recovering step. Optionally, the determining step is performed after the chromatography step. In various embodiments, the chromatography step is a Protein A chromatography step. In various examples of the disclosed methods, the one or more downstream process steps include a dilution step, a filling step, a filtration step, a formulation step, a chromatography step, a viral filtration step, a viral inactivation step, or a combination thereof. Optionally, the chromatography step is an ion exchange chromatography step, optionally a cation exchange chromatography step or an anion exchange chromatography step.

[0019] In various embodiments of the present disclosure, each antibody of the antibody composition is an IgG, and optionally, each antibody of the antibody composition is an IgG1. In exemplary cases, each antibody of the antibody composition binds to a tumor-associated antigen. In exemplary embodiments, the tumor-associated antigen comprises the amino acid sequence of SEQ ID NO: 3. The tumor-associated antigen is at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 3. In exemplary embodiments, each antibody of the antibody composition is an anti-CD20 antibody. In various examples, each antibody of the antibody composition comprises: (i) a light chain (LC) CDR1 comprising the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 4, or a variant amino acid sequence of SEQ ID NO: 4 with one or two amino acid substitutions; (ii) a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 5, or a variant amino acid sequence of SEQ ID NO: 5 with one or two amino acid substitutions; (iii) a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 6, or a variant amino acid sequence of SEQ ID NO: 6 with one or two amino acid substitutions; (iv) a heavy chain (HC) CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, or a variant amino acid sequence of SEQ ID NO: 7 with one or two amino acid substitutions; (v) a HC CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 8, or a variant amino acid sequence of SEQ ID NO: 8 with one or two amino acid substitutions. and / or (vi) a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 9, or a variant amino acid sequence of SEQ ID NO: 9 having one or two amino acid substitutions.

[0020] In an exemplary embodiment, each antibody of the antibody composition comprises an LC variable region comprising the amino acid sequence of SEQ ID NO: 10, an amino acid sequence at least 90% identical to SEQ ID NO: 10, or a variant amino acid sequence of SEQ ID NO: 10 with 1 to 10 amino acid substitutions. Optionally, each antibody of the antibody composition comprises an HC variable region comprising the amino acid sequence of SEQ ID NO: 11, an amino acid sequence at least 90% identical to SEQ ID NO: 11, or a variant amino acid sequence of SEQ ID NO: 11 with 1 to 10 amino acid substitutions. In an exemplary embodiment, each antibody of the antibody composition comprises a light chain comprising the amino acid sequence of SEQ ID NO: 12, an amino acid sequence at least 90% identical to SEQ ID NO: 12, or a variant amino acid sequence of SEQ ID NO: 12 with 1 to 10 amino acid substitutions. In an exemplary case, each antibody of the antibody composition comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 13, an amino acid sequence at least 90% identical to SEQ ID NO: 13, or a variant amino acid sequence of SEQ ID NO: 13 with 1 to 10 amino acid substitutions.

[0021] In an exemplary embodiment, the tumor-associated antigen comprises the amino acid sequence of SEQ ID NO: 14. It is also preferred that the antibody composition comprises a VH region selected from the group consisting of the VH regions set forth in SEQ ID NO: 14. In an exemplary embodiment, each antibody of the antibody composition is an anti-TNFα antibody, and optionally infliximab or a biosimilar thereof. In an exemplary embodiment, each antibody of the antibody composition comprises an LC variable region comprising the amino acid sequence of SEQ ID NO: 15, an amino acid sequence at least 90% identical to SEQ ID NO: 15, or a variant amino acid sequence of SEQ ID NO: 15 having 1 to 10 amino acid substitutions. Optionally, each antibody of the antibody composition comprises an HC variable region comprising the amino acid sequence of SEQ ID NO: 16, an amino acid sequence at least 90% identical to SEQ ID NO: 16, or a variant amino acid sequence of SEQ ID NO: 16 having 1 to 10 amino acid substitutions.

[0022] The present disclosure further provides a method for producing an antibody composition within a target ADCC% range, the method comprising the steps of: (i) measuring the ADCC% of a series of samples comprising various glycoforms of an antibody; (ii) determining the total nonfucosylated (TAF) glycan% for each of the series of samples; (iii) determining, for each of the series of samples, a linear equation for a best-fit line of a graph plotting the ADCC% measured in step (i) as a function of the TAF glycan% determined in step (ii); (iv) determining the TAF% of the antibody composition and then calculating the ADCC% using the linear equation of step (iii); and (v) selecting the antibody composition for one or more downstream processing steps if the ADCC% calculated in step (iv) is within the target ADCC% range.

[0023] A method for producing an antibody composition having a TAF glycan content within a target range is provided, the method comprising: (i) measuring the ADCC activity level of a series of samples containing various glycoforms of an antibody; (ii) determining the % TAF glycan for each of the series of samples; (iii) creating a model correlating the ADCC activity level to the TAF glycan content; (iv) determining the ADCC activity level of the antibody composition and then calculating the TAF glycan content using the model; or determining the TAF glycan content of the antibody composition and calculating the ADCC activity level using the model; and (v) selecting the antibody composition for one or more downstream process steps if the TAF glycan content calculated in step (iv) is within the target range of TAF glycan content or if the ADCC activity level calculated in step (iv) is within the target range of ADCC activity levels.

[0024] A method for producing an antibody composition having a TAF% within a target range is provided, the method comprising the steps of: (i) measuring the ADCC% of a series of samples comprising various glycoforms of an antibody; (ii) determining the total nonfucosylated (TAF) glycan% for each of the series of samples; (iii) determining a linear equation for a best-fit line of a graph plotting the ADCC% measured in step (i) as a function of the TAF glycan% determined in step (ii) for each of the series of samples; (iv) determining the ADCC% of the antibody composition and then calculating the TAF% using the linear equation of step (iii); and (v) selecting the antibody composition for one or more downstream processing steps if the TAF% calculated in step (iv) is within the target TAF% range. Also provided is a method for producing an antibody composition within a target TAF% range, the method comprising the steps of: (i) generating a linear equation for a best-fit line by plotting the ADCC% and TAF glycan% of a series of at least five reference antibody compositions produced under cell culture conditions (each reference antibody composition has the same amino acid sequence as the antibody composition), (ii) selecting a target TAF glycan% range based on the linear equation generated in step (i) and a desired ADCC activity%, (iii) culturing the antibody composition under cell culture conditions, (iv) purifying the antibody composition, (v) sampling the antibody composition to determine the TAF%, and (vi) determining whether the TAF% of the antibody composition is within the target %TAF% range of step (ii). In an exemplary embodiment, the method further comprises selecting the antibody composition for one or more downstream process steps if the TAF% calculated in step (v) is within the target TAF% range.

[0025] Also provided is a method for determining the antibody-dependent cellular cytotoxicity (ADCC) % of an antibody composition, the method comprising: (i) determining the total nonfucosylated (TAF) glycan % of the antibody composition; and (ii) determining the ADCC % of the antibody composition by Equation A: Y=2.6+24.1×X [Equation A] where Y is the ADCC % and X is the TAF glycan % determined in step (i). The method includes calculating the ADCC% of the antibody composition based on the TAF% using the following:

[0026] Further provided is a method for determining the antibody-dependent cellular cytotoxicity (ADCC) % of an antibody composition, the method comprising: (i) determining the high mannose glycan % and the non-fucosylated glycan % of the antibody composition; and (ii) determining the ADCC % of the antibody composition by Equation B: Y = (0.24 + 27 × HM + 22.1 × AF) [Equation B] where Y is ADCC %, HM is high mannose glycan % determined in step (i), and AF is non-fucosylated glycan % determined in step (i). The method includes calculating the % ADCC of the antibody composition based on the % high mannose glycans and the % nonfucosylated glycans using the method.

[0027] In exemplary cases, the method further includes selecting the antibody composition for one or more downstream processing steps if Y is within the target ADCC % range. [Brief explanation of the drawings]

[0028] [Figure 1A] 1 is a diagram of the three types of N-glycans (oligomannose, complex, and hybrid) and the commonly used symbols for such saccharides. [Figure 1B] 1 is an illustration of exemplary glycan structures. [Figure 2A] Representative glycan map chromatogram (full scale view). [Figure 2B] Representative glycan map chromatogram (enlarged scale view). [Figure 3] FIG. 1 is a schematic diagram of the NK92 ADCC assay described in Example 2. [Figure 4] Representative dose-response curve for NK92 ADCC assay. Each dose point is the mean ± standard deviation of triplicates. Assay signal = fluorescence. [Figure 5A-5B][Figure 5A] Graph of actual ADCC (%) plotted as a function of TAF (%). A best-fit line is shown. [Figure 5B] Table of statistical parameters of the best-fit line of Figure 5A. [Figure 5C-5D] [Figure 5C] Graph of actual ADCC (%) (determined by the assay described in Example 2) plotted as a function of predicted ADCC (%) calculated using the predictive equation shown in Figure 5B. [Figure 5D] Graph of Figure 5A showing 95% confidence bands (shaded in gray). [Figure 5E] Graphs of the 95% confidence intervals for both the y-intercept and slope of Equation 1 are provided. [Figures 6A-6B] [Figure 6A] Graph of actual ADCC (%) plotted as a function of HM (%). A best-fit line is shown. [Figure 6B] Graph of actual ADCC (%) plotted as a function of AF (%). A best-fit line is shown. [Figure 6C-6D] [Figure 6A] Table of statistical parameters of the best-fit lines shown in Figures 6A and 6B. [Figure 6D] Graph of actual ADCC (%) (determined by the assay described in Example 2) plotted as a function of predicted ADCC (%) calculated using the prediction equation shown in Figure 6C. [Figures 7A-7B] [Figure 7A] Graph of actual ADCC (%) plotted as a function of galactosylation (%). The best-fit line is shown in red. [Figure 7B] Graph of actual ADCC (%) (determined by the assay described in Example 2) plotted as a function of predicted ADCC (%) calculated using a predictive equation (not shown) correlating ADCC with galactosylation. [Figure 8A-8B] [Figure 8A] Graph of actual ADCC (%) plotted as a function of TAF (%). Best-fit lines are shown. [Figure 8B] Table of statistical parameters of the best-fit lines of Figure 8A. [Figure 8C-8D] [Figure 8C] Graph of actual ADCC (%) (determined by the assay described in Example 2) plotted as a function of predicted ADCC (%) calculated using the predictive equation shown in Figure 8B. [Figure 8D] Graph of Figure 8A showing 95% confidence bands (shaded in gray). [Figure 8E] Graphs of the 95% confidence regions for both the y-intercept and slope of Equation 3 are shown. [Figure 9A-9B] [Figure 9A] Graph of actual ADCC (%) plotted as a function of HM (%). A best-fit line is shown. [Figure 9B] Graph of actual ADCC (%) plotted as a function of AF (%). A best-fit line is shown. [Figure 9C-9D] [Figure 9C] Table of statistical parameters of the best-fit lines shown in Figures 9A and 9B. [Figure 9D] Graph of actual ADCC (%) (determined by the assay described in Example 2) plotted as a function of predicted ADCC (%) calculated using the prediction equation shown in Figure 9C. [Figures 10A-10B] 10A and 10B are graphs correlating the y-intercept-free predictions of the ADCC-HM / AF model with the y-intercept-free predictions of the ADCC-TAF model for anti-CD20 antibodies (FIG. 10A) and anti-TNF-alpha antibodies (FIG. 10B). DETAILED DESCRIPTION OF THE INVENTION

[0029] Provided herein for the first time are data demonstrating a statistically significant correlation between the ADCC level of an antibody composition and the TAF glycan level of that antibody composition. Also provided herein for the first time are data demonstrating a statistically significant correlation between the ADCC level of an antibody composition and the high mannose glycan and nonfucosylated glycan levels of that antibody composition. As further described herein, Equation A and Equation B relate the % ADCC of an antibody composition to the % TAF glycan (Equation A) or the % high mannose glycan and nonfucosylated glycan (Equation B) of the antibody composition. These correlations and equations, and others, of the present disclosure are useful in methods for predicting the ADCC level of an antibody composition based on glycan levels. In various aspects, the predicted ADCC level serves as a marker for identifying an antibody composition as acceptable in terms of meeting a therapeutic threshold and therefore to be used in one or more downstream manufacturing process steps, or for identifying an antibody composition as unacceptable and not to proceed through such manufacturing processes. The correlations and equations disclosed herein are also useful in identifying the glycoprofile of a desired antibody composition. Using the relationships and equations provided herein, and given a target ADCC level, a glycoprofile (e.g., a profile of TAF glycans, HM glycans, and nonfucosylated glycans) of an antibody composition having a target ADCC level can be identified. Using the identified profile of TAF glycans, HM glycans, and nonfucosylated glycans of an antibody composition having a target ADCC level, a manufacturing process, e.g., a cell culture step, can be performed to target the identified profile.

[0030] Thus, the present disclosure provides a method for determining the product quality of an antibody composition, wherein at least one of the acceptance criteria for the antibody composition is the ADCC activity level. A method for monitoring the product quality of an antibody composition is also provided. The present disclosure further provides methods for producing an antibody composition, and provided herein are, for example, a method for producing an antibody composition having a target ADCC%, a method for producing an antibody composition having an ADCC% within a target ADCC% range or a specified ADCC%, and a method for producing an antibody composition within a target TAF% range.

[0031] Glycosylation, glycans, and methods for measuring glycans Many secreted proteins are post-translationally glycosylated, a process in which sugar moieties (e.g., glycans, sugars) are covalently attached to specific amino acids of the protein. In eukaryotic cells, two types of glycosylation occur: (1) N-linked glycosylation, in which the glycan is linked to an asparagine at the recognition sequence Asn-X-Thr / Ser (where "X" is any amino acid except proline), and (2) O-linked glycosylation, in which the glycan is linked to a serine or threonine. Regardless of the type of glycosylation (N-linked or O-linked), there is a wide range of glycan structures attached to each site (O or N), resulting in microheterogeneity of protein glycoforms.

[0032] All N-glycans share a common core sugar sequence: Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-X-Ser / Thr (Man3GlcNAc2Asn) and are classified into one of three types: (A) high-mannose (HM) or oligomannose (OM) types, consisting of two N-acetylglucosamine (GluNAc) moieties and multiple (e.g., 5, 6, 7, 8, or 9) mannose (Man) residues; (B) complex types, containing three or more GlcNAc moieties and any number of other sugar types; or (C) hybrid types, containing a Man residue on one branch and a GlcNAc at the base of the complex branch. Figure 1A (Stanley et al., Chapter 8: N-Glycans, Essentials of Glycobiology, 2014).nd ed., Cold Spring Harbor Laboratory Press; 2009) shows three types of N-glycans:

[0033] N-linked glycans generally contain one or more monosaccharides: galactose (Gal), N-acetylgalactosamine (GalNAc), galactosamine (GalN), glucose (GLc), N-acetylglucosamine (GlcNAc), glucosamine (GlcN), mannose (Man), N-acetylmannosamine (ManNAc), mannosamine (ManN), xylose (Xyl), N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxynonanoic acid (Kdn), fucose (Fuc), glucuronic acid (GLcA), iduronic acid (IdoA), galacturonic acid (GalA), and mannuronic acid (ManA). Commonly used symbols for such saccharides are shown in Figure 1A. Exemplary glycans and their individual properties are shown in Figure 1B.

[0034] N-linked glycosylation is initiated in the endoplasmic reticulum (ER) and results in a complex series of reactions that culminate in the attachment of a core glycan structure, essentially composed of two GlcNAc and three Man residues. The glycan complexes formed in the ER are then modified by enzymes in the Golgi apparatus. If the sugars are relatively inaccessible to enzymes, they generally remain in their original HM form. If the sugars are accessible to enzymes, many of the Man residues are cleaved off and the sugars are further modified, resulting in complex N-glycan structures. For example, mannosidase-1, located in the cis-Golgi, can cleave or hydrolyze HM glycans, while fucosyltransferase FUT-8, located in the medial-Golgi, fucosylates the glycans (Harue Imai-Nishiya (2007), BMC Biotechnology, 7:84).

[0035] Thus, the sugar composition and structural configuration of glycan structures varies depending, inter alia, on the glycosylation machinery in the ER and Golgi apparatus, the accessibility of the glycan structure to the enzymes of that machinery, the order of action of each enzyme, and the stage at which the protein is released from the glycosylation machinery.

[0036] Various methods are known in the art for assessing the glycans present in a glycoprotein-containing composition or for determining, detecting, or measuring the glycoform profile (e.g., glycoprofile) of a particular sample containing a glycoprotein. Suitable methods include, but are not limited to, cationic MALDI-TOF analysis, anionic MALDI-TOF analysis, weak anion exchange (WAX) chromatography, normal phase chromatography (NP-HPLC), exoglycosidase digestion, Bio-Gel P-4 chromatography, anion exchange chromatography, and one-dimensional nmr spectroscopy, and combinations thereof. See, for example, Mattu et al., JBC 273:2260-2272 (1998); Field et al., Biochem J 299(Pt 1):261-275 (1994); Yoo et al., MAbs 2(3):320-334 (2010); Wuhrer M. et al., Journal of Chromatography B, 2005, Vol. 825, Issue 2, pp. 124-133; Ruhaak LR, Anal Bioanal Chem, 2010, Vol. 397:3457-3481, and Geoffrey, R. Get. al. Analytical Biochemistry 1996, Vol. 240, pages 210-226. Example 1 herein also describes a suitable method for evaluating glycans present in a glycoprotein-containing composition, e.g., an antibody composition. The method of Example 1 describes an assay in which glycans attached to a glycosylated protein of a composition, e.g., an antibody of an antibody composition, are enzymatically cleaved from the protein (e.g., an antibody). The glycans are then separated by hydrophilic interaction liquid chromatography (HILIC), producing a chromatogram with several peaks. Each peak in the chromatogram represents the average distribution (abundance) of a different glycan. Two representative HILIC chromatograms containing peaks for different glycans are provided in Figures 2A and 2B. For these purposes, % peak area = peak area / total peak area × 100%, and % total peak area = total area of ​​sample / total area of ​​standard × 100%.Thus, the level of a particular glycan (or group of glycans) is reported as a %. For example, if an antibody composition is characterized as having a Man6 level of 30%, this means that 30% of all glycans cleaved from the antibodies in the composition are Man6.

[0037] The present disclosure, including the relationships and equations presented herein, relates to total nonfucosylated glycans, high mannose glycans, and nonfucosylated glycans of an antibody composition. As used herein, "total nonfucosylated glycans" or "TAF glycans" refers to the combined amount of high mannose (HM) glycans and nonfucosylated glycans. As used herein, the term "high mannose glycans" or "HM glycans" encompasses glycans containing 5, 6, 7, 8, or 9 mannose residues, abbreviated as Man5, Man6, Man7, Man8, and Man9, respectively. In various embodiments, the level of HM glycans is obtained by summing Man5%, Man6%, Man7%, Man8%, and Man9%. As used herein, the term "nonfucosylated glycans" or "AF glycans" refers to glycans lacking core fucose, e.g., α1,6-linked fucose on GlcNAc residues involved in amide bonds with Asn at N-glycosylation sites. Non-fucosylated glycans include, but are not limited to, A1G0, A2G0, A2G1a, A2G1b, A2G2, and A1G1M5. Additional non-fucosylated glycans include, for example, A1G1a, G0[H3N4], G0[H4N4], G0[H5N4], and FO-N[H3N3]. See, e.g., Reusch and Tejada, Glycobiology 25(12):1325-1334 (2015). The level of nonfucosylated glycans is, in various embodiments, obtained by summing A1G0%, A2G0%, A2G1A%, A2G1b%, A2G2, A1G1M5%, A1G1A%, G0[H3N4]%, G0[H4N4]%, G0[H5N4]%, and FO-N[H3N3]%.

[0038] In exemplary embodiments, the glycan level (e.g., glycan content, optionally expressed as a percentage, e.g., TAF glycan %, HM glycan %, AF glycan %) is determined (e.g., measured) by any of a variety of methods known in the art for assessing the glycans present in a glycoprotein-containing composition or for determining, detecting, or measuring the glycoform profile (e.g., glycoprofile) of a particular sample containing a glycoprotein. In exemplary cases, the glycan level (e.g., TAF glycan %, HM glycan %, AF glycan %) of an antibody composition is determined by measuring the level of such glycans in a sample of the antibody composition by a chromatography-based method, e.g., HILIC, and the glycan level is expressed as a percentage, as described herein. See, e.g., Example 1. In exemplary cases, the glycan level of an antibody composition is expressed as a percentage of all glycans cleaved from the antibodies of the composition. In various aspects, % TAF glycans are determined by calculating the sum of % high mannose glycans and % nonfucosylated glycans, where % high mannose glycans and % nonfucosylated glycans are determined by hydrophilic interaction chromatography (e.g., as described in Example 1). In various embodiments, glycan levels (e.g., % TAF glycans, % HM glycans, % AF glycans) are determined (e.g., measured) by measuring the levels of such glycans in samples of the antibody composition. In exemplary cases, at least five, at least six, at least seven, at least eight, or at least nine samples of the antibody composition are obtained, and the glycan levels (e.g., % TAF glycans, % HM glycans, % AF glycans) are determined (e.g., measured) for each sample. In various embodiments, the mean or average of % TAF glycans, % HM glycans, and / or % AF glycans is determined.

[0039] In exemplary embodiments, glycan levels (eg, % TAF glycans, % HM glycans, % AF glycans) are calculated using Equation A or Equation B, as further described herein.

[0040] ADCC The present disclosure, including the relationships and equations presented herein, relates the % total nonfucosylated glycans, or % high mannose glycans and % nonfucosylated glycans, of an antibody composition to the ADCC activity level (e.g., ADCC%) of the antibody composition.

[0041] The terms "ADCC" or "antibody-dependent cell-mediated cytotoxicity" or "antibody-dependent cellular cytotoxicity" refer to a mechanism by which effector cells of the immune system (e.g., natural killer cells (NK cells), macrophages, neutrophils, eosinophils) actively lyse target cells that have specific antibodies bound to their surface antigens. ADCC is part of the adaptive immune response and occurs when an antigen-specific antibody (1) binds to a surface antigen on a target cell via its antigen-binding region and (2) binds to an Fc receptor on the surface of an effector cell via its Fc region. Binding of the antibody's Fc region to the Fc receptor causes the effector cell to release cytotoxic factors that result in the death of the target cell (e.g., via cell lysis or degranulation).

[0042] Fc receptors are receptors found on the surface of B lymphocytes, follicular dendritic cells, NK cells, macrophages, neutrophils, eosinophils, basophils, platelets, and mast cells that bind to the Fc region of antibodies. Fc receptors are classified into different classes based on the type of antibody they bind to. For example, Fc gamma receptors are receptors for the Fc region of IgG antibodies, Fc alpha receptors are receptors for the Fc region of IgA antibodies, and Fc epsilon receptors are receptors for the Fc region of IgE antibodies.

[0043] The term "FcγR" or "Fc-gamma receptor" refers to a protein belonging to the IgG superfamily that is involved in inducing phagocytosis of opsonized cells or microorganisms. See, e.g., Fridman WH. Fc receptors and immunoglobulin binding factors. FASEB Journal. 5(12):2684-90 (1991). Members of the Fc gamma receptor family include FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b). The sequences of FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA and FcγRIIIB can be found in many sequence databases, for example the Uniprot database (www.uniprot.org) under accession numbers P12314 (FCGR1_HUMAN), P12318 (FCG2A_HUMAN), P31994 (FCG2B_HUMAN), P08637 (FCG3A_HUMAN) and P08637 (FCG3A_HUMAN), respectively.

[0044] "ADCC activity," "ADCC level," or "ADCC activity level" refers to the degree to which ADCC is activated or stimulated. Methods for measuring or determining the ADCC level of an antibody composition (including commercially available assays and kits for measuring or determining the ADCC level) are described in Yamashita et al., Scientific Reports 6: article number 19772 (2016), doi:10.1038 / srep19772; Kantakamalakul et al., "A novel EGFP-CEM-NKr flow cytometric method for measuring antibody dependent cell-mediated-cytotoxicity (ADCC) activity in HIV-1 infected individuals", J Immunol Methods 315 (Issues 1-2): 1-10; (2006); Gomez-Roman et al., "A simplified method for the rapid fluorometric assessment of antibody-dependent cell-mediated cytotoxicity", J Immunol Methods 308 (Issues 1-2): 53-67 (2006); Schnueriger et al., "Development of a quantitative, cell-line based assay to measure ADCC activity mediated by therapeutic antibodies”, Molec Immunology 38(Issues 12-13):1512-1517 (2011); and Mata et al., “Effects of cryopreservation on effector cells for antibody dependent cell-mediated cytotoxicity (ADCC) and natural killer (NK) cell activity in 51Cr-release and CD107a assays,” J Immunol Methods 406:1-9 (2014), all of which are incorporated herein by reference for all purposes. The term “ADCC assay” or “FcγR reporter gene assay” refers to an assay, kit, or method useful for determining the ADCC activity of an antibody. Exemplary methods for measuring or determining the ADCC activity of an antibody composition in the methods described herein include the ADCC assay described in Example 2 or the ADCC reporter assay commercially available from Promega (catalog numbers G7010 and G7018). In some embodiments, ADCC activity is measured or determined using a calcein release assay comprising one or more of the following: FcγRIIIa(158V)-expressing NK92(M1) cells as effector cells and HCC2218 cells or WIL2-S cells as target cells labeled with calcein-AM.

[0045] In exemplary aspects, the ADCC level of an antibody composition is determined by a quantitative cell-based assay that measures the ability of the antibodies of the antibody composition to mediate dose-dependent cytotoxicity in cells that express the antibody's antigen and engage the Fc-gamma RIIIA receptor on effector cells via the antibody's Fc domain. In various embodiments, the method involves the use of target cells bearing a detectable label that is released upon lysis of the target cells by the effector cells. The amount of detectable label released from the target cells is a measure of the ADCC activity of the antibody composition. In some aspects, the amount of detectable label released from the target cells is compared to a baseline. The ADCC level may also be reported as an ADCC % relative to a control ADCC %. In various aspects, the ADCC % is a relative ADCC %, which is optionally relative to a control ADCC %. In various aspects, the control ADCC % is the ADCC % of a reference antibody. In various aspects, the reference antibody is rituximab. In exemplary cases, the control ADCC % is within the range of about 60% to about 130%. Optionally, the % ADCC is determined by the assay described in Example 2.

[0046] The present disclosure relates to the TAF glycan content, HM glycan content, and / or AF glycan content of an antibody composition relative to the ADCC activity level of the antibody composition. As demonstrated herein, the TAF glycan %, HM glycan %, and / or AF glycan % of an antibody composition is related to the ADCC activity of the antibody composition. In various embodiments, based on a first model correlating TAF glycan content to ADCC activity level, (a) the ADCC activity level is calculated based on the TAF glycan content (e.g., measuring the TAF glycan content), or (b) the TAF glycan content is calculated based on the ADCC activity level (e.g., measuring the ADCC activity level). In various examples, given that a particular antibody of the antibody composition is to be produced, the target ADCC activity level or target range of ADCC activity levels is known. For example, the antibody may be a biosimilar of a reference antibody, and the target ADCC activity level or range is known for the reference antibody. In exemplary embodiments, the target TAF glycan content or the target range of TAF glycan content can be calculated based on a first model. In various examples, the first model is a linear regression model. In various examples, the first model is a simplified version of a linear regression model without a y-intercept. In various embodiments, the first model correlating ADCC with TAF glycan content is statistically significant, as evidenced by its low p-value. In various embodiments, the p-value is less than 0.0001.

[0047] In various embodiments, the first model correlates the ADCC activity level of the antibody composition to about 13.5%±0.5% for every 1% TAF glycan content present in the antibody composition (optionally, the antibodies of the antibody composition bind to an antigen comprising only one antibody binding site). In various embodiments, the first model correlates the ADCC activity level of the antibody composition to about 24.74%±0.625% for every 1% TAF glycan content present in the antibody composition, where optionally, the antibodies of the antibody composition bind to an antigen comprising only two antibody binding sites. In an exemplary embodiment, the first model correlates the ADCC activity level of the antibody composition to about 12%±1.5%×Q for every 1% TAF glycan content present in the antibody composition (where Q is the number of antibody binding sites present on the antigen). In an exemplary case, Q is 1, and optionally, the antibody is infliximab or a biosimilar thereof. Alternatively, Q is 2 and optionally the antibody is rituximab or a biosimilar thereof.

[0048] In various embodiments, a target range of ADCC activity levels is known, preselected, or predetermined, and the first model allows for calculation of a target range of TAF glycan content based on this target range of ADCC activity levels. In an exemplary case, the target range of TAF glycan content is m to n, where m is [ADCC min / 12Q] and ADCC min is the minimum of the target range of ADCC activity levels for the reference antibody, and n is [ADCC max ] / 12Q] and ADCC max is the maximum of the target range of ADCC activity levels for the reference antibody). In various examples, Q is 2. In various examples, the ADCC activity level predicted by the first model is about 24×TAF%. In various examples, the target range of TAF glycan content is m to n (where m is the maximum of the target range of ADCC activity levels for the reference antibody). min / 24], and n is [ADCC max] / 24). In various examples, Q is 1. In various embodiments, the ADCC activity level predicted by the first model is about 12×TAF%. In various examples, the target range of TAF glycan content is m to n (where m is [ADCC min / 12], and n is [ADCC max In various embodiments, the target range for TAF glycan content is m° to n°, where m° is [[ADCC min -y] / x] (where ADCC min is the minimum of the target range of ADCC activity levels), and n° is [[ADCC max -y] / x] (where ADCC max (where x is the maximum of the target range for ADCC activity levels). Optionally, x is about 20.4 to about 27.7 and y is about -11.4 to about 16.7. Alternatively, x is about 9.7 to about 15.2 and y is about -15.6 to about 34.2. In various embodiments, the target range for TAF glycan content is m' to n', where m' is the maximum of the target range for ADCC activity levels. min / x'] (where ADCC min is the minimum of the target range of ADCC activity levels), and n' is [ADCC max ] / x' (where ADCC max (where x' is the maximum of the target range of ADCC activity levels). Optionally, x' is about 24.1 to about 25.4. Alternatively, x' is about 13.0 to about 13.95. In various examples, the ADCC activity level of the antibody composition is about 13.5% ± 0.5% for every 1% of TAFs present in the antibody composition, where, optionally, the antibodies of the antibody composition bind to an antigen that comprises only one antibody binding site. In various embodiments, the ADCC activity level of the antibody composition is about 24.74% ± 0.625% for every 1% of TAFs present in the antibody composition, where, optionally, the antibodies of the antibody composition bind to an antigen that comprises only two antibody binding sites. In an exemplary embodiment, the ADCC activity level of the antibody composition is about 12% ± 1.5% × Q for every 1% of TAFs present in the antibody composition, where Q is the number of antibody binding sites present on the antigen. In an exemplary case, the reference antibody is infliximab. In an exemplary embodiment, the reference antibody is rituximab.

[0049] ADCC activity or % ADCC can be calculated using an equation relating % TAF glycans, % HM glycans, and / or % AF glycans to % ADCC activity of a given antibody composition. In various embodiments, the equation relates % TAF glycans to % ADCC. In an exemplary embodiment, the equation is: Equation A: Y=2.6+24.1×X [Equation A] where Y is ADCC % and X is TAF glycan %. is.

[0050] In various examples, an equation relates the % HM glycans and % AF glycans to the % ADCC of an antibody composition. In an exemplary embodiment, the equation is Equation B: Y = (0.24 + 27 × HM + 22.1 × AF) [Equation B] where Y is ADCC %, HM is high mannose glycan %, and AF is non-fucosylated glycan %. is.

[0051] In exemplary embodiments, the method includes determining (e.g., measuring) the TAF glycan %, and the determined (e.g., measured) TAF glycan % can be used to calculate the ADCC % according to Equation A. Thus, in exemplary cases, the method includes calculating the ADCC % of the antibody composition based on the determined (e.g., measured) TAF glycan % using Equation A. In various embodiments, the ADCC % calculated in such a manner is useful because it avoids the need to experimentally determine (e.g., measure) the ADCC % of the antibody composition.

[0052] In exemplary embodiments, the method includes determining (e.g., measuring) the % HM glycans and % AF glycans, and the determined (e.g., measured) % HM glycans and % AF glycans can be used to calculate the % ADCC according to Equation B. Thus, in exemplary cases, the method includes calculating the % ADCC of the antibody composition based on the determined (e.g., measured) % HM glycans and % AF glycans using Equation B. In various embodiments, the % ADCC calculated in this manner is useful because it avoids the need to experimentally determine (e.g., measure) the % ADCC of the antibody composition.

[0053] In various embodiments, the equations disclosed herein relating to ADCC % and TAF glycan %, HM glycan %, and / or AF glycan % can be re-expressed, e.g., such that the equations can be used to determine TAF glycan %. For example, Equation A can be re-expressed as follows: X=(Y-2.6) / 24.1 (where Y is ADCC % and X is TAF glycan %).

[0054] Alternatively, equation B can be re-expressed as: (Y-0.24)=27×HM+22.1×AF, or [(Y-0.24)-22.1×AF] / 27=HM, or [(Y-0.24)-27×HM] / 22.1=AF (where Y is ADCC %, HM is high mannose glycan %, and AF is non-fucosylated glycan %).

[0055] In an exemplary embodiment, the ADCC% is determined (e.g., measured), and the TAF% associated with the determined ADCC% can be calculated using the determined ADCC% in the reformulated Equation A. The TAF% calculated using Equation A and the determined ADCC% is useful for identifying a target TAF% to achieve a particular ADCC%. Also, in an exemplary embodiment, the ADCC% is determined (e.g., measured), and the determined ADCC% can be used in the reformulated Equation B to calculate the HM glycan% or AF glycan%.

[0056] In various embodiments, the ADCC% is a target ADCC%, and the method uses the target ADCC level to identify a target TAF glycan%. In various embodiments, the method includes maintaining glycosylation-competent cells in cell culture to produce an antibody composition having a target TAF% level, as calculated using Equation A. The method can include performing one or more downstream process steps with the antibody composition once the antibody composition achieves the target TAF level. In various embodiments, the method optionally includes confirming the actual TAF% of the antibody composition.

[0057] In various embodiments, the method includes selecting the antibody composition for one or more downstream processing steps if the Y calculated using the % TAF glycan determined using Equation A, or the Y calculated using the % HM glycan and % AF glycan determined using Equation B, is within the target ADCC range.

[0058] Methods for determining and / or monitoring product quality Based on these correlations, the product quality of the antibody composition can be determined and / or monitored. Thus, the present disclosure provides a method for determining the product quality of an antibody composition, wherein the ADCC activity level of the antibody composition is a criterion on which the product quality of the antibody composition is based. In an exemplary embodiment, the method includes (i) determining the total nonfucosylated (TAF) glycan content of a sample of the antibody composition; and (ii) determining that the product quality is acceptable and / or that the ADCC activity level standard is achieved if the TAF glycan content determined in (i) is within a target range. In an exemplary aspect, the target range of the TAF glycan content is based on (1) a target range of the ADCC activity level of a reference antibody and (2) a first model correlating the ADCC activity level of the antibody composition with the TAF glycan content of the antibody composition. In exemplary embodiments, the ADCC predicted by the first model is about 95% to about 105% of the ADCC predicted by the second model, where the second model correlates the ADCC activity level of the antibody composition with the HM glycan content of the antibody composition and the AF glycan content of the antibody composition.

[0059] Advantageously, the ADCC predicted by the first model is statistically significantly similar to the ADCC predicted by the second model. For example, the ADCC activity level predicted by the first model is about 95% to about 105% of the ADCC activity level predicted by the second model. The ADCC activity level predicted by the first model is optionally about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 101%, about 102%, about 103%, about 104%, or about 105% of the ADCC activity level predicted by the second model. In various examples, the ADCC activity level predicted by the first model is about 100% of the ADCC predicted by the second model. In certain embodiments, there is a one-to-one correspondence between the ADCC predicted by the first model and the ADCC predicted by the second model. In various examples, the first model and / or the second model are statistically significant. For example, the first model has a p-value of less than 0.0001 and / or the second model has a p-value of less than 0.0001. Optionally, each of the first model and the second model has a p-value of less than 0.0001.

[0060] In an exemplary embodiment, the ADCC activity level predicted by the first model is about 12Q x TAF%, where Q is the number of antibody binding sites on the antigen to which the antibody binds, and TAF% is the TAF glycan content of the antibody composition. In an exemplary case, the target range for TAF glycan content is m to n, where m is the [ADCC min / 12Q] (where ADCC min is the minimum of the target range of ADCC activity levels for the reference antibody), and n is [ADCC max ] / 12Q (where ADCC max is the maximum of the target range of ADCC activity levels for the reference antibody). In various examples, Q is 2. In various examples, the ADCC activity level predicted by the first model is about 24×TAF%. In various examples, the target range of TAF glycan content is m to n (where m is the maximum of the target range of ADCC activity levels for the reference antibody). min / 24], and n is [ADCC max] / 24). In various examples, the ADCC activity level predicted by the second model is about 27×HM% + about 22×AF%, where AF% is the AF glycan content of the antibody composition and HM% is the HM glycan content of the antibody composition. In various examples, Q is 1. In various embodiments, the ADCC activity level predicted by the first model is about 12×TAF%. In various examples, the target range for TAF glycan content is m to n, where m is [ADCC min / 12], and n is [ADCC max ] / 12). In various examples, the ADCC activity level predicted by the second model is about 14.8×HM% + about 12.8×AF%. Suitable alternative first and second models are described herein. In exemplary cases, the first model is any one of the models (e.g., formulas) described herein that correlate ADCC with TAF glycan content, including, but not limited to, formulas 1, 3, 5, and 7, and formula A. In exemplary cases, the second model is any one of the models (e.g., formulas) described herein that correlate ADCC with HM glycan content and AF glycan content, including, but not limited to, formulas 2, 4, 6, and 8, and formula B. For example, in various embodiments, the target range for TAF glycan content is m° to n°, where m° is [[ADCC min -y] / x] (where ADCC min is the minimum value of the target range of ADCC activity levels), and n° is [[ADCC max -y] / x] (where ADCC max is the maximum value of the target range for ADCC activity levels). Optionally, x is about 20.4 to about 27.7 and y is about -11.4 to about 16.7. Alternatively, x is about 9.7 to about 15.2 and y is about -15.6 to about 34.2. In various embodiments, the target range for TAF glycan content is m' to n', where m' is the maximum value of the target range for ADCC activity levels. min / x'] (where ADCC min is the minimum of the target range of ADCC activity levels), and n' is [ADCC max ] / x' (where ADCCmax (where x' is the maximum of the target range of ADCC activity levels). Optionally, x' is about 24.1 to about 25.4. Alternatively, x' is about 13.0 to about 13.95. In various examples, the ADCC activity level of the antibody composition is about 13.5% ± 0.5% for every 1% of TAFs present in the antibody composition, where, optionally, the antibodies of the antibody composition bind to an antigen that comprises only one antibody binding site. In various embodiments, the ADCC activity level of the antibody composition is about 24.74% ± 0.625% for every 1% of TAFs present in the antibody composition, where, optionally, the antibodies of the antibody composition bind to an antigen that comprises only two antibody binding sites. In an exemplary embodiment, the ADCC activity level of the antibody composition is about 12% ± 1.5% × Q for every 1% of TAFs present in the antibody composition, where Q is the number of antibody binding sites present on the antigen.

[0061] In an exemplary embodiment, the antibody binds to an antigen that comprises only one antibody binding site. In an exemplary case, the reference antibody is infliximab. In an exemplary embodiment, the antibody binds to an antigen that comprises only two antibody binding sites. In an exemplary embodiment, the reference antibody is rituximab.

[0062] In exemplary embodiments, the method is a quality control (QC) assay. In exemplary embodiments, the method is an in-process QC assay. In various embodiments, the sample is a sample of in-process material. In various examples, the TAF glycan content is determined before or after harvest. In exemplary cases, the TAF glycan content is determined after a chromatography step. Optionally, the chromatography step includes capture chromatography, intermediate chromatography, and / or polishing chromatography. In some embodiments, the TAF glycan content is determined after viral inactivation and neutralization, viral filtration, or buffer exchange. In various examples, the method is a lot release assay. In some embodiments, the sample is a sample of a manufacturing lot.

[0063] In various embodiments, the method further comprises selecting the antibody composition for downstream processing if the TAF glycan content determined in (i) is within the target range. If the TAF glycan content determined in (i) is not within the target range, in various embodiments, modifying one or more conditions of the cell culture to obtain an altered cell culture. In some embodiments, the method further comprises determining the TAF glycan content of a sample of the antibody composition obtained after modifying one or more conditions of the cell culture, for example, determining the TAF glycan content of a sample of the antibody composition of the altered cell culture. In various embodiments, if the TAF glycan content determined in (i) is not within the target range, the method further comprises (iii) modifying one or more conditions of the cell culture to obtain an altered cell culture, and (iv) determining the TAF glycan content of a sample of the antibody composition obtained from the altered cell culture. In an exemplary embodiment, if the TAF glycan content determined in (i) is not within the target range, the method further includes steps (iii) and (iv) until the TAF glycan content determined in (iv) is within the target range. In an exemplary case, an assay directly measuring the ADCC activity of the antibody composition is performed on the antibody composition only if the TAF glycan content determined in (i) is not within the target range, e.g., outside the target range. An assay directly measuring ADCC activity includes, for example, a cell-based assay that measures the release of a detectable agent upon lysis of antigen-expressing cells containing the detectable agent by effector cells that bind to an antibody that binds to both the antigen-expressing cells and the effector cells. In an exemplary case, an assay directly measuring the ADCC activity of the antibody composition is not performed on the antibody composition. In various embodiments, determining the TAF glycan content is the only step required to determine product quality with respect to the ADCC activity level standard. Without being bound by theory, the statistically significant correlation between the first model and the second model allows the TAF glycan content to indicate the ADCC activity level, and therefore an assay to directly measure the ADCC activity level is not required. Thus, directly measuring the ADCC activity level of the antibody composition is not necessary and is therefore not performed in various embodiments of the methods disclosed herein.

[0064] In various embodiments, the methods determine product quality with respect to an ADCC activity level criterion. In various embodiments, the ADCC activity level criterion is one of the acceptance criteria for the antibody composition. In various embodiments, the methods of the present disclosure are intended to ensure that a batch of drug product meets appropriate specifications and appropriate statistical quality control criteria, respectively, as a condition of approval and sale, pursuant to 21 CFR 211.165. In various embodiments, the methods of the present disclosure for determining product quality meet statistical quality control criteria, including appropriate acceptance levels and / or appropriate rejection levels. Terms including, but not limited to, "acceptance criteria," "lot," and "in-process" are ascribed their meanings in accordance with Title 21 Code of Federal Regulations (CFR), Section 210.3.

[0065] The present disclosure also provides a method for monitoring the product quality of an antibody composition, wherein the ADCC activity level of the antibody composition is a criterion on which the product quality of the antibody composition is based. In an exemplary embodiment, the method includes determining the product quality of the antibody composition using a first sample obtained at a first time point and a second sample taken at a second time point different from the first time point according to the method of the present disclosure. In various examples, each of the first sample and the second sample is a sample of in-process material. In various aspects, the first sample is a sample of in-process material, and the second sample is a sample of a manufacturing lot. Optionally, the first sample is a sample obtained before modifying one or more conditions of the cell culture, and the second sample is a sample obtained after modifying one or more conditions of the cell culture. In an exemplary case, the TAF glycan content is determined for each of the first sample and the second sample. Additional samples can be obtained to determine the product quality of the antibody composition and to determine the TAF glycan content. The product quality of the antibody composition depends on whether the TAF glycan content is within a target range. In an exemplary embodiment, the target range of TAF glycan content is based on (1) a target range of ADCC activity levels of a reference antibody and (2) a first model correlating the ADCC activity level of an antibody composition with the TAF glycan content of the antibody composition. In an exemplary embodiment, the ADCC predicted by the first model is about 95% to about 105% of the ADCC predicted by a second model, where the second model correlates the ADCC activity level of an antibody composition with the HM glycan content of the antibody composition and the AF glycan content of the antibody composition.

[0066] Methods for producing antibody compositions The present disclosure provides methods for producing an antibody composition. In exemplary embodiments, the method includes determining the product quality of the antibody composition, wherein the product quality of the antibody composition is determined according to the methods of the present disclosure. Optionally, the method includes determining the TAF glycan content of a sample of the antibody composition, where the sample is a sample of in-process material. In various examples, the method includes determining the product quality of the antibody composition as acceptable and / or achieving the ADCC activity level standard if the TAF glycan content determined in (i) is within a target range as defined herein. In exemplary embodiments, the target range of the TAF glycan content is based on (1) a target range of the ADCC activity level of a reference antibody and (2) a first model correlating the ADCC activity level of the antibody composition with the TAF glycan content of the antibody composition. In exemplary embodiments, the ADCC predicted by the first model is about 95% to about 105% of the ADCC predicted by the second model, where the second model correlates the ADCC activity level of the antibody composition with the HM glycan content of the antibody composition and the AF glycan content of the antibody composition. In various embodiments, if the TAF glycan content determined in (i) is not within the target range, the method further includes (iii) modifying one or more conditions of the cell culture to obtain an modified cell culture, and (iv) determining the TAF glycan content of a sample of the antibody composition obtained from the modified cell culture, optionally repeating steps (iii) and (iv) until the TAF glycan content is within the target range. In various examples, the sample is a sample of a cell culture containing cells expressing an antibody of the antibody composition. In various examples, one or more conditions of the cell culture are modified to modify the TAF glycan content. In various embodiments, the TAF glycan content of the antibody composition is achieved by modifying the AF glycan content. In exemplary embodiments, one or more conditions of cell culture are modified to modify the AF glycan content of the antibody composition.In exemplary embodiments, one or more conditions mainly modify the AF glycan content.In various examples, one or more conditions modify the AF glycan content and do not modify the HM glycan content.In exemplary embodiments, the method includes achieving a TAF glycan content of the antibody composition by modifying the HM glycan content. Optionally, one or more conditions of the cell culture are modified to modify the HM glycan content of the antibody composition. In some examples, the one or more conditions modify primarily the HM glycan content. In some embodiments, the one or more conditions modify the HM glycan content but not the AF glycan content. In various examples, the method includes repeating the modification of the nonfucosylated (AF) glycan content and / or repeating the modification of the high mannose (HM) glycan content until the TAF glycan content is within a target range.

[0067] In an exemplary embodiment, a method for producing an antibody composition includes (i) determining the total nonfucosylated (TAF) glycan content of a sample of the antibody composition; and (ii) selecting the antibody composition for downstream processing based on the TAF glycan content determined in (i). In various aspects, the sample is taken from a cell culture containing cells expressing an antibody of the antibody composition. In various examples, the method further includes modifying the TAF glycan content of the antibody composition and determining the modified TAF glycan content. Optionally, one or more conditions of the cell culture are modified to modify the TAF glycan content. In an exemplary aspect, the method includes repeating the modification until the TAF glycan content falls within a target range. In an exemplary case, the target range is based on a target range of ADCC activity levels of the antibody. Without being bound by theory, TAF glycan content correlates with the ADCC activity level of an antibody composition; therefore, the ADCC activity level of an antibody composition can be predicted based on the TAF glycan content of the antibody composition. The ADCC activity level of an antibody composition can be a criterion worth considering when determining whether the antibody composition should be selected for downstream processing. Thus, in various embodiments, the method includes: (i) determining the TAF glycan content of a sample of the antibody composition; (ii) determining the ADCC activity level of the antibody composition based on the TAF glycan content determined in (i); and, optionally, (iii) selecting the antibody composition for downstream processing if the ADCC level of the antibody composition determined in (ii) is within a target range of ADCC activity levels. In various embodiments, the target range of ADCC activity levels is known for the antibodies of the antibody composition. In various embodiments, the antibodies of the antibody composition are biosimilars of a reference antibody. In various examples, the target range of TAF glycan content is based on or determined (e.g., calculated) based on a known target range of ADCC activity levels. Thus, in an exemplary embodiment, the method includes: (i) determining the TAF glycan content of a sample of an antibody composition; and (ii) selecting the antibody composition for downstream processing if the TAF glycan content determined in (i) is within a target range.When the method further comprises modifying the TAF glycan content of the antibody composition, the method, in various examples, comprises modifying the nonfucosylated (AF) glycan content to modify the TAF glycan content. Optionally, one or more conditions of the cell culture are modified to modify the AF glycan content of the antibody composition, which then modifies the TAF glycan content. Alternatively, or in addition, when the method comprises modifying the TAF glycan content of the antibody composition, the method, in various examples, comprises modifying the high mannose (HM) glycan content to modify the TAF glycan content. Optionally, one or more conditions of the cell culture are modified to modify the HM glycan content of the antibody composition, which then modifies the TAF glycan content. In exemplary embodiments, one or more conditions primarily modify the AF glycan content. In exemplary cases, one or more conditions primarily modify the HM glycan content. In exemplary embodiments, one or more conditions modify the AF glycan content but not the HM glycan content. In exemplary cases, the one or more conditions modify HM glycan content but do not modify AF glycan content. The method optionally includes repeating the modification of nonfucosylated (AF) glycan content and / or repeating the modification of high mannose (HM) glycan content until the TAF glycan content is within the target range. In exemplary embodiments, the antibody of the antibody composition is an IgG, optionally an IgG1. In various embodiments, the target range for TAF glycan content is m to n, where m is [[ADCC. min -y] / x] (where ADCC min is the minimum of the target range of ADCC activity levels), and n is [[ADCC max -y] / x] (where ADCC max (where x is the maximum of the target range for ADCC activity levels). Optionally, x is about 20.4 to about 27.7 and y is about -11.4 to about 16.7. Alternatively, x is about 9.7 to about 15.2 and y is about -15.6 to about 34.2. In various embodiments, the target range for TAF glycan content is m' to n', where m' is the maximum of the target range for ADCC activity levels. min / x'] (where ADCC min is the minimum of the target range of ADCC activity levels), and n' is [ADCC max] / x' (where ADCC max (where x' is the maximum of the target range of ADCC activity levels). Optionally, x' is about 24.1 to about 25.4. Alternatively, x' is about 13.0 to about 13.95. In various examples, the ADCC activity level of the antibody composition is about 13.5%±0.5% for every 1% of TAFs present in the antibody composition, and optionally, the antibodies of the antibody composition bind to an antigen that comprises only one antibody binding site. In various embodiments, the ADCC activity level of the antibody composition is about 24.74%±0.625% for every 1% of TAFs present in the antibody composition, and optionally, the antibodies of the antibody composition bind to an antigen that comprises only two antibody binding sites. In an exemplary embodiment, the ADCC activity level of the antibody composition is about 12%±1.5%×Q for every 1% of TAFs present in the antibody composition, where Q is the number of antibody binding sites present on the antigen. In an exemplary case, Q is 1, and optionally, the antibody is infliximab or a biosimilar thereof. Optionally, Q is 2, and optionally the antibody is rituximab or a biosimilar thereof.

[0068] The disclosed methods for producing an antibody composition include modifying the total nonfucosylated (TAF) glycan content of an antibody composition produced by cells in a cell culture. In various examples, one or more conditions of the cell culture are modified to modify the TAF glycan content. In various aspects, the method includes determining the modified TAF glycan content. Optionally, the modification is repeated until the determined TAF glycan content is within a target TAF range. Without being bound by theory, TAF glycan content can be modified by changing the nonfucosylated (AF) glycan content or the high mannose (HM) content, or a combination thereof, as each affects TAF glycan content. Thus, these methods advantageously allow for achieving a target range of TAF glycan content in multiple ways. For example, one or more conditions of the cell culture are modified to modify the AF glycan content to modify the TAF glycan content. Alternatively, one or more conditions of the cell culture are modified to modify the HM glycan content to modify the TAF glycan content. In various examples, one or more conditions of the cell culture are modified to modify the AF glycan content and the HM glycan content to modify the TAF glycan content. Accordingly, the present disclosure further provides a method for modifying the total nonfucosylated (TAF) glycan content of an antibody composition produced by cells of a cell culture. In exemplary embodiments, the method includes modifying the AF glycan content. In exemplary embodiments, the method includes modifying the HM glycan content. In various aspects, the method includes (i) determining the nonfucosylated (AF) glycan content and the high mannose (HM) glycan content of a sample of the antibody composition; (ii) determining a target range for the AF glycan content based on a target range of ADCC activity levels of antibodies in the antibody composition, assuming that the HM glycan content is constant; and (iii) selecting the antibody composition for downstream processing if the AF glycan content is within the target range of AF glycan content.In various examples, the method includes (i) determining the nonfucosylated (AF) glycan content and high mannose (HM) glycan content of a sample of an antibody composition; (ii) determining a target range of HM glycan content based on a target range of ADCC activity levels of antibodies in the antibody composition, assuming the AF glycan content is constant; and (iii) selecting the antibody composition for downstream processing if the HM glycan content is within the target range of HM glycan content. In various examples, the method includes (i) determining the AF glycan content and HM glycan content of a sample of the antibody composition, and (ii) determining a target range of AF glycan content based on the HM glycan content determined in (i), and (iii) modifying the AF glycan content until the AF glycan content is within the target range of AF glycan content, wherein the HM glycan content is not modified. Alternatively, the method includes (i) determining the AF glycan content and HM glycan content of a sample of an antibody composition; (ii) determining a target range for the HM glycan content based on the AF glycan content determined in (i); and (iii) modifying the HM glycan content until the HM glycan content falls within the target range for the HM glycan content, wherein the AF glycan content is not modified. In an exemplary embodiment, a model correlating the ADCC activity level of an antibody composition with the TAF glycan content of the antibody composition predicts an ADCC activity level substantially the same as the level predicted by a model correlating ADCC with HM and AF glycan content. Suitable methods for modifying the AF glycan content and / or the HM glycan content are known in the art. For example, WO 2019 / 191150 teaches a method for modifying the level of nonfucosylated glycans in an antibody composition and a method for modifying the level of high mannose glycans in an antibody composition. In such methods, one or more conditions of the cell culture, eg, pH, fucose concentration, glucose concentration, are modified to achieve a desired level of AF glycans and / or HM glycans.Also, WO 2013 / 114164, WO 2016 / 089919, WO 2013 / 114245, WO 2015 / 128793, and WO 2013 / 114167, U.S. Patent Application Publication No. 2014 / 0356910, and Konno et al., Cytotech 64:249-265 (2012) each teach methods for obtaining increased defucosylated glycans.

[0069] In an exemplary embodiment, the method of producing an antibody composition includes (i) determining the % total nonfucosylated (TAF) glycans of the antibody composition, (ii) determining the % total nonfucosylated (TAF) glycans of the antibody composition based on Equation A: Y=2.6+24.1×X [Equation A] where Y is the ADCC % and X is the TAF glycan % determined in step (i). calculating the antibody-dependent cellular cytotoxicity (ADCC) % of the antibody composition based on the TAF % using the (iii) if Y is within the target ADCC % range, selecting the antibody composition for one or more downstream processing steps.

[0070] In an exemplary embodiment, the method of producing an antibody composition includes (i) determining the % high mannose glycans and % non-fucosylated glycans of the antibody composition; (ii) calculating the % high mannose glycans and % non-fucosylated glycans of the antibody composition based on Equation B: Y = (0.24 + 27 × HM + 22.1 × AF) [Equation B] where Y is ADCC %, HM is high mannose glycan % determined in step (i), and AF is non-fucosylated glycan % determined in step (i). calculating the % antibody-dependent cellular cytotoxicity (ADCC) of the antibody composition based on the % high mannose glycans and the % non-fucosylated glycans using the following: (iii) if Y is within the target ADCC % range, selecting the antibody composition for one or more downstream processing steps.

[0071] In an exemplary embodiment, the method for producing an antibody composition with a target ADCC % comprises: (i) calculating the ADCC percentage of an antibody composition according to Equation A: Y=2.6+24.1×X [Equation A] where Y is the target ADCC % and X is the target TAF glycan %. calculating the target total nonfucosylated (TAF) glycan % for the target ADCC % using (ii) maintaining glycosylation-competent cells in cell culture to produce an antibody composition having a target TAF glycan %, X.

[0072] In an exemplary embodiment, the method for producing an antibody composition having a target ADCC % comprises: (i) reacting an antibody composition of Formula B: Y = (0.24 + 27 × HM + 22.1 × AF) [Equation B] where Y is the target ADCC %, HM is the target high mannose glycan %, and AF is the target non-fucosylated glycan %. calculating the target nonfucosylated glycan % and the target high mannose glycan % for the target ADCC % using the and (iii) maintaining the glycosylation-competent cells in cell culture to produce an antibody composition having a target % high mannose glycans and a target % nonfucosylated glycans.

[0073] In exemplary embodiments, the target ADCC % is within a target ADCC % range. Optionally, the target ADCC % range is greater than or equal to about 40 and less than about 170 or about 175. For example, the target ADCC % range may be about 40 to about 175, about 50 to about 175, about 60 to about 175, about 70 to about 175, about 80 to about 175, about 90 to about 175, about 100 to about 175, about 110 to about 175, about 120 to about 175, about 130 to about 175, about 140 to about 175, about 150 to about 175, or about 160 to about 175. 5, or about 170 to about 175, or about 40 to about 170, about 40 to about 160, about 40 to about 150, about 40 to about 140, about 40 to about 130, about 40 to about 120, about 40 to about 110, about 40 to about 100, about 40 to about 90, about 40 to about 80, about 40 to about 70, about 40 to about 60, or about 40 to about 50. In some embodiments, the target ADCC% range is about 44 or more and less than about 165 (e.g., about 45 to about 165, about 50 to about 165, about 60 to about 165, about 100 to about 165, about 45 to about 100, about 45 to about 60, about 100 to about 150, about 100 to about 125, about 125 to about 150). In various examples, the target ADCC % range is from about 60 to about 130.

[0074] In exemplary cases, the target ADCC % range depends on Y in Equation A or Equation B. For example, in some embodiments, the target ADCC % range is Y±20, optionally Y±17 or Y±18. In some embodiments, the target ADCC % range is Y±17 in Equation A and Y±18 in Equation B.

[0075] The target ADCC % range can be any one of those described for the antibody composition, see e.g., composition.

[0076] In exemplary embodiments, the method of producing an antibody composition optionally having an ADCC%, Y, of about 40 or greater and about 170 or less further comprises (i) determining the % total nonfucosylated (TAF) glycans, X, of the antibody composition, and (ii) selecting the antibody composition for one or more downstream processing steps if X is equal to (Y-2.6) / 24.1. In various aspects, X is greater than or equal to about 1.55 and less than about 6.95, optionally between about 1.6 and about 6.9, or between about 1.6 and about 6.5, between about 1.6 and about 6.0, between about 1.6 and about 5.5, between about 1.6 and about 5.0, between about 1.6 and about 4.5, between about 1.6 and about 4.0, between about 1.6 and about 3.5, between about 1.6 and about 3.0, between about 1.6 and about 4.5, between about 1.6 and about 5.0, between about 1.6 and about 6.5 ... Y is about 2.5, about 1.6 to about 2.0, about 2.0 to about 6.95, about 2.5 to about 6.95, about 3.0 to about 6.95, about 3.5 to about 6.95, about 4.0 to about 6.95, about 4.5 to about 6.95, about 5.0 to about 6.95, about 5.5 to about 6.95, about 6.0 to about 6.95, or about 6.5 to about 6.95. In various embodiments, Y is about 44 or greater and about 165 or less, and optionally, X is about 1.72 to about 6.74.

[0077] In an exemplary embodiment, a method is for producing an antibody composition having an ADCC %, Y, the method comprising: (i) determining the % total nonfucosylated (TAF) glycans, X, of the antibody composition; and (ii) selecting the antibody composition for one or more downstream processing steps if X is equal to (Y-2.6) / 24.1, where, optionally, X is greater than or equal to about X-0.4 and less than or equal to about X+0.4, and the ADCC % is greater than or equal to about Y-17 and less than or equal to Y+17. In various examples, X is X±0.3, X±0.2, or X±0.1, and / or Y is Y±16, Y±15, Y±12, Y±9, Y±6, Y±3, ​​Y±2, or Y±1.

[0078] In an exemplary embodiment, a method is a method of producing an antibody composition having an ADCC %, the method comprising the steps of (i) determining the % nonfucosylated glycans and % high mannose glycans of the antibody composition, and (ii) determining whether AF and HM are soluble in Equation B: Y = (0.24 + 27 × HM + 22.1 × AF) [Equation B] where Y is ADCC %, HM is high mannose glycan % determined in step (i), and AF is non-fucosylated glycan % determined in step (i). selecting the antibody composition for one or more downstream process steps if the antibody composition is related to Y according to

[0079] In exemplary cases, Y is greater than or equal to about 40 and less than about 175, or any subrange described herein, optionally from about 41 to about 171. In some embodiments, AF is from about 1 to about 4, or from about 1 to about 3, or from about 1 to about 2, and HM is from about 40 to about 175, or any subrange thereof. Optionally, Y is from about 30 to about 185, optionally from about 32 to about 180, HM is from about 1 to about 4, and AF is from about 30 to about 185. In exemplary embodiments, the ADCC % of the antibody composition is within a range defined by Y. Optionally, the ADCC % of the antibody composition is within a range of Y ± 18. In exemplary embodiments, AF is from about 1 to about 4. In some embodiments, the % high mannose glycans is within a range defined by HM, optionally within a range of HM ± 1. Optionally, HM is from about 1 to about 4. In some examples, the % nonfucosylated glycans is a value within a range defined by AF, optionally, the range is AF±1.

[0080] Methods for producing an antibody composition having a TAF glycan content within a target range are provided, the methods comprising: (i) measuring the ADCC activity level of a series of samples containing various glycoforms of an antibody; (ii) determining the % TAF glycan for each of the series of samples; (iii) creating a model correlating the ADCC activity level to the TAF glycan content; (iv) determining the ADCC activity level of the antibody composition and then calculating the TAF glycan content using the model; or determining the TAF glycan content of the antibody composition and calculating the ADCC activity level using the model; and (v) selecting the antibody composition for one or more downstream processing steps if the TAF glycan content calculated in step (iv) is within the target range of TAF glycan content or if the ADCC activity level calculated in step (iv) is within the target range of ADCC activity levels. In some embodiments, the ADCC activity level is measured essentially as described in Example 2. In some embodiments, the TAF glycan content is measured essentially as described in Example 1. The model can be created by any method known in the art. In various embodiments, the model is a linear regression model, constructed essentially as described in Example 3 and / or Example 5.

[0081] A method for producing an antibody composition having an ADCC % within a target range is provided, the method comprising: i. measuring the ADCC% of a series of samples containing various glycoforms of the antibody; ii. determining the % total nonfucosylated (TAF) glycans for each sample in the series; iii. determining the linear equation for a best-fit line of a graph plotting the % ADCC measured in step (i) as a function of the % TAF glycan determined in step (ii) for each sample in the series; iv. determining the TAF% of the antibody composition and then calculating the ADCC% using the linear equation of step (iii); and v. If the ADCC % calculated in step (iv) is within the target ADCC % range, selecting the antibody composition for one or more downstream processing steps. Includes.

[0082] Also provided is a method for producing an antibody composition having a TAF% within a target range, the method comprising: i. measuring the ADCC% of a series of samples containing various glycoforms of the antibody; ii. determining the % total nonfucosylated (TAF) glycans for each sample in the series; ii. determining the % total nonfucosylated (TAF) glycans for each sample in the series; iii. determining the linear equation for a best-fit line of a graph plotting the % ADCC measured in step (i) as a function of the % TAF glycan determined in (ii) for each sample in the series; iv. determining the ADCC% of the antibody composition and then calculating the TAF% using the linear equation of step (iii); and v. If the TAF% calculated in step (iv) is within the target TAF% range, selecting the antibody composition for one or more downstream processing steps. Includes.

[0083] An exemplary method for carrying out the first three steps is described in further detail in Example 3.

[0084] The present disclosure further provides methods of producing an antibody composition having a TAF glycan content within a target range, the method comprising determining a target range of TAF glycan content, and selecting the antibody composition for one or more downstream process steps if the TAF glycan content is within the target range of TAF glycan content. In various embodiments, the target range of TAF glycan content is m to n, where m is [[ADCC min -y] / x] (where ADCC min is the minimum of the target range of ADCC activity levels), and n is [[ADCC max -y] / x] (where ADCC max(where x is the maximum of the target range for ADCC activity levels). Optionally, x is about 20.4 to about 27.7 and y is about -11.4 to about 16.7. Alternatively, x is about 9.7 to about 15.2 and y is about -15.6 to about 34.2. In various embodiments, the target range for TAF glycan content is m' to n', where m' is the maximum of the target range for ADCC activity levels. min / x'] (where ADCC min is the minimum of the target range of ADCC activity levels), and n' is [ADCC max ] / x' (where ADCC max is the maximum of the target range of ADCC activity levels. Optionally, x' is from about 24.1 to about 25.4. Alternatively, x' is from about 13.0 to about 13.95.

[0085] The present disclosure further provides a method for producing an antibody composition having a TAF% within a target range, the method comprising the steps of: (i) generating a linear equation for a best-fit graph by plotting the ADCC% and TAF glycan% of a series of at least five reference antibody compositions produced under cell culture conditions (each reference antibody composition has the same amino acid sequence as the antibody composition), (ii) selecting a target TAF glycan% range based on the linear equation generated in step (i) and the desired ADCC activity%, (iii) culturing the antibody composition under cell culture conditions, (iv) purifying the antibody composition, (v) sampling the antibody composition to determine the TAF%, and (vi) determining whether the TAF% of the antibody composition is within the target TAF% range of step (ii). In an exemplary embodiment, the method further comprises selecting the antibody composition for one or more downstream process steps if the TAF% calculated in step (v) is within the target TAF% range.

[0086] The present disclosure also provides a method for determining the % antibody-dependent cellular cytotoxicity (ADCC) of an antibody composition.

[0087] In an exemplary embodiment, the method comprises: i. determining the % total nonfucosylated (TAF) glycans of the antibody composition; ii.Equation A: Y=2.6+24.1×X [Equation A] where Y is the ADCC % and X is the TAF glycan % determined in step (i). calculating the ADCC% of the antibody composition based on the TAF% using Includes.

[0088] Further provided is a method for determining the antibody-dependent cellular cytotoxicity (ADCC) % of an antibody composition. In an exemplary embodiment, the method comprises: i. determining the % high mannose glycans and % nonfucosylated glycans of the antibody composition; ii.Equation B: Y = (0.24 + 27 × HM + 22.1 × AF) [Equation B] where Y is ADCC %, HM is high mannose glycan % determined in step (i), and AF is non-fucosylated glycan % determined in step (i). calculating the ADCC % of the antibody composition based on the % high mannose glycans and the % non-fucosylated glycans using Includes.

[0089] In various embodiments, the method further comprises selecting the antibody composition for one or more downstream processing steps if Y is within the target ADCC % range.

[0090] process steps The % total nonfucosylated (TAF) glycans, % high mannose glycans, and / or % nonfucosylated glycans can be determined (e.g., measured) to provide more complete information about the % antibody-dependent cell-mediated cytotoxicity (ADCC) of the antibody composition. The determining step (e.g., measuring step) can be performed at any step during manufacturing. In particular, the measurement can be performed before harvesting or after harvesting, at any stage during downstream processing, such as following any chromatographic unit operation, including capture chromatography, intermediate chromatography, and / or polish chromatography unit operations; viral inactivation and neutralization, viral filtration; and / or final formulation. In various embodiments, the % total nonfucosylated (TAF) glycans, % high mannose glycans, and / or % nonfucosylated glycans are determined (e.g., measured) in real time, near real time, and / or post-hoc. Monitoring and measuring can be performed using known techniques and commercially available equipment.

[0091] In various embodiments of the present disclosure, the step of determining (e.g., measuring) the % total nonfucosylated (TAF) glycans, % high mannose glycans, and / or % nonfucosylated glycans is performed after the harvesting step. As used herein, the term "harvest" refers to a step in which the cell culture medium containing the recombinant protein of interest is collected and separated from at least the cells of the cell culture. Harvesting can be performed continuously. In some embodiments, harvesting is performed using centrifugation and may further include precipitation, filtration, etc. In various embodiments, the determining step is performed after a chromatography step, optionally after Protein A chromatography. In various embodiments, the determining step is performed after harvesting and after a chromatography step, optionally after Protein A chromatography.

[0092] In various embodiments, the antibody composition of the present disclosure is selected for further processing steps, e.g., one or more downstream processing steps, where the selection is based on a particular parameter, e.g., ADCC %, % total nonfucosylated (TAF) glycans, % high mannose glycans, and / or % nonfucosylated glycans. In various examples, the methods disclosed herein include using the antibody composition in further processing steps, e.g., one or more downstream processing steps, based on a particular parameter, e.g., ADCC %, % total nonfucosylated (TAF) glycans, % high mannose glycans, and / or % nonfucosylated glycans. In various examples, the methods disclosed herein include performing further processing steps, e.g., one or more downstream processing steps, using the antibody composition based on a particular parameter, e.g., ADCC %, % total nonfucosylated (TAF) glycans, % high mannose glycans, and / or % nonfucosylated glycans.

[0093] In exemplary cases, the one or more downstream process steps are any process steps that occur after (or downstream of) the process step that determines (e.g., measures) the % total nonfucosylated (TAF) glycans, % high mannose glycans, and / or % nonfucosylated glycans. For example, if the % total nonfucosylated (TAF) glycans, % high mannose glycans, and / or % nonfucosylated glycans are determined (e.g., measured) at the time of harvest, the one or more downstream process steps are any process steps that occur after (or downstream of) the harvesting step, and in various embodiments include: a dilution step, a loading step, a filtration step, a formulation step, a chromatography step, a viral filtration step, a viral inactivation step, or a combination thereof. Also, for example, if the % total nonfucosylated (TAF) glycans, % high mannose glycans, and / or % nonfucosylated glycans are determined (e.g., measured) after a chromatography step, e.g., after Protein A chromatography, the one or more downstream process steps are any process steps that occur after (or downstream of) the chromatography step, and in various embodiments include: a dilution step, a loading step, a filtration step, a formulation step, a further chromatography step, a viral filtration step, a viral inactivation step, or a combination thereof. In exemplary cases, the further chromatography step is an ion exchange chromatography step (e.g., a cation exchange chromatography step or an anion exchange chromatography step).

[0094] Chromatography steps / types used during downstream processing include capture or affinity chromatography, which are used to separate the recombinant product from other proteins, aggregates, DNA, viruses, and other such impurities. In an exemplary case, the first chromatography step is performed using Protein A (e.g., Protein A bound to a resin). In various embodiments, intermediate and polish chromatography further purify the recombinant protein to remove bulk contaminants, adventitious viruses, trace impurities, aggregates, isoforms, etc. Chromatography can be performed in a bind-and-elute mode, where the recombinant protein of interest binds to the chromatography medium and impurities flow through, or in a flow-through mode, where impurities bind and the recombinant protein flows through. Examples of such chromatographic methods include ion exchange chromatography (IEX), such as anion exchange chromatography (AEX) and cation exchange chromatography (CEX); hydrophobic interaction chromatography (HIC); mixed-mode or multimodal chromatography (MM), hydroxyapatite chromatography (HA); reversed-phase chromatography and gel filtration.

[0095] In various embodiments, the downstream process is a viral inactivation process. Enveloped viruses have capsids surrounded by a lipoprotein membrane or "envelope" and are therefore susceptible to inactivation. In various examples, viral inactivation processes include heat inactivation / pasteurization, pH inactivation, UV and gamma irradiation, the use of high-intensity broad-spectrum white light, the addition of chemical inactivators, surfactants, and solvent / detergent treatments.

[0096] In various embodiments, the downstream process is a virus filtration process. In various embodiments, the virus filtration process comprises removing non-enveloped viruses. In various embodiments, the virus filtration process comprises the use of a microfilter or nanofilter.

[0097] In various embodiments, downstream process steps include one or more formulation steps. In various embodiments, after completion of the chromatography steps, the purified recombinant protein is buffer exchanged into a formulation buffer. In an exemplary embodiment, buffer exchange is performed using ultrafiltration and diafiltration (UF / DF). In an exemplary embodiment, the recombinant protein is buffer exchanged into a desired formulation buffer using diafiltration and concentrated to a desired final formulation concentration using ultrafiltration. In various embodiments, additional stability-enhancing excipients are added following the UF / DF formulation step.

[0098] Recombinant glycosylated proteins The methods of the disclosure relate to compositions comprising a recombinant glycosylated protein. In various aspects, the recombinant glycosylated protein has the formula: Asn-Xaa1-Xaa2 (wherein Xaa1 is any amino acid except Pro, and Xaa2 is Ser or Thr) The amino acid sequence includes one or more N-glycosylation consensus sequences.

[0099] In exemplary embodiments, the recombinant glycosylated protein comprises a crystallizable fragment (Fc) polypeptide. The term "Fc polypeptide," as used herein, includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Truncated forms of such polypeptides containing the hinge region that promotes dimerization are also included. Fusion proteins comprising an Fc portion (and oligomers formed therefrom) offer the advantage of easy purification by affinity chromatography using a Protein A or Protein G column. In exemplary embodiments, the recombinant glycosylated protein comprises the Fc of an IgG, e.g., a human IgG. In exemplary aspects, the recombinant glycosylated protein comprises the Fc of an IgG1 or IgG2. In exemplary aspects, the recombinant glycosylated protein is an antibody, an antibody protein product, a peptibody, or an Fc-fusion protein.

[0100] In an exemplary embodiment, the recombinant glycosylated protein is an antibody. As used herein, the term "antibody" refers to a protein having the conventional immunoglobulin structure, including heavy and light chains, and including a variable region and a constant region. For example, an antibody may be an IgG, which is a "Y-shaped" structure consisting of two identical pairs of polypeptide chains, each pair having one "light" chain (generally having a molecular weight of about 25 kDa) and one "heavy" chain (generally having a molecular weight of about 50-70 kDa). An antibody has a variable region and a constant region. In the IgG structure, the variable region is generally about 100-110 or more amino acids, contains three complementarity-determining regions (CDRs), is primarily responsible for antigen recognition, and differs substantially among other antibodies that bind to different antigens. See, e.g., Janeway et al., "Structure of the Antibody Molecule and the Immunoglobulin Genes," Immunobiology: The Immune System in Health and Disease, 4 th ed. Elsevier Science Ltd. / Garland Publishing, (1999).

[0101] Briefly, in an antibody framework, CDRs are embedded within the framework of the heavy and light chain variable regions, where they constitute the regions that play a major role in antigen binding and recognition. A variable region comprises at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md.; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; see also Chothia et al., 1989, Nature 342:877-883), which are located within framework regions (referred to as framework regions 1 to 4, FR1, FR2, FR3, and FR4, by Kabat et al., 1991; see also Chothia and Lesk, 1987, supra).

[0102] Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, which define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses (e.g., but not limited to, IgG1, IgG2, IgG3, and IgG4). IgM has subclasses (e.g., but not limited to, IgM1 and IgM2). Embodiments of the present disclosure include all such classes or isotypes of antibodies. The light chain constant region can be, for example, a kappa- or lambda-type light chain constant region, such as a human kappa- or lambda-type light chain constant region. The heavy chain constant region can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region, such as a human alpha-, human delta-, human epsilon-, human gamma-, or human mu-type heavy chain constant region. Thus, in exemplary embodiments, the antibody is of the isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgG1, IgG2, IgG3, and IgG4.

[0103] In various aspects, the antibody can be a monoclonal or polyclonal antibody. In exemplary cases, the antibody is a mammalian antibody, such as a mouse antibody, rat antibody, rabbit antibody, goat antibody, horse antibody, chicken antibody, hamster antibody, pig antibody, human antibody, etc. In certain aspects, the recombinant glycosylated protein is a monoclonal human antibody.

[0104] In various embodiments, antibodies are cleaved into fragments by enzymes such as, for example, papain and pepsin. Papain cleaves antibodies to generate two Fab fragments and one Fc fragment. Pepsin cleaves antibodies to generate an F(ab')2 fragment and a pFc' fragment. In exemplary embodiments, the recombinant glycosylated protein is an antibody fragment, e.g., Fab, Fc, F(ab')2, or pFc', that retains at least one glycosylation site. For the methods of the present disclosure, an antibody may lack a specific portion of the antibody and may be an antibody fragment. In various embodiments, the antibody fragment contains a glycosylation site. In some embodiments, the fragment is a "glycosylated Fc fragment" that contains at least a portion of the Fc region of the antibody that is glycosylated by post-translational modification in eukaryotic cells. In various examples, the recombinant glycosylated protein is a glycosylated Fc fragment.

[0105] Antibody structures have been utilized to expand the range of alternative antibody formats, spanning a molecular weight range of at least or about 12-150 kDa and valencies (n) ranging from monomers (n=1), dimers (n=2) and trimers (n=3) to tetramers (n=4) and potentially higher, and such alternative antibody formats are referred to herein as "antibody protein products" or "antibody binding proteins."

[0106] Antibody protein products may be antibody fragments, such as scFv, Fab, and VHH / VH-based antigen-binding formats, that retain complete antigen-binding ability. The smallest antigen-binding fragment that retains its complete antigen-binding site is the Fv fragment, which consists entirely of the variable (V) region. Soluble and flexible amino acid peptide linkers are used to link the V region to scFv (single-chain variable fragment) fragments to stabilize the molecule, or constant (C) domains are added to the V region to generate Fab fragments (fragments, antigen-binding). Both scFv and Fab are widely used fragments that can be easily produced in prokaryotic hosts. Other antibody protein products include disulfide-stabilized scFv (ds-scFv), single-chain Fab (scFab), and dimeric and multimeric antibody formats, such as diabodies, triabodies, and tetrabodies, or minibodies (miniAbs), which contain different formats consisting of scFvs linked to oligomerization domains. The smallest fragments are the VHH / VH of camelid heavy chain Abs and single domain Abs (sdAbs). The building blocks most frequently used to generate novel antibody types are single variable (V) domain antibody fragments (scFv), which contain V domains (VH and VL domains) from heavy and light chains linked by a peptide linker of approximately 15 amino acid residues. Peptibodies or peptide-Fc fusions are yet another antibody protein product. The peptibody structure consists of a biologically active peptide grafted onto an Fc domain. Peptibodies have been well described in the art. See, for example, Shimamoto et al., mAbs 4(5):586-591 (2012).

[0107] Other antibody protein products include single-chain antibodies (SCAs), diabodies, triabodies, tetrabodies, and bispecific or triabodies. Bispecific antibodies can be classified into five major classes: BsIgG, adducted IgG, BsAb fragments, bispecific fusion proteins, and BsAb conjugates. See, e.g., Spiess et al., Molecular Immunology 67(2) Part A:97-106 (2015).

[0108] In exemplary aspects, the recombinant glycosylated protein comprises any one of these antibody protein products (e.g., scFv, Fab VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibody, multimeric antibody (e.g., diabody, triabody, tetrabody), miniAb, camelid heavy chain antibody peptibody VHH / VH, sdAb, diabody; triabody; tetrabody; bispecific or triabody, BsIgG, adduct IgG, BsAb fragment, bispecific fusion protein, and BsAb conjugate) and comprises one or more N-glycosylation consensus sequences, and optionally one or more Fc polypeptides. In various aspects, the antibody fragment comprises a glycosylation site. In exemplary aspects, the antibody protein product may be a glycosylated Fc fragment conjugated to an antibody binding fragment (a "glycosylated Fc fragment antibody product").

[0109] The recombinant glycosylated protein can be an antibody protein product in monomeric form or in polymeric, oligomeric, or multimeric form. In certain embodiments, where an antibody comprises two or more distinct antigen-binding region fragments, the antibody is considered bispecific, trispecific, or multispecific, or bivalent, trivalent, or multivalent, depending on the number of distinct epitopes recognized and bound by the antibody.

[0110] In various embodiments, the recombinant glycosylated protein is a chimeric or humanized antibody. The term "chimeric antibody" is used herein to refer to an antibody that contains a constant domain from one species and a variable domain from a second species, or more commonly, a stretch of amino acid sequence from at least two species. The term "humanized," when used in reference to an antibody, refers to an antibody having at least the CDR regions from a non-human source that have been engineered to have a structure and immunological function more similar to that of a true human antibody than the original antibody. For example, humanization can involve grafting CDRs from a non-human antibody, such as a murine antibody, onto a human antibody. Humanization can also involve the selection of amino acid substitutions to make the non-human sequence appear more human.

[0111] In an exemplary embodiment, the antibody of the antibody composition binds to an antigen that comprises only one antibody binding site, and optionally, the level of ADCC activity of the antibody composition is about 13.5%±0.5% for every 1% of TAFs present in the antibody composition. In various embodiments, the antibody of the antibody composition binds to an antigen that comprises only two antibody binding sites, and optionally, the level of ADCC activity of the antibody composition is about 24.74%±0.625% for every 1% of TAFs present in the antibody composition. In an exemplary embodiment, the level of ADCC activity of the antibody composition is about 12%±1.5%×Q for every 1% of TAFs present in the antibody composition, where Q is the number of antibody binding sites present on the antigen. In an exemplary case, Q is 1, and optionally, the antibody is infliximab or a biosimilar thereof. Optionally, Q is 2, and optionally, the antibody is rituximab or a biosimilar thereof. In various embodiments, Q is 3, and thus the antibody composition has an ADCC activity level of about 36% to about 40.5% for every 1% TAF glycan content present in the antibody composition, and in some instances, Q is 4, and thus the antibody composition has an ADCC activity level of about 48% to about 54% for every 1% TAF glycan content present in the antibody composition.

[0112] Advantageously, the method is not limited by the antigen specificity of the antibody, glycosylated Fc fragment, antibody protein product, chimeric antibody, or humanized antibody. Thus, the antibody, glycosylated Fc fragment, antibody protein product, chimeric antibody, or humanized antibody can have virtually any binding specificity for any antigen. In exemplary embodiments, the antibody binds to a hormone, growth factor, cytokine, cell surface receptor, or any of their ligands. In exemplary embodiments, the antibody binds to a protein expressed on the cell surface of an immune cell. In exemplary embodiments, the antibody is selected from the group consisting of CD1a, CD1b, CD1c, CD1d, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11A, CD11B, CD11C, CDw12, CD13, CD14, CD15, CD15s, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31,CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD6 1, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75 , CD76, CD79α, CD79β, CD80, CD81, CD82, CD83, CDw84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CDw92, CD93, CD94, CD95, CD96, CD97, C D98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CDw108, CD109, CD114, CD115, CD116, CD117, CD118, CD 119, CD120a, CD120b, CD121a, CDw121b, CD122, CD123, CD124, CD125, CD126, CD127, CDw128, CD129, CD130, CDw131, CD132, CD134, CD13 5, CDw136, CDw137, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD144, CD145, CD146, CD147, CD148, CD150, CD151, CD152, CD153, CD154, CD155, CD156, CD157, CD158a, CD158b, CD161, CD162, CD163, CD164, CD165, CD166, and CD182.

[0113] In exemplary embodiments, the antibody, glycosylated Fc fragment, antibody protein product, chimeric antibody, or humanized antibody is selected from the group consisting of antibodies described in U.S. Pat. No. 7,947,809 and U.S. Patent Application Publication No. 20090041784 (glucagon receptor), U.S. Pat. No. 7,939,070, U.S. Pat. No. 7,833,527, U.S. Pat. No. 7,767,206 and U.S. Pat. No. 7,786,284 (IL-17 receptor A), U.S. Pat. No. 7,872,106 and U.S. Pat. No. 7,592,429 (sclerostin), U.S. Pat. No. 7,871,611, U.S. Pat. Japanese Patent No. 7815907, U.S. Patent No. 7037498, U.S. Patent No. 7700742 and U.S. Patent Application Publication No. 20100255538 (IGF-1 receptor), U.S. Patent No. 7868140 (B7RP1), U.S. Patent No. 7807159 and U.S. Patent Application Publication No. 20110091455 (myostatin), U.S. Patent No. 7736644, U.S. Patent No. 7628986, U.S. Patent No. 7524496 and U.S. Patent Application Publication No. 20100111979 (epidermal growth factor receptor Deletion mutants of SARS coronavirus), U.S. Patent No. 7,728,110 (SARS coronavirus), U.S. Patent No. 7,718,776 and U.S. Patent Application Publication No. 20100209435 (OPGL), U.S. Patent No. 7,658,924 and U.S. Patent No. 7,521,053 (angiopoietin 2), U.S. Patent No. 7,601,818, U.S. Patent No. 7,795,413, U.S. Patent Application Publication No. 20090155274, U.S. Patent Application Publication No. 20110040076 (NGF), U.S. Patent No. 7,579,186 (TGF-β type II receptor No. 7,541,438 (connective tissue growth factor), U.S. Pat. No. 7,438,910 (IL1-R1), U.S. Pat. No. 7,423,128 (properdin), U.S. Pat. No. 7,411,057, U.S. Pat. No. 7,824,679, U.S. Pat. No. 7,109,003, U.S. Pat. No. 6,682,736, U.S. Pat. No. 7,132,281 and U.S. Pat. No. 7,807,797 (CTLA-4), U.S. Pat. No. 7,084,257, U.S. Pat. No. 7,790,859, U.S. Pat. No. 7,335,743,U.S. Patent No. 7,084,257 and U.S. Patent Application Publication No. 20110045537 (interferon-gamma), U.S. Patent No. 7,932,372 (MAdCAM), U.S. Patent No. 7,906,625, U.S. Patent Application Publication No. 20080292639 and U.S. Patent Application Publication No. 20110044986 (amyloid), U.S. Patent No. 7,815,907 and U.S. Patent No. 7,700,742 (insulin-like growth factor I), U.S. Patent No. 7,566,772 and U.S. Patent No. 7,964,193 (Interleukin-1β), U.S. Patent Nos. 7,563,442, 7,288,251, 7,338,660, 7,626,012, 7,618,633, and U.S. Patent Application Publication No. 20100098694 (CD40), U.S. Patent No. 7,498,420 (c-Met), U.S. Patent Nos. 7,326,414, 7,592,430, and 7,728,113 (M-CSF), U.S. Patent No. 6,924,360, U.S. Patent No. 7 Nos. 067131 and 7090844 (MUC18), U.S. Pat. Nos. 6235883, 7807798 and U.S. Patent Application Publication No. 20100305307 (epidermal growth factor receptor), U.S. Pat. Nos. 6716587, 7872113, 7465450, 7186809, 7317090 and 7638606 (interleukin-4 receptor), U.S. Patent Application Publication No. 201101356 No. 57 (beta-Klotho), U.S. Pat. Nos. 7,887,799 and 7,879,323 (fibroblast growth factor-like polypeptides), U.S. Pat. No. 7,867,494 (IgE), U.S. Patent Application Publication No. 20100254,975 (alpha-4 beta-7), U.S. Patent Application Publication Nos. 20100197,005 and 7,537,762 (activin receptor-like kinase 1), U.S. Pat. No. 7,585,500 and 20100047,253 (IL-13),U.S. Patent Application Publication No. 20090263383 and U.S. Patent No. 7,449,555 (CD148), U.S. Patent Application Publication No. 20090234106 (activin A), U.S. Patent Application Publication No. 20090226447 (angiopoietin 1 and angiopoietin 2), U.S. Patent Application Publication No. 20090191212 (angiopoietin 2), U.S. Patent Application Publication No. 20090155164 (C-FMS), U.S. Patent No. 7,537,762 (activin receptor-like kinase 1), U.S. Patent No. 7,371,381 (galanin), U.S. Patent Application Publication No. 20070196376 (insulin-like growth factors), U.S. Patent Nos. 7,267,960 and 7,741,115 (LDCAM), U.S. Patent No. 7,265,212 (CD45RB), U.S. Patent No. 7,709,611, U.S. Patent Application Publication Nos. 20060127,393 and 20100040619 (DKK1), U.S. Patent No. 7,807,795, U.S. Patent Application Publication Nos. 20030103978 and 7,923,008 (osteoprotease inhibitors), Phosphorin), U.S. Patent Application Publication No. 20090208489 (OV064), U.S. Patent Application Publication No. 20080286284 (PSMA), U.S. Patent Application Publication No. 7888482, U.S. Patent Application Publication No. 20110165171 and U.S. Patent Application Publication No. 20110059063 (PAR2), U.S. Patent Application Publication No. 20110150888 (hepcidin), U.S. Patent Application Publication No. 7939640 (B7L-1), U.S. Patent Application Publication No. 7915391 (c-Kit), U.S. Patent Application Publication No. 7807796, U.S. Patent Application Publication No. 7193058 and the specifications of U.S. Patent No. 7,427,669 (ULBP), U.S. Patent No. 7,786,271, U.S. Patent No. 7,304,144 and U.S. Patent Application Publication No. 20090238823 (TSLP), U.S. Patent No. 7,767,793 (SIGIRR), U.S. Patent No. 7,705,130 (HER-3), U.S. Patent No. 7,704,501 (ataxin 1-like polypeptide), U.S. Patent No. 7,695,948 and U.S. Patent No. 7,199,224 (TNF-α converting enzyme), U.S. Patent Application Publication No. 20090234106 (activin A),U.S. Patent Application Publication No. 20090214559 and U.S. Patent No. 7,438,910 (IL1-R1), U.S. Patent No. 7,579,186 (TGF-β type II receptor), U.S. Patent No. 7,569,387 (TNF receptor-like molecule), U.S. Patent No. 7,541,438 (connective tissue growth factor), U.S. Patent No. 7,521,048 (TRAIL receptor 2), U.S. Patent No. 6,319,499, U.S. Patent No. 7,081,523 and U.S. Patent Application Publication No. 200801 No. 82976 (erythropoietin receptor), U.S. Patent Application Publication No. 20080166352 and U.S. Patent No. 7435796 (B7RP1), U.S. Patent No. 7423128 (properdin), U.S. Patent Nos. 7422742 and 7141653 (interleukin-5), U.S. Patent Nos. 6740522 and 7411050 (RANKL), U.S. Patent No. 7378091 (carbonic anhydrase IX (CA) IX) Tumor antigens), U.S. Pat. Nos. 7,318,925 and 7,288,253 (parathyroid hormone), U.S. Pat. No. 7,285,269 (TNF), U.S. Pat. Nos. 6,692,740 and 7,270,817 (ACPL), U.S. Pat. No. 7,202,343 (monocyte chemotactic protein 1), U.S. Pat. No. 7,144,731 (SCF), U.S. Pat. Nos. 6,355,779 and 7,138,500 (4-1BB), U.S. Pat. No. 7,135,174 (PDGFD), U.S. Pat. No. 6,630,143 and U.S. Patent No. 7,045,128 (Flt-3 ligand), U.S. Patent No. 6,849,450 (metalloprotease inhibitor), U.S. Patent No. 6,596,852 (LERK-5), U.S. Patent No. 6,232,447 (LERK-6), U.S. Patent No. 6,500,429 (brain-derived neurotrophic factor), U.S. Patent No. 6,184,359 (epithelial-derived T-cell factor), U.S. Patent No. 6,143,874 (neurotrophic factor NNT-1), U.S. Patent Application Publication No. 20110027287 (proprotein convertase subtilisin kexin type 9 (PCSK9)),and antibodies described in U.S. Patent Application Publication No. 20110014201 (IL-18 RECEPTOR) and U.S. Patent Application Publication No. 20090155164 (C-FMS). The above-mentioned patents and published patent applications are incorporated herein by reference in their entirety for the purposes of their disclosure of variable domain polypeptides, nucleic acids encoding variable domains, host cells, vectors, methods of making polypeptides encoding said variable domains, pharmaceutical compositions, and methods of treating diseases associated with the respective targets of the variable domain-containing antigen binding proteins or antibodies.

[0114] In exemplary embodiments, the glycosylated Fc fragment, antibody protein product, chimeric antibody, or humanized antibody is selected from the group consisting of muromonab-CD3 (a product commercially available under the trade name Orthoclone Okt3®), abciximab (a product commercially available under the trade name Reopro®), rituximab (a product commercially available under the trade name MabThera®, Rituxan®), basiliximab (a product commercially available under the trade name Simulect®), daclizumab (a product commercially available under the trade name Zenapax®), palivizumab (a product commercially available under the trade name Synagis®), infliximab (a product commercially available under the trade name Remicade®), trastuzumab ( products marketed under the trade name Herceptin®), alemtuzumab (products marketed under the trade name MabCampath®, Campath-1H®), adalimumab (products marketed under the trade name Humira®), tositumomab-I131 (products marketed under the trade name Bexxar®), efalizumab (products marketed under the trade name Raptiva®), cetuximab (products marketed under the trade name Erbitux®), ibritumomab tiuxetan (products marketed under the trade name Zevalin®), (products marketed under the trade name Xolair®), bevacizumab (products marketed under the trade name Avastin®), natalizumab (products marketed under the trade name Tysabri®), ranibizumab (products marketed under the trade name Lucentis®), panitumumab (products marketed under the trade name Vectibix®), eculizumab (products marketed under the trade name Soliris®), certolizumab pegol (products marketed under the trade name Cimzia®) , golimumab (products marketed under the trade name Simponi®), canakinumab (products marketed under the trade name Ilaris®), catumaxomab (products marketed under the trade name Removab®), ustekinumab (products marketed under the trade name Stelara®), tocilizumab (products marketed under the trade name RoActemra®, Actemra®), ofatumumab (products marketed under the trade name Arzerra®), denosumab (products marketed under the trade name Prolia®),The antibody is one of belimumab (a product marketed under the trade name Benlysta®), raxibacumab, ipilimumab (a product marketed under the trade name Yervoy®), and pertuzumab (a product marketed under the trade name Perjeta®). In exemplary embodiments, the antibody is one of an anti-TNF-alpha antibody, such as adalimumab, infliximab, etanercept, golimumab, and certolizumab pegol; an anti-IL1.beta antibody, such as canakinumab; an anti-IL12 / 23(p40) antibody, such as ustekinumab and briakinumab; and an anti-IL2R antibody, such as daclizumab.

[0115] In exemplary embodiments, the antibody binds to tumor-associated antigen and is an anti-cancer antibody.Examples of suitable anti-cancer antibodies include but are not limited to anti-BAFF antibody such as belimumab; anti-CD20 antibody such as rituximab; anti-CD22 antibody such as epratuzumab; anti-CD25 antibody such as daclizumab; anti-CD30 antibody such as iratumumab, anti-CD33 antibody such as gemtuzumab, anti-CD52 antibody such as alemtuzumab; anti-CD152 antibody such as ipilimumab; anti-EGFR antibody such as cetuximab; anti-HER2 antibody such as trastuzumab and pertuzumab; anti-IL6 antibody such as siltuximab; and anti-VEGF antibody such as bevacizumab; anti-IL6 receptor antibody such as tocilizumab.

[0116] In an exemplary embodiment, the tumor-associated antigen is CD20, and the antibody is an anti-CD20 antibody, e.g., an anti-CD20 monoclonal antibody. In an exemplary embodiment, the tumor-associated antigen comprises SEQ ID NO: 3. In an exemplary case, the antibody comprises the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2. In various embodiments, the IgG1 antibody is rituximab, or a biosimilar thereof. The term rituximab refers to an IgG1 kappa chimeric mouse / human monoclonal antibody that binds to the CD20 antigen (CAS No.: 174722-31-7; DrugBank-DB00073; see Kyoto Encyclopedia of Genes and Genomes (KEGG) entry D02994). In an exemplary embodiment, the antibody comprises a light chain comprising CDR1, CDR2, and CDR3 as set forth in Table A. In an exemplary embodiment, the antibody comprises a heavy chain comprising CDR1, CDR2, and CDR3 as set forth in Table A. In various examples, the antibody comprises a VH and VL listed in Table A, or comprises a VH-IgG1 and VL-IgGkappa sequence.

[0117] [Table A]

[0118] In various embodiments, the antibody comprises: i. a light chain (LC) CDR1 comprising the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 4, or a variant amino acid sequence of SEQ ID NO: 4 having one or two amino acid substitutions; ii. an LC CDR2 comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 5, or a variant amino acid sequence of SEQ ID NO: 5 having one or two amino acid substitutions; iii. an LC CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 6, or a variant amino acid sequence of SEQ ID NO: 6 having one or two amino acid substitutions; iv. a heavy chain (HC) CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 7, or a variant amino acid sequence of SEQ ID NO: 7 having one or two amino acid substitutions; v. HC CDR2 comprising the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 8, or a variant amino acid sequence of SEQ ID NO: 8 having one or two amino acid substitutions; vi. HC CDR3 comprising the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 9, or a variant amino acid sequence of SEQ ID NO: 9 having one or two amino acid substitutions.

[0119] In various examples, the antibody comprises an LC variable region comprising the amino acid sequence of SEQ ID NO: 10, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 10, or a variant amino acid sequence of SEQ ID NO: 10 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2) amino acid substitutions.

[0120] In exemplary embodiments, the antibody comprises an HC variable region comprising the amino acid sequence of SEQ ID NO: 11, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 11, or a variant amino acid sequence of SEQ ID NO: 11 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0121] In exemplary cases, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 12, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 12, or a variant amino acid sequence of SEQ ID NO: 12 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2) amino acid substitutions.

[0122] In various embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 13, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 13, or a variant amino acid sequence of SEQ ID NO: 13 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0123] In an exemplary embodiment, the antibody's antigen is TNFα, and the antibody is an anti-TNFα antibody (which for brevity may also be referred to simply as an "anti-TNF" antibody), e.g., an anti-TNFα monoclonal antibody. In an exemplary embodiment, the antibody's antigen comprises SEQ ID NO: 14. In various embodiments, the IgG1 antibody is infliximab, or a biosimilar thereof. The term infliximab refers to a chimeric monoclonal IgG1 kappa antibody composed of a human constant region and a mouse variable region and binding to the TNFα antigen (see CAS No.: 170277-31-3, DrugBank Accession No. DB00065). Infliximab, also known as the chimeric antibody cA2, was derived from a murine monoclonal antibody called A2 (Knight et al., Molec Immunol 30(16):1443-1453 (1993)). The variable region of the cA2 light chain is published in WO 2006 / 065975. In an exemplary embodiment, the antibody comprises a light chain comprising CDR1, CDR2, and CDR3 of the light chain variable region of infliximab as set forth in Table B. In an exemplary embodiment, the antibody comprises a heavy chain comprising CDR1, CDR2, and CDR3 of the heavy chain variable region of infliximab as set forth in Table B. In various examples, the antibody comprises the VH and VL of infliximab or comprises the VH-IgG1 and VL-IgGkappa sequences.

[0124] [Table B]

[0125] In various examples, the antibody comprises an LC variable region comprising the amino acid sequence of SEQ ID NO: 15, an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 15, or a variant amino acid sequence of SEQ ID NO: 15 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2) amino acid substitutions. In exemplary embodiments, the antibody comprises an HC variable region comprising the amino acid sequence of SEQ ID NO: 16, an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 16, or a variant amino acid sequence of SEQ ID NO: 16 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2) amino acid substitutions.

[0126] composition The disclosed methods relate to compositions comprising recombinant glycosylated proteins. In various embodiments, the composition comprises only one type of recombinant glycosylated protein. In various examples, the composition comprises recombinant glycosylated proteins, wherein each recombinant glycosylated protein of the composition comprises the same or substantially the same amino acid sequence. In various embodiments, the composition comprises recombinant glycosylated proteins, wherein each recombinant glycosylated protein of the composition comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of every other recombinant glycosylated protein of the composition. In various embodiments, the composition comprises recombinant glycosylated proteins, wherein each recombinant glycosylated protein of the composition comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of every other recombinant glycosylated protein of the composition. In various embodiments, a composition comprises recombinant glycosylated proteins, and each recombinant glycosylated protein of the composition comprises an amino acid sequence that is identical or substantially identical (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of every other recombinant glycosylated protein of the composition), although the glycoprofiles of the recombinant glycosylated proteins of the composition can differ from one another.

[0127] In an exemplary embodiment, the recombinant glycosylated protein is an antibody fragment, and thus the composition can be an antibody fragment composition.

[0128] In an exemplary embodiment, the recombinant glycosylated protein is an antibody protein product, and therefore the composition can be an antibody protein product composition.

[0129] In an exemplary embodiment, the recombinant glycosylated protein is a glycosylated Fc fragment, and thus the composition may be a glycosylated Fc fragment composition.

[0130] In an exemplary embodiment, the recombinant glycosylated protein is a glycosylated Fc fragment antibody product, and thus the composition may be a glycosylated Fc fragment antibody product composition.

[0131] In an exemplary embodiment, the recombinant glycosylated protein is a chimeric antibody, and therefore the composition may be a chimeric antibody composition.

[0132] In an exemplary embodiment, the recombinant glycosylated protein is a humanized antibody, and thus the composition may be a humanized antibody composition.

[0133] In exemplary embodiments, the recombinant glycosylated protein is an antibody, and the composition is an antibody composition. In various embodiments, the composition comprises only one type of antibody. In various examples, the composition comprises antibodies, and each antibody of the antibody composition comprises the same or substantially identical amino acid sequence. In various embodiments, the antibody composition comprises antibodies, and each antibody of the antibody composition comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of every other antibody in the antibody composition. In various embodiments, the antibody composition comprises antibodies, and each antibody of the antibody composition comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of every other antibody in the antibody composition. In various embodiments, the antibody composition comprises antibodies, and each recombinant glycosylated protein of the composition comprises an amino acid sequence that is identical or substantially identical (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of every other antibody protein in the antibody composition), but the glycoprofiles of the antibodies in the antibody composition can differ from one another. In exemplary embodiments, an antibody composition comprises a heterogeneous mixture of different glycoforms of an antibody. In various examples, an antibody composition can be characterized for its TAF glycan content, HM glycan content, and / or its AF glycan content. In various embodiments, an antibody composition is described in terms of % TAF glycans, % HM glycans, and / or % nonfucosylated glycans. Optionally, an antibody composition can be characterized for its content of other types of glycans, such as galactosylated glycoforms, fucosylated glycoforms, etc.

[0134] In various embodiments, each antibody of the antibody composition is an IgG, optionally an IgG1. In exemplary cases, each antibody of the antibody composition binds to a tumor-associated antigen, such as CD20. In various embodiments, CD20 comprises the amino acid sequence of SEQ ID NO: 3. In exemplary embodiments, each antibody of the antibody composition is an anti-CD20 antibody. In various examples, each antibody of the antibody composition comprises: i. a light chain (LC) CDR1 comprising the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 4, or a variant amino acid sequence of SEQ ID NO: 4 having one or two amino acid substitutions; ii. an LC CDR2 comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 5, or a variant amino acid sequence of SEQ ID NO: 5 having one or two amino acid substitutions; iii. an LC CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 6, or a variant amino acid sequence of SEQ ID NO: 6 having one or two amino acid substitutions; iv. a heavy chain (HC) CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 7, or a variant amino acid sequence of SEQ ID NO: 7 having one or two amino acid substitutions; v. HC CDR2 comprising the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 8, or a variant amino acid sequence of SEQ ID NO: 8 having one or two amino acid substitutions; and / or vi. HC CDR3 comprising the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 9, or a variant amino acid sequence of SEQ ID NO: 9 having one or two amino acid substitutions.

[0135] In various examples, each antibody of the antibody composition comprises an LC variable region comprising the amino acid sequence of SEQ ID NO: 10, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 10, or a variant amino acid sequence of SEQ ID NO: 10 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0136] In exemplary embodiments, each antibody of the antibody composition comprises an HC variable region comprising the amino acid sequence of SEQ ID NO: 11, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 11, or a variant amino acid sequence of SEQ ID NO: 11 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0137] In exemplary cases, each antibody of the antibody composition comprises a light chain comprising the amino acid sequence of SEQ ID NO: 12, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 12, or a variant amino acid sequence of SEQ ID NO: 12 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0138] In various embodiments, each antibody of the antibody composition comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 13, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 13, or a variant amino acid sequence of SEQ ID NO: 13 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0139] In various embodiments, each antibody of the antibody composition is an IgG, optionally an IgG1. In various examples, each antibody of the antibody composition binds to a tumor-associated antigen, such as TNF-alpha.

[0140] In various embodiments, the TNF-alpha comprises the amino acid sequence of SEQ ID NO: 14. In exemplary embodiments, each antibody of the antibody composition is an anti-TNF-alpha antibody. In various examples, each antibody of the antibody composition comprises an LC variable region comprising the amino acid sequence of SEQ ID NO: 15, an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 15, or a variant amino acid sequence of SEQ ID NO: 15 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0141] In exemplary embodiments, each antibody of the antibody composition comprises an HC variable region comprising the amino acid sequence of SEQ ID NO: 16, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 16, or a variant amino acid sequence of SEQ ID NO: 16 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0142] In exemplary embodiments, an antibody composition comprises a heterogeneous mixture of different glycoforms of an antibody. In various examples, an antibody composition can be characterized for its TAF glycan content, HM glycan content, and / or its AF glycan content. In various embodiments, an antibody composition is described in terms of % TAF glycans, % HM glycans, and / or % nonfucosylated glycans. Optionally, an antibody composition can be characterized for its content of other types of glycans, such as galactosylated glycoforms, fucosylated glycoforms, etc.

[0143] In exemplary embodiments, the antibody composition has a TAF glycan % calculated using Equation A. In exemplary embodiments, the antibody composition has a TAF glycan % within the range defined by X in Equation A. In exemplary cases, the TAF glycan % is within X ± 0.4. In exemplary embodiments, the antibody composition has a TAF glycan % determined (e.g., measured) in the determining step of the methods disclosed herein. In exemplary embodiments, the TAF glycan % is determined by hydrophilic interaction chromatography, optionally by the method described in Example 1. Illustratively, the antibody composition in various embodiments has less than about 50% TAF glycans (e.g., less than about 40%, less than about 30%, less than about 25%, less than about 20%, less than about 15%). In exemplary embodiments, the antibody composition has less than about 10% TAF glycans (e.g., about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less). In exemplary embodiments, the antibody composition has about 4% to about 10% TAF glycans. In exemplary embodiments, the antibody composition has about 2% to about 6% TAF glycans. In exemplary embodiments, the antibody composition has about 2.5% to about 5% TAF glycans. In exemplary embodiments, the antibody composition has about 4% or less TAF glycans. In further exemplary embodiments, the antibody composition has about 4% or less to about 2% or more TAF glycans. In various embodiments, the TAF glycan percentage is about 1.55% to about 6.95%, or about 1.72% to about 6.74%.

[0144] In exemplary embodiments, the antibody composition has a % nonfucosylated glycan calculated using Equation B. In exemplary embodiments, the antibody composition has a % nonfucosylated glycan within the range defined by AF of Equation B. In exemplary cases, the % nonfucosylated glycan is within AF ± 1. In exemplary embodiments, the antibody composition has a % nonfucosylated glycan determined (e.g., measured) in the determining step of the methods disclosed herein. In exemplary embodiments, the % nonfucosylated glycan is determined by hydrophilic interaction chromatography, optionally by the method described in Example 1. Illustratively, the antibody composition in various embodiments has about 5% or less nonfucosylated glycans. In exemplary embodiments, the % nonfucosylated glycans is about 1 to about 4. In exemplary embodiments, the antibody composition has about 4% or less nonfucosylated glycans. In exemplary embodiments, the antibody composition has about 3.5% or less nonfucosylated glycans.

[0145] In exemplary embodiments, the antibody composition has a % high mannose glycan calculated using Equation B. In exemplary embodiments, the antibody composition has a % high mannose glycan within the range defined by HM of Equation B. In exemplary cases, the % high mannose glycan is within HM ± 1. In exemplary embodiments, the antibody composition has a % high mannose glycan determined (e.g., measured) in the determining step of the methods disclosed herein. In exemplary embodiments, the % high mannose glycan is determined by hydrophilic interaction chromatography, optionally by the method described in Example 1. By way of example, the antibody composition in exemplary embodiments has about 5% or less high mannose glycans. In exemplary embodiments, the % high mannose glycans is about 1 to about 4. In exemplary embodiments, the antibody composition has about 4% or less high mannose glycans. In exemplary embodiments, the antibody composition has about 3.5% or less high mannose glycans.

[0146] In exemplary embodiments, the antibody composition has an ADCC% calculated using Equation A or Equation B. In exemplary embodiments, the antibody composition has an ADCC% determined (e.g., measured) in the determining step. In exemplary embodiments, the ADCC% is determined by a quantitative cell-based assay, such as the method described in Example 2, that measures the ability of antibodies of the antibody composition to mediate cytotoxicity in a dose-dependent manner in cells that express the antibody's antigen and engage the Fc gamma RIIIA receptor on effector cells via the antibody's Fc domain. For example, the antibody composition in various embodiments has an ADCC% of about 40% to about 175%, or about 40% to about 170%, or about 44% to about 165%. In exemplary embodiments, the antibody composition has an ADCC% of about 40 or more and about 175 or less, or about 170 or less, optionally about 41 to about 171. In exemplary embodiments, the antibody composition has an ADCC% of about 30 to about 185, optionally about 32 to about 180. In various embodiments, the ADCC % is greater than or equal to about 60 and less than or equal to about 130. In exemplary embodiments, the antibody composition has an ADCC % within the range defined by Y of Equation A or Equation B. In various embodiments, the ADCC % is within Y±20, e.g., Y±19, Y±18, or Y±17.

[0147] With respect to the TAF glycan %, X, and ADCC %, Y, of Equation A, in some embodiments, Y is greater than or equal to about 40 and less than or equal to about 170, and X is greater than or equal to about 1.55% and less than or equal to about 6.95%. In various examples, Y is greater than or equal to about 44% and less than or equal to about 165%, and optionally, X is between about 1.72% and about 6.74%.

[0148] With respect to % nonfucosylated glycans, AF, and % high mannose glycans, HM, and % ADCC, Y, of Equation B, in some embodiments, Y is greater than or equal to about 40 and less than about 175, optionally about 41 to about 171, AF is about 1 to about 4, and HM is about 40 to about 175. In various examples, Y is about 30 to about 185, optionally about 32 to about 180, HM is about 1 to about 4, and AF is about 30 to about 185.

[0149] In exemplary embodiments, the composition is combined with a pharmaceutically acceptable carrier, diluent, or excipient. Thus, provided herein are pharmaceutical compositions comprising a recombinant glycosylated protein composition described herein (e.g., an antibody composition or an antibody-binding protein composition) and a pharmaceutically acceptable carrier, diluent, or excipient. As used herein, the term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate-buffered saline, water, emulsions such as oil / water or water / oil emulsions, and various types of wetting agents.

[0150] In an exemplary embodiment, the antibody composition is produced by glycosylation-competent cells in cell culture, as described herein.

[0151] Additional processes The methods disclosed herein, in various aspects, include additional steps. For example, in some aspects, the methods include one or more upstream or downstream steps involved in the production, purification, and formulation of a recombinant glycosylated protein (e.g., an antibody). Optionally, the downstream step is any one of the downstream process steps described herein or known in the art. See, e.g., process steps. In exemplary embodiments, the methods include steps for generating a host cell that expresses the recombinant glycosylated protein (e.g., an antibody). The host cell, in some aspects, is a prokaryotic host cell, e.g., E. coli or Bacillus subtilis, or the host cell, in some aspects, is a eukaryotic host cell, e.g., a yeast cell, a filamentous fungal cell, a protozoan cell, an insect cell, or a mammalian cell (e.g., a CHO cell). Such host cells are described in the art. See, e.g., Frenzel, et al., Front Immunol 4:217 (2013) and the "Cells" section herein. For example, the methods, in some instances, include introducing into a host cell a vector containing a nucleic acid that includes a nucleotide sequence encoding the recombinant glycosylated protein, or a polypeptide chain thereof.

[0152] In exemplary embodiments, the method includes maintaining cells, e.g., glycosylation-competent cells, in cell culture. Thus, the method can include performing any one or more of the steps described in the section herein on maintaining cells in cell culture.

[0153] In exemplary embodiments, the methods disclosed herein include isolating and / or purifying a recombinant glycosylated protein (e.g., a recombinant antibody) from a culture. In exemplary aspects, the methods include one or more chromatography steps, including, but not limited to, affinity chromatography (e.g., Protein A affinity chromatography), ion exchange chromatography, and / or hydrophobic interaction chromatography. In exemplary aspects, the methods include generating a crystalline biomolecule from a solution containing the recombinant glycosylated protein.

[0154] The methods of the present disclosure, in various embodiments, include one or more steps for producing a composition, e.g., in some embodiments, a pharmaceutical composition comprising a purified recombinant glycosylated protein, such as those described herein.

[0155] Maintenance of cells in cell culture Regarding the method for producing an antibody composition of the present disclosure, the antibody composition can be produced by maintaining cells in cell culture. The cell culture can be maintained according to any set of conditions suitable for the production of recombinant glycosylated proteins. For example, in some embodiments, the cell culture is maintained at a specific pH, temperature, cell density, culture volume, dissolved oxygen level, pressure, osmolality, etc. In an exemplary embodiment, the pre-seeded cell culture is shaken (e.g., 70 rpm) in a CO2 incubator under standard humidified conditions at 5% CO2. In an exemplary embodiment, about 10 cells in 1.5 L of medium are cultured. 6 Cell cultures are seeded at a seeding density of 10 cells / mL.

[0156] In exemplary embodiments, the methods of the disclosure include maintaining glycosylation-competent cells in a cell culture medium at a pH of about 6.85 to about 7.05, e.g., in various embodiments, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, about 6.90, about 6.91, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.00, about 7.01, about 7.02, about 7.03, about 7.04, or about 7.05.

[0157] In an exemplary aspect, the method includes maintaining the cell culture at a temperature between 30° C. and 40° C. In exemplary embodiments, the temperature is from about 32° C. to about 38° C. or from about 35° C. to about 38° C.

[0158] In exemplary embodiments, the methods include maintaining an osmolality of about 200 mOsm / kg to about 500 mOsm / kg. In exemplary embodiments, the methods include maintaining an osmolality of about 225 mOsm / kg to about 400 mOsm / kg or about 225 mOsm / kg to about 375 mOsm / kg. In exemplary embodiments, the methods include maintaining an osmolality of about 225 mOsm / kg to about 350 mOsm / kg. In various embodiments, the osmolality (mOsm / kg) is maintained at about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, or about 500.

[0159] In exemplary embodiments, the methods include maintaining the dissolved oxygen (DO) level of the cell culture at about 20% to about 60% oxygen saturation during the initial cell culture period. In exemplary cases, the methods include maintaining the DO level of the cell culture at about 30% to about 50% (e.g., about 35% to about 45%) oxygen saturation during the initial cell culture period. In exemplary cases, the methods include maintaining the DO level of the cell culture at about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% oxygen saturation during the initial cell culture period. In exemplary embodiments, the DO level is about 35 mmHg to about 85 mmHg, or about 40 mmHg to about 80 mmHg, or about 45 mmHg to about 75 mmHg.

[0160] The cell culture is maintained in any one or more culture media. In exemplary embodiments, the cell culture is maintained in a medium suitable for cell growth and / or provided with one or more feed media according to any suitable feeding schedule. In exemplary embodiments, the method includes maintaining the cell culture in a medium comprising glucose, fucose, lactate, ammonia, glutamine, and / or glutamate. In exemplary embodiments, the method includes maintaining the cell culture in a medium comprising manganese at a concentration of about 1 μM or less during the initial cell culture period. In exemplary embodiments, the method includes maintaining the cell culture in a medium comprising about 0.25 μM to about 1 μM manganese. In exemplary embodiments, the method includes maintaining the cell culture in a medium comprising negligible amounts of manganese. In exemplary embodiments, the method includes maintaining the cell culture in a medium comprising copper at a concentration of about 50 ppb or less during the initial cell culture period. In exemplary embodiments, the method includes maintaining the cell culture in a medium comprising copper at a concentration of about 40 ppb or less during the initial cell culture period. In an exemplary embodiment, the method comprises maintaining the cell culture in a medium containing copper at a concentration of about 30 ppb or less during the initial cell culture period. In an exemplary embodiment, the method comprises maintaining the cell culture in a medium containing copper at a concentration of about 20 ppb or less during the initial cell culture period. In an exemplary embodiment, the medium contains copper at a concentration of about 5 ppb or more or about 10 ppb or more. In an exemplary embodiment, the cell culture medium contains mannose. In an exemplary embodiment, the cell culture medium does not contain mannose.

[0161] In exemplary embodiments, the type of cell culture is fed-batch or continuous perfusion, although the methods of the present disclosure are advantageously not limited to any particular type of cell culture.

[0162] The cells maintained in cell culture can be glycosylation-competent cells. In exemplary embodiments, the glycosylation-competent cells are eukaryotic cells, including, but not limited to, yeast cells, filamentous fungal cells, protozoan cells, algae cells, insect cells, or mammalian cells. Such host cells are described in the art. See, for example, Frenzel, et al., Front Immunol 4:217 (2013). In exemplary embodiments, the eukaryotic cells are mammalian cells. In exemplary embodiments, the mammalian cells are non-human mammalian cells. In some embodiments, the cells are selected from the group consisting of Chinese hamster ovary (CHO) cells and their derivatives (e.g., CHO-K1, CHO pro-3), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2 / 0), cells engineered to lack dihydrofolate reductase (DHFR) activity (e.g., DUKX-X11, DG44), human embryonic kidney 293 (HEK293) cells or their derivatives (e.g., HEK293T, HEK293-EBNA), African green monkey kidney cells (e.g., COS cells, VERO cells), human cervical carcinoma cells (e.g., HeLa), human bone osteosarcoma epithelial cells U2-OS, adenocarcinoma human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonic carcinoma cells P19, and mouse embryonic fibroblast cells NIH. 3T3, mouse fibroblast L929, mouse neuroblastoma N2a, human breast cancer MCF-7, retinoblastoma Y79, human retinoblastoma SO-Rb50, human hepatocarcinoma Hep G2, mouse B myeloma J558L, or baby hamster kidney (BHK) cells (Gaillet et al. 2007; Khan, Adv Pharm Bull 3(2):257-263(2013)).

[0163] Cells that are not glycosylation competent can also be transformed into glycosylation competent cells, for example, by introducing into them genes encoding the relevant enzymes required for glycosylation. Exemplary enzymes include, but are not limited to, oligosaccharyltransferase, glycosidase, glucosidase I, glucosidase II, calnexin / calreticulin, glycosyltransferase, mannosidase, GlcNAc transferase, galactosyltransferase, and sialyltransferase.

[0164] In an exemplary embodiment, the glycosylation-competent cells are not genetically modified to alter the activity of enzymes in the de novo pathway or the salvage pathway. These two pathways of fucose metabolism are shown in FIG. 2. In an exemplary embodiment, the glycosylation-competent cells are not genetically modified to alter the activity of any one or more of fucosyltransferases (FUTs, e.g., FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9), fucose kinase, GDP-fucose pyrophosphorylase, GDP-D-mannose-4,6-dehydratase (GMD), and GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase (FX). In an exemplary embodiment, the glycosylation-competent cells are not genetically modified to knock out the gene encoding FX.

[0165] In exemplary embodiments, the glycosylation-competent cells are not genetically modified to alter the activity of β(1,4)-N-acetylglucosaminyltransferase III (GNTIII) or GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD). In exemplary aspects, the glycosylation-competent cells are not genetically modified to overexpress GNTIII or an RMD.

[0166] Illustrative Embodiments Exemplary embodiments of the present disclosure are provided below. E1. A method for producing an antibody composition, said method comprising: i. determining the % total nonfucosylated (TAF) glycans of the antibody composition; ii.Equation A: Y=2.6+24.1×X [Equation A] where Y is the ADCC % and X is the TAF glycan % determined in step (i). calculating the antibody-dependent cellular cytotoxicity (ADCC) % of the antibody composition based on the TAF % using the (iii) if Y is within the target ADCC % range, selecting the antibody composition for one or more downstream processing steps; A method comprising: E2. A method for producing an antibody composition, said method comprising: i. determining the % high mannose glycans and % nonfucosylated glycans of the antibody composition; ii.Equation B: Y = (0.24 + 27 × HM + 22.1 × AF) [Equation B] where Y is ADCC %, HM is high mannose glycan % determined in step (i), and AF is non-fucosylated glycan % determined in step (i). calculating the % antibody-dependent cellular cytotoxicity (ADCC) of the antibody composition based on the % high mannose glycans and the % non-fucosylated glycans using the following: iii. If Y is within the target ADCC % range, selecting the antibody composition for one or more downstream processing steps. A method comprising: E3. A method for producing an antibody composition having a target ADCC %, said method comprising: i. Equation A: Y=2.6+24.1×X [Equation A] where Y is the target ADCC % and X is the target TAF glycan %. calculating the target total nonfucosylated (TAF) glycan % for the target ADCC % using ii. Maintaining glycosylation-competent cells in cell culture to produce an antibody composition having a target TAF glycan %, X. A method comprising: E4. A method for producing an antibody composition having a target ADCC %, said method comprising: i. Equation B: Y = (0.24 + 27 × HM + 22.1 × AF) [Equation B] where Y is the target ADCC %, HM is the target high mannose glycan %, and AF is the target non-fucosylated glycan %. calculating the target nonfucosylated glycan % and the target high mannose glycan % for the target ADCC % using the ii. Maintaining glycosylation-competent cells in cell culture to produce an antibody composition having a target % high mannose glycans and a target % nonfucosylated glycans. A method comprising: E5. The method of embodiment 3 or 4, wherein the target ADCC% is within a target ADCC% range. E6. The method of any one of embodiments 1, 2, and 5, wherein the target ADCC % range is from about 40 to about 170. E7. The method of embodiment 6, wherein the target ADCC% range is from about 44 to about 165. E8. The method of embodiment 7, wherein the target ADCC% range is from about 60 to about 130. E9. The method of any one of embodiments 1-4, wherein the target ADCC % range is Y±20. E10. The method of embodiment 1 or embodiment 3, wherein the target ADCC % range is Y±17. E11. The method of embodiment 2 or embodiment 4, wherein the target ADCC % range is Y±18. E12. Optionally, a method of producing an antibody composition having an ADCC%, Y, of about 40 to about 170, the method comprising: i. determining the % total nonfucosylated (TAF) glycans, X, of the antibody composition; and ii. If X is equal to (Y-2.6) / 24.1, selecting the antibody composition for one or more downstream processing steps. A method comprising: The method of embodiment 13, wherein E13.X is greater than or equal to about 1.55% and less than or equal to about 6.95%. E14. The method of embodiment 13 or 14, wherein Y is greater than or equal to about 44% and less than or equal to about 165%, and optionally, X is between about 1.72% and about 6.74%. E15. A method for producing an antibody composition having ADCC%, Y, said method comprising: i. determining the % total nonfucosylated (TAF) glycans, X, of the antibody composition; and ii. if X is equal to (Y-2.6) / 24.1 (where, optionally, X is greater than or equal to about X-0.4 and less than or equal to about X+0.4 and the ADCC% is greater than about Y-17 and less than or equal to about Y+17), selecting the antibody composition for one or more downstream processing steps; A method comprising: E16. A method for producing an antibody composition having an ADCC%, the method comprising: i. determining the % nonfucosylated glycans and % high mannose glycans of the antibody composition; and ii.Equation B: Y = (0.24 + 27 × HM + 22.1 × AF) [Equation B] where Y is ADCC %, HM is high mannose glycan % determined in step (i), and AF is non-fucosylated glycan % determined in step (i). selecting the antibody composition for one or more downstream process steps if it is related to Y according to A method comprising: E17. The method of embodiment 16, wherein Y is from about 40 to about 175, optionally from about 41 to about 171; AF is from about 1 to about 4; and HM is from about 40 to about 175. E18. The method of embodiment 16, wherein Y is from about 30 to about 185, optionally from about 32 to about 180, HM is from about 1 to about 4, and AF is from about 30 to about 185. E19. The method of embodiment 16, wherein the ADCC% of the antibody composition is within the range defined by Y. E20. The method of embodiment 19, wherein the ADCC% of the antibody composition is within the range of Y±18. E21. The method of any one of embodiments 16, 19 and 20, wherein AF is from about 1 to about 4. E22. The method of embodiment 21, wherein the % high mannose glycans is a value within a range defined by HM, optionally the range is HM±1. The method of any one of embodiments 16, 19 and 20, wherein E23.HM is about 1 to about 4. E24. The method of embodiment 24, wherein the % nonfucosylated glycans is a value within a range defined by AF, optionally the range is AF±1. E25. The method according to any one of embodiments 1 to 24, wherein % TAF glycans is determined by calculating the sum of % high mannose glycans and % non-fucosylated glycans. E26. The method of any one of embodiments 1-25, wherein the % high mannose glycans and % nonfucosylated glycans are determined by hydrophilic interaction chromatography. E27. The method of claim 26, wherein the % high mannose glycans and % nonfucosylated glycans are determined by the method described in Example 1. E28. The method of any one of embodiments 1 to 27, wherein ADCC% is determined by a quantitative cell-based assay that measures the ability of antibodies of the antibody composition to mediate cytotoxicity in a dose-dependent manner in cells that express the antibody's antigen and engage Fc gamma RIIIA receptors on effector cells via the antibody's Fc domain. E29. The method of embodiment 28, wherein ADCC % is determined by the assay described in Example 2. E30. The method of any one of embodiments 1, 2 and 5-19, wherein the determining step is performed after the recovering step. E31. The method of embodiment 30, wherein the determining step is performed after the chromatography step. E32. The method of embodiment 31, wherein the chromatography step is a Protein A chromatography step. E33. The method of any one of embodiments 1-32, wherein the one or more downstream processing steps comprise a dilution step, a filling step, a filtration step, a formulation step, a chromatography step, a viral filtration step, a viral inactivation step, or a combination thereof. E34. The method of embodiment 33, wherein the chromatography step is an ion exchange chromatography step, optionally a cation exchange chromatography step or an anion exchange chromatography step. E35. The method of any one of embodiments 1-34, wherein each antibody of the antibody composition is an IgG. E36. The method of embodiment 35, wherein each antibody of the antibody composition is an IgG1. E37. The method of any one of embodiments 1 to 36, wherein each antibody of the antibody composition binds to a tumor-associated antigen. E38. The method of embodiment 37, wherein the tumor-associated antigen comprises the amino acid sequence of SEQ ID NO:3. E39. The method of any one of embodiments 1-38, wherein each antibody of the antibody composition is an anti-D20 antibody. E40. Each antibody of the antibody composition is i. a light chain (LC) CDR1 comprising the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 4, or a variant amino acid sequence of SEQ ID NO: 4 having one or two amino acid substitutions; ii. LC CDR2 comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 5, or a variant amino acid sequence of SEQ ID NO: 5 having one or two amino acid substitutions; iii. an LC CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 6, or a variant amino acid sequence of SEQ ID NO: 6 having one or two amino acid substitutions; iv. a heavy chain (HC) CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, or a variant amino acid sequence of SEQ ID NO: 7 having one or two amino acid substitutions; v. HC CDR2 comprising the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 8, or a variant amino acid sequence of SEQ ID NO: 8 having one or two amino acid substitutions; and vi. HC CDR3 comprising the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 9, or a variant amino acid sequence of SEQ ID NO: 9 having one or two amino acid substitutions. 40. The method of any one of embodiments 1 to 39, comprising: E41. The method of any one of embodiments 1 to 40, wherein each antibody of the antibody composition comprises an LC variable region comprising the amino acid sequence of SEQ ID NO: 10, an amino acid sequence that is at least 90% identical to SEQ ID NO: 10, or a variant amino acid sequence of SEQ ID NO: 10 having 1 to 10 amino acid substitutions. E42. The method of any one of embodiments 1 to 41, wherein each antibody of the antibody composition comprises an HC variable region comprising the amino acid sequence of SEQ ID NO: 11, an amino acid sequence that is at least 90% identical to SEQ ID NO: 11, or a variant amino acid sequence of SEQ ID NO: 11 having 1 to 10 amino acid substitutions. E43. The method of any one of embodiments 1 to 42, wherein each antibody of the antibody composition comprises a light chain comprising the amino acid sequence of SEQ ID NO: 12, an amino acid sequence that is at least 90% identical to SEQ ID NO: 12, or a variant amino acid sequence of SEQ ID NO: 12 having 1 to 10 amino acid substitutions. E44. The method of any one of embodiments 1 to 43, wherein each antibody of the antibody composition comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 13, an amino acid sequence that is at least 90% identical to SEQ ID NO: 13, or a variant amino acid sequence of SEQ ID NO: 13 having 1 to 10 amino acid substitutions. E45. A method for producing an antibody composition having an ADCC% within a target range, the method comprising: i. measuring the ADCC% of a series of samples containing various glycoforms of the antibody; ii. determining the % total nonfucosylated (TAF) glycans for each sample in the series; iii. determining the linear equation for a best-fit line of a graph plotting the % ADCC measured in step (i) as a function of the % TAF glycan determined in step (ii) for each sample in the series; iv. determining the TAF% of the antibody composition and then calculating the ADCC% using the linear equation of step (iii); and v. If the ADCC % calculated in step (iv) is within the target ADCC % range, selecting the antibody composition for one or more downstream processing steps. A method comprising: E46. A method for producing an antibody composition having a % Total Nonfucosylated (TAF) within a target range, the method comprising: i. measuring the ADCC% of a series of samples containing various glycoforms of the antibody; ii. determining the % total nonfucosylated (TAF) glycans for each sample in the series; iii. determining the linear equation for a best-fit line of a graph plotting the % ADCC measured in step (i) as a function of the % TAF glycan determined in step (ii) for each sample in the series; iv. determining the ADCC% of the antibody composition and then calculating the TAF% using the linear equation of step (iii); and v. If the TAF% calculated in step (iv) is within the target TAF% range, selecting the antibody composition for one or more downstream processing steps. A method comprising: E47. A method for determining the antibody-dependent cellular cytotoxicity (ADCC) % of an antibody composition, said method comprising: i. determining the % total nonfucosylated (TAF) glycans of the antibody composition; ii.Equation A: Y=2.6+24.1×X [Equation A] where Y is the ADCC % and X is the TAF glycan % determined in step (i). calculating the ADCC% of the antibody composition based on the TAF% using A method comprising: E48. A method for determining the antibody-dependent cellular cytotoxicity (ADCC) % of an antibody composition, said method comprising: i. determining the % high mannose glycans and % nonfucosylated glycans of the antibody composition; ii.Equation B: Y = (0.24 + 27 × HM + 22.1 × AF) [Equation B] where Y is ADCC %, HM is high mannose glycan % determined in step (i), and AF is non-fucosylated glycan % determined in step (i). calculating the ADCC % of the antibody composition based on the % high mannose glycans and the % non-fucosylated glycans using A method comprising: The method of embodiment 47 or 48, further comprising the step of selecting the antibody composition for one or more downstream processing steps if E49.Y is within the target ADCC % range. A method for producing an antibody composition having an E50.TAF% within a target range, comprising the steps of: i. generating a linear equation of a best-fit graph by plotting ADCC % and TAF glycan % for a series of at least five reference antibody compositions produced under cell culture conditions, each reference antibody composition having the same amino acid sequence as the antibody composition; ii. selecting a target TAF glycan % range based on the linear equation generated in step (i) and the desired % ADCC activity; iii: culturing the antibody composition under cell culture conditions; iv. purifying the antibody composition; v. Sampling the antibody composition to determine the TAF%, and vi. Determining whether the TAF% of the antibody composition is within the target TAF% range of step (ii). E51. The method of embodiment 50, further comprising the step of selecting the antibody composition for one or more downstream process steps if the TAF% calculated in step (v) is within the target TAF% range.

[0167] The following examples are presented merely to illustrate the present invention and are not intended to limit the scope of the present invention in any way. [Example]

[0168] Example 1 This example describes an exemplary method for determining the N-linked glycosylation profile of an antibody.

[0169] The purpose of this analytical method is to determine the N-linked glycosylation profile of a specific antibody in an antibody-containing sample by hydrophilic interaction chromatography. This glycan mapping method is a quantitative purity analysis of the N-linked glycan distribution of an antibody. Briefly, N-linked glycans are enzymatically released using N-glycosidase F (PNGase F), and the terminal N-acetylglucosamine (GlcNAc) is derivatized with a fluorophore. The labeled glycans are then separated using a hydrophilic interaction column (HILIC). The analytical method consists of the following steps: (1) releasing and labeling N-linked glycans from reference and test samples using PNGase F and a fluorophore that can specifically derivatize the released glycans; (2) loading the sample within the effective linear range onto the HILIC column and separating the labeled N-linked glycans using a gradually decreasing organic solvent gradient; and (3) monitoring the elution of glycan species using a fluorescence detector.

[0170] Standards and test samples are prepared by performing the following steps: (1) diluting the samples and controls with water; (2) adding GNase F to the samples and controls and incubating to release N-linked glycans; (3) mixing with a fluorophore-labeled solution using a fluorophore such as 2-aminobenzoic acid, vortexing the samples and controls, and incubating; (4) centrifuging to precipitate proteins and removing the supernatant; and (5) drying and reconstituting the labeled glycans in injection solution.

[0171] The reagents used in this assay are Mobile Phase A (100 mM ammonium formate, target pH 3.0) and Mobile Phase B (acetonitrile). The equipment used to carry out the steps of this method has the following capabilities:

[0172] [Table B-1]

[0173] The HPLC instrument settings using a 1.7 μm column for hydrophilic interaction analysis, 2.1 mm inner diameter x 150 mm, are as follows:

[0174] [Table B-2]

[0175] The recommended gradients are as follows:

[0176] [Table B-3]

[0177] The system conforms to the following:

[0178] [Table B-4]

[0179] Results are reported as follows:

[0180] [Table B-5]

[0181] Representative glycan map chromatograms are shown in Figure 2A (full scale) and Figure 2B (enlarged scale).

[0182] Example 2 This example describes an exemplary assay to assess the ADCC activity of anti-CD20 antibodies using engineered effector cells.

[0183] The purpose of this analytical method is to determine the antibody-dependent cellular cytotoxicity (ADCC) level (expressed as a %) of an antibody. This ADCC bioassay is a quantitative cell-based assay that measures the ability of an anti-CD20 antibody to mediate cytotoxicity in CD20-expressing B lymphocytes in a dose-dependent manner by binding to the CD20 antigen on WIL2-S (human B lymphocytes) and engaging the FcγRIIIA (158V) receptor on NK92-M1 effector cells via the antibody's Fc domain. This activates the effector cells and leads to tumor cell destruction via exocytosis of the cytolytic granule complex perforin / granzyme. A schematic of the ADCC assay is shown in Figure 3, and a representative dose-response curve for the ADCC assay is shown in Figure 4. In Figure 4, each dose point is the mean ± standard deviation of three replicates, and the assay signal = fluorescence.

[0184] The method comprises the following steps:

[0185] [Table B-6]

[0186] Standards and test samples are prepared by diluting the reference standards, assay controls, and samples to cover the validated dose range.

[0187] The reagents used in this assay include the following, with the composition indicated:

[0188] [Table B-7]

[0189] Some steps of this method require a microplate reader with fluorescence capability.

[0190] System compatibility is as follows:

[0191] [Table B-8]

[0192] Results are reported as relative ADCC %.

[0193] Example 3 This example describes the studies that led to the establishment of a model linking ADCC to glycan levels.

[0194] Representative samples (N=41) of anti-CD20 antibodies produced in small-scale bioreactors were assessed for the levels of the following glycoforms: high mannose, nonfucosylated, and galactosylated glycoforms using the exemplary method described in Example 1. The % total nonfucosylated (TAF%) is the sum of the % high mannose and % nonfucosylated. The ADCC level of each representative sample of anti-CD20 antibody was determined by the assay described in Example 2. The results are shown in Table 1.

[0195] [Table 1-1]

[0196] [Table 1-2]

[0197] The data in Table 1 were analyzed using the JMP suite of computer programs for statistical analysis (SAS Institute, Cary, NC). A regression plot of the data is shown in Figure 5A. The line of best fit for the plotted data is shown in this figure and can be described by the following linear equation, Equation 1: ADCC%=2.6129696497+24.071940292×TAF% [Equation 1].

[0198] Additional statistical parameters are shown in Figure 5B. As shown in this figure, the significance of the association between ADCC and TAF was determined by r 2 value (r 2 =0.88) and p-value (p<0.0001).

[0199] Using Equation 1 and the TAF values ​​in Table 1, the predicted ADCC% value was calculated for each sample in Table 1. In Figure 5C, the actual ADCC% (listed in Table 1) was plotted against the predicted ADCC%. The results confirmed that there was a positive correlation between total nonfucosylation and ADCC, with higher levels of total nonfucosylation associated with higher ADCC activity.

[0200] Figure 5D is the same graph as Figure 5A, but with a graphical depiction of the 95% confidence interval (shown as the light blue region). As shown in Figure 5D, most of the data points fall within the 95% confidence interval. Figure 5E shows a graph of the 95% confidence region for both the y-intercept and slope of Equation 1.

[0201] The data in Table 1 for the individual components of TAF (nonfucosylated (AF) and high mannose (HM)) were also analyzed using the JMP suite and showed a similar correlation with ADCC. Figures 6A and 6B show regression plots for these data for high mannose and nonfucosylated. The best-fit lines for the plotted data are shown in Figures 6A and 6B, respectively, and can be described by the following linear equation, Equation 2: ADCC%=0.2358435425+27.030822634×HM%+22.12397042×AF% [Equation 2].

[0202] Additional statistical parameters are shown in Figure 6C. As shown in this figure, the significance of the association between ADCC and TAF was determined by r 2 value (r 2 =0.88) and p-value (p<0.0001).

[0203] Using Equation 2 and the high mannose and nonfucosylation values ​​from Table 1, the predicted ADCC % was calculated for each sample in Table 1. In Figure 6D, the actual ADCC % (listed in Table 1) is plotted against the predicted ADCC %. The results confirmed a positive correlation between nonfucosylated glycans, high mannose, and ADCC, with higher levels of nonfucosylated glycans and high mannose associated with higher ADCC activity. Nonfucosylated glycans and high mannose contributed equally to ADCC activity.

[0204] The association between ADCC and HM and AF (or TAF) was specific to these glycans, as galactosylation did not show a statistically significant association. Figure 7A shows a regression plot between ADCC and galactosylation levels. The lack of statistical significance is due to the r 2 value (r 2 = 0.02) and p-value (p < 0.3715). Figure 7B is a graph of the % actual ADCC (listed in Table 1) plotted as a function of predicted ADCC. As shown in these figures, only a very weak association was observed between ADCC and galactosylation.

[0205] Statistical analysis confirmed that TAFs contributed most significantly to ADCC activity. The correlation between TAF levels and ADCC activity levels was significantly different from the relationship between ADCC% and other glycans.

[0206] Example 4 This example describes studies validating a model linking ADCC to TAF.

[0207] The model relating ADCC to TAF described in Example 3 was validated using large-scale production samples of the same antibody as the large-scale bioreactor samples in Table 1. Each large-scale sample (N=13) was characterized for TAF levels by measuring high mannose and non-fucosylation levels according to the method described in Example 1 and then summing the two percentages to obtain the % TAF level. The experimental ADCC level for each large-scale sample was determined by performing the assay described in Example 2, repeating twice to obtain three values ​​per sample, and then recording the average of the three values. The predicted ADCC was calculated using Equation 1. The results are shown in Table 2 below.

[0208] [Table 2]

[0209] As shown by the data in Table 2, the predicted ADCC results generated by Equation 1 are in good agreement with the reported experimental results. Therefore, a reliable and accurate model related to ADCC and TAF was established.

[0210] Example 5 This example describes a novel glycan model that provides a basis for predicting ADCC of anti-CD20 antibodies.

[0211] Anti-CD20 antibodies are being developed as biosimilars of rituximab. They are recombinant chimeric mouse / human IgG1 monoclonal antibodies that specifically bind to the CD20 antigen expressed on B cells and promote B cell killing through multiple mechanisms, with ADCC being one of their key mechanisms of action. It has been established that the absence of core fucose leads to increased ADCC activity, while galactosylation and high mannose content may also play a role. A systematic evaluation of the contribution of N-glycans to the ADCC activity of anti-CD20 antibodies was performed through glycoengineering studies, and it was confirmed that there was a positive correlation between nonfucosylated glycans, high mannose content, and ADCC, with higher levels of nonfucosylated glycans and high mannose content associated with increased ADCC activity. However, the glycan profiles of samples generated by glycoengineering may not fully represent the full range of glycan attributes of anti-CD20 antibodies. Therefore, a statistical evaluation of small-scale bioreactor datasets of anti-CD20 antibodies was performed to establish a representative glycan ADCC model by capturing the full range in the anti-CD20 antibody manufacturing process. This approach revealed that nonfucosylation and high mannose showed similar correlations with ADCC. A novel methodology was applied to the glycan model to predict anti-CD20 antibody ADCC using total nonfucosylation (the sum of nonfucosylation and high mannose). A prediction equation (ADCC = 2.6 + 24.1 × total nonfucosylation) was established and validated using large-scale manufacturing data. The predicted ADCC results obtained by the equation were in good agreement with reported ADCC assay results. Therefore, the correlation between total nonfucosylation and ADCC was established as a glycan-ADCC model, enabling the process of monitoring ADCC using glycan measurements as an orthogonal method.

[0212] The results of this study identified the basis for the correlation of glycans with ADCC and functional assays between anti-CD20 antibodies and orthogonal methods (HPLC glycan methods). This data enabled Amgen to advance an attribute-focused development approach and identified mechanisms to consider results and provide novel attribute analyses for market applications.

[0213] Approaches used included HPLC, ADCC assays and cross-functional collaboration

[0214] Example 6 This example presents studies that led to the establishment of a model linking ADCC to glycan levels for second antibodies.

[0215] Example 3 describes studies that led to the establishment of a model linking ADCC to glycan levels for IgG1 that binds to CD20. This study evaluates the relationship between ADCC and glycan levels for a chimeric monoclonal IgG1 kappa antibody composed of a human constant region and a mouse variable region that binds to the TNFα antigen.

[0216] Representative samples of a second antibody (anti-TNFα antibody) produced in a small-scale bioreactor were assessed for the levels of the following glycoforms: high mannose and non-fucosylation using the exemplary method described in Example 1. The percentage of total non-fucosylation (TAF%) is the sum of high mannose % and non-fucosylation %. The ADCC level of each representative sample of anti-TNFα antibody was determined by the assay described in Example 2. The data were analyzed using the JMP suite of computer programs for statistical analysis (SAS Institute, Cary, NC). A regression plot of the data is shown in Figure 8A. The line of best fit for the plotted data is shown in this figure and can be described by the following linear equation, Equation 3: ADCC%=9.3+12.47×TAF% [Equation 3].

[0217] Additional statistical parameters are shown in Figure 8B. As shown in this figure, the significance of the association between ADCC and TAF was determined by r 2 value (r 2 =0.80) and p-value (p<0.0001).

[0218] Using Equation 3 and the measured TAF values, the predicted ADCC% was calculated for each sample. In Figure 8C, the actual ADCC% (measured as described in Example 2) is plotted against the predicted ADCC%. The results confirmed a positive correlation between total nonfucosylation and ADCC, with higher levels of total nonfucosylation associated with higher ADCC activity.

[0219] Figure 8D is the same graph as Figure 8A, but with a graphical depiction of the 95% confidence interval (shown as the gray shaded area). As shown in Figure 8D, most of the data points fall within the 95% confidence interval. Figure 8E shows a graph of the 95% confidence interval for both the y-intercept and slope of Equation 3.

[0220] Data from the individual components of TAF (nonfucosylated (AF) and high mannose (HM)) were also analyzed using the JMP suite and showed a similar correlation to ADCC. Figures 9A and 9B show regression plots of these data for high mannose and nonfucosylated, respectively. The best-fit lines of the plotted data are shown in Figures 9A and 9B, respectively, and can be described by the following linear equation, Equation 4: ADCC%=8.66+12.86×HM%+12.37×AF% [Equation 4].

[0221] Additional statistical parameters are shown in Figure 9C. As shown in this figure, the significance of the association between ADCC and TAF was determined by r 2 value (r 2 =0.8) and p-value (p<0.0001).

[0222] Using Equation 4 and the measured high mannose and nonfucosylation values, the predicted ADCC % was calculated for each sample. In Figure 9D, the actual ADCC % (measured as described in Example 2) is plotted against the predicted ADCC %. The results confirmed a positive correlation between nonfucosylated glycans, high mannose, and ADCC, with higher levels of nonfucosylated glycans and high mannose corresponding to higher ADCC activity. Nonfucosylated glycans and high mannose contributed equally to ADCC activity.

[0223] This example demonstrated that for a second antibody (anti-TNFα antibody), statistical analysis confirmed that TAFs showed a highly significant contribution to ADCC activity.

[0224] Example 7 This example demonstrates a second set of models linking ADCC to TAF, HM and / or AF glycans.

[0225] Examples 3 and 6 each establish a linear regression model relating ADCC to TAF glycan content or ADCC to HM and AF glycan content for two antibodies, an anti-CD20 antibody and an anti-TNF alpha antibody: in an exemplary embodiment, the CD20 antibody is an anti-TNF alpha antibody. The models are mathematically described in Equations 1-4. For each of these equations, the significance of the y-intercept was assessed by analyzing the p-value of the y-intercept for each equation. Table 3 shows the p-values ​​of the y-intercepts for each of Equations 1-4.

[0226] [Table 3]

[0227] Because each p-value was greater than 0.05, each y-intercept in Equations 1-4 was considered close to zero and could be removed from the equation.

[0228] Taking the above into consideration, the measured ADCC data and the measured glycan data were refitted to a "y-intercept-free model" and the statistical significance of these models was assessed. Table 4 lists the equations for the y-intercept-free model that describe the relationship between ADCC and TAF glycans, or ADCC and HM and AF glycans, for the two antibodies.

[0229] [Table 4]

[0230] As shown in Table 4, the no y-intercept model was statistically significant and represents an alternative model correlating ADCC to TAF glycan content or ADCC to HM and AF glycan content.

[0231] Table 5 shows the slopes of the linear regression model and the model without a y-intercept.

[0232] [Table 5]

[0233] As shown in Table 5, the two models show high agreement with each other. The x-intercepts for TAF in the linear regression model and the no-y-intercept model (24.07070 vs. 24.73579) were very close in value. The same was observed for HM (27.03082 vs. 27.14941) and AF (22.12397 vs. 22.12018) glycans, respectively.

[0234] Example 8 This example demonstrates that the ADCC-TAF and ADCC-HM / AF models are interchangeable.

[0235] The predicted ADCC was calculated using Equation 6 in Table 4, which correlates ADCC to HM and AF glycan content. The predicted ADCC was plotted against the predicted ADCC calculated according to Equation 5 in Table 4, which correlates ADCC to TAF glycan content. The results are shown in the graph in Figure 10A. The same procedure was performed for Equations 7 and 8 in Table 4 and graphed in Figure 10B. The equations for the best-fit lines are shown below each graph. As shown in these figures and equations, the models show high agreement with each other (p<0.0001). The slopes are near 1.0 (0.97 or 0.98). These data support that the ADCC of antibody compositions can be predicted based on one glycan type (TAF glycan) versus two glycan types (HM and AF). These data also suggest that for antibody compositions with a target ADCC, a target TAF can be calculated and either the HM or AF can be modified to achieve the target TAF. The method of modifying the HM or AF of an antibody composition is simpler than the combined method of modifying both the HM and AF.

[0236] All references cited herein (e.g., publications, patent applications, and patents) are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0237] In connection with the description of this disclosure (and particularly in connection with the claims which follow), use of the terms "a," "an," and "the," and similar referents should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprise," "have," "include," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise specified.

[0238] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each of the separate values ​​within that range, and each of the endpoints, unless otherwise indicated herein, and each separate value and endpoint is incorporated herein as if it were individually recited herein.

[0239] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any examples or exemplary language (e.g., "etc.") provided herein is intended merely to further clarify the disclosure and does not impose limitations on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0240] Preferred embodiments of the present disclosure have been described herein, including the best mode known to the inventors for carrying out the disclosure. Variations of these preferred embodiments will be apparent to those skilled in the art upon reading the foregoing description. The inventors expect that those skilled in the art will adopt such variations as appropriate, and the inventors intend for the present disclosure to be practiced in forms other than those specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, any combination of the above-described elements in all possible variations thereof is encompassed by the present disclosure unless otherwise indicated herein or clearly contradicted by context. The present invention provides, for example, the following items. (Item 1) 1. A method for determining the product quality of an antibody composition, wherein the ADCC activity level of the antibody composition is a basis for the product quality of the antibody composition, the method comprising: i. determining the total nonfucosylated (TAF) glycan content of a sample of the antibody composition; and ii. determining the product quality of the antibody composition as acceptable and / or achieving ADCC activity level criteria if the TAF glycan content determined in (i) is within a target range. Including, the target range of TAF glycan content is based on (1) a target range of ADCC activity levels of a reference antibody, and (2) a first model correlating the ADCC activity level of the antibody composition with the TAF glycan content of the antibody composition; The method, wherein the ADCC predicted by the first model is about 95% to about 105% of the ADCC predicted by the second model, and wherein the second model correlates the ADCC activity level of the antibody composition with the high mannose (HM) glycan content of the antibody composition and the nonfucosylated (AF) glycan content of the antibody composition. (Item 2) 2. The method of claim 1, wherein the ADCC predicted by the first model is about 100% of the ADCC predicted by the second model. (Item 3) 3. The method according to item 1 or 2, wherein the p-value of the first model is less than 0.0001 and / or the p-value of the second model is less than 0.0001. (Item 4) 4. The method of any one of items 1 to 3, wherein the ADCC activity level predicted by the first model is about 12Q x TAF%, where Q is the number of antibody binding sites on the antigen to which the antibody binds, and TAF% is the TAF glycan content of the antibody composition. (Item 5) Item 5. The method of item 4, wherein Q is 2. (Item 6) 6. The method according to any one of items 1 to 5, wherein the ADCC activity level predicted by the first model is about 24×TAF%. (Item 7) 7. The method of any one of items 1 to 6, wherein the ADCC activity level predicted by the second model is about 27×HM%+about 22×AF%, where AF% is the AF glycan content of the antibody composition and HM% is the HM glycan content of the antibody composition. (Item 8) Item 5. The method of item 4, wherein Q is 1. (Item 9) 9. The method according to any one of items 1 to 4 and 8, wherein the ADCC activity level predicted by the first model is about 12×TAF%. (Item 10) 9. The method according to any one of items 1 to 3, 7 and 8, wherein the ADCC activity level predicted by the second model is about 14.8×HM%+about 12.8×AF%. (Item 11) 11. The method of any one of items 1 to 10, wherein the reference antibody is rituximab. (Item 12) 12. The method of any one of items 1 to 11, wherein the reference antibody is infliximab. (Item 13) 13. The method of any one of items 1 to 12, wherein the method is a quality control (QC) assay. (Item 14) 14. The method according to any one of items 1 to 13, wherein the method is an in-process QC assay. (Item 15) 15. The method according to any one of items 1 to 14, wherein the sample is a sample of in-process material. (Item 16) 16. The method according to any one of items 1 to 15, wherein the TAF glycan content is determined before or after collection. (Item 17) 17. The method according to any one of items 1 to 16, wherein the TAF glycan content is determined after a chromatography step. (Item 18) Item 18. The method according to item 17, wherein the chromatography step comprises capture chromatography, intermediate chromatography, and / or polish chromatography. (Item 19) 19. The method of claim 17 or 18, wherein the TAF glycan content is determined after viral inactivation and neutralization, viral filtration, or buffer exchange. (Item 20) 20. The method according to any one of items 1 to 19, wherein the method is a lot release assay. (Item 21) 21. The method according to any one of items 1 to 20, wherein the sample is obtained from a manufacturing lot. (Item 22) 22. The method of any one of items 1 to 21, further comprising selecting the antibody composition for downstream processing if the TAF glycan content is within a target range. (Item 23) 23. The method of any one of items 1 to 22, wherein if the TAF glycan content determined in (i) is not within the target range, one or more conditions of the cell culture are modified to obtain a modified cell culture. (Item 24) 24. The method of claim 23, further comprising determining the TAF glycan content of a sample of the antibody composition obtained after modifying one or more conditions of the cell culture. (Item 25) 25. The method of any one of items 1 to 24, wherein if the TAF glycan content determined in (i) is not within the target range, the method further comprises: (iii) modifying one or more conditions of the cell culture to obtain an engineered cell culture; and (iv) determining the TAF glycan content of a sample of the antibody composition obtained from the engineered cell culture. (Item 26) 26. The method of claim 25, wherein if the TAF glycan content determined in (i) is not within the target range, the method further comprises (iii) and (iv) until the TAF glycan content determined in (iv) is within the target range. (Item 27) 27. The method of any one of items 1 to 26, wherein an assay that directly measures the ADCC activity of the antibody composition is performed on the antibody composition only if the TAF glycan content is outside the target range. (Item 28) 28. The method of any one of items 1 to 27, wherein an assay that directly measures the ADCC activity of the antibody composition is not performed on the antibody composition if the TAF glycan content is within the target range. (Item 29) 25. The method of item 23 or 24, wherein the assay for directly measuring ADCC activity of the antibody composition is a cell-based assay that measures the release of a detectable agent upon lysis of antigen-expressing cells containing the detectable agent by effector cells that bind to an antibody that binds to both the antigen-expressing cells and the effector cells. (Item 30) 30. A method for monitoring the product quality of an antibody composition, comprising determining the product quality of the antibody composition using a first sample obtained at a first time point and a second sample collected at a second time point different from the first time point according to the method of any one of items 1 to 29. (Item 31) Item 31. The method of item 30, wherein each of the first sample and the second sample is a sample of an in-process material. (Item 32) 31. The method of claim 30, wherein the first sample is a sample of in-process material and the second sample is a sample of a manufacturing lot. (Item 33) 31. The method of claim 30, wherein the first sample is a sample obtained before modifying one or more conditions of the cell culture, and the second sample is a sample obtained after modifying one or more conditions of the cell culture. (Item 34) 30. A method for producing an antibody composition, comprising determining a product quality of the antibody composition, wherein the product quality of the antibody composition is determined according to the method of any one of items 1 to 29, wherein the sample is in-process material, and if the TAF glycan content determined in (i) is not within the target range, the method further comprises (iii) modifying one or more conditions of the cell culture to obtain an engineered cell culture, and (iv) determining the TAF glycan content of a sample of the antibody composition obtained from the engineered cell culture, optionally repeating steps (iii) and (iv) until the TAF glycan content is within the target range. (Item 35) 35. The method of claim 34, wherein one or more conditions of the cell culture are modified to primarily modify the HM glycan content to achieve the target range for the TAF glycan content. (Item 36) 35. The method of claim 34, wherein one or more conditions of the cell culture are modified to primarily modify the AF glycan content to achieve the target range for the TAF glycan content.

Claims

1. 1. A method for producing an antibody composition, comprising: i. determining the product quality of the antibody composition, wherein the antibody-dependent cell-mediated cytotoxicity (ADCC) activity level of the antibody composition is a criterion on which the product quality of the antibody composition is based, and the product quality of the antibody composition is determined by determining the total nonfucosylated (TAF) glycan content of a sample of the antibody composition, the sample being a sample of in-process material of a cell culture comprising cells expressing the antibodies of the antibody composition; ii. if the TAF glycan content is not within the target range, modifying one or more conditions of the cell culture to obtain an engineered cell culture; and iii. Determining the TAF glycan content of a sample of the antibody composition obtained from the engineered cell culture; iv. Repeating (ii) and (iii) as necessary until the TAF glycan content is within the target range. wherein the target range of TAF glycan content is based on (1) a target range of ADCC activity levels of a reference antibody, and (2) a first model correlating the ADCC activity level of the antibody composition with the TAF glycan content of the antibody composition; the ADCC activity level predicted by the first model is 95% to 105% of the ADCC activity level predicted by the second model, wherein the second model correlates the ADCC activity level of the antibody composition with each of the high mannose (HM) glycan content and the non-fucosylated (AF) glycan content of the antibody composition; A method wherein an assay that directly measures the ADCC activity of said antibody composition is not performed on said antibody composition.

2. 2. The method of claim 1, further comprising modifying one or more conditions of the cell culture to primarily alter HM glycan content to achieve the target range of TAF glycan content.

3. 2. The method of claim 1, further comprising modifying one or more conditions of the cell culture to primarily alter AF glycan content to achieve the target range of TAF glycan content.

4. The method of any one of claims 1 to 3, further comprising selecting the antibody composition for downstream processing if the TAF glycan content is within a target range.

5. 1. A method for monitoring the product quality of an antibody composition, wherein the antibody-dependent cell-mediated cytotoxicity (ADCC) activity level of the antibody composition is a fundamental measure of the product quality of the antibody composition, the method comprising: i. determining the total nonfucosylated (TAF) glycan content of a first sample of the antibody composition obtained at a first time point; ii. if the TAF glycan content is not within the target range, modifying one or more conditions of the cell culture to obtain an engineered cell culture; and iii. Determining the TAF glycan content of a second sample taken at a second time point different from the first time point, wherein the second sample is a sample of the antibody composition obtained from the modified cell culture; iv. Repeating (ii) and (iii) as necessary until the TAF glycan content is within the target range. wherein the target range of TAF glycan content is based on (1) a target range of ADCC activity levels of a reference antibody, and (2) a first model correlating the ADCC activity level of the antibody composition with the TAF glycan content of the antibody composition; the ADCC activity level predicted by the first model is 95% to 105% of the ADCC activity level predicted by the second model, wherein the second model correlates the ADCC activity level of the antibody composition with each of the high mannose (HM) glycan content and the non-fucosylated (AF) glycan content of the antibody composition; A method wherein an assay that directly measures the ADCC activity of said antibody composition is not performed on said antibody composition.

6. The method of claim 5 , wherein each of the first sample and the second sample is a sample of an in-process material.

7. 7. The method of claim 6, wherein each of the first sample and the second sample is a sample of in-process material from a cell culture containing cells expressing an antibody of the antibody composition.

8. The method of any one of claims 5 to 7, comprising modifying one or more conditions of the cell culture to primarily alter HM glycan content to achieve the target range of TAF glycan content.

9. The method of any one of claims 5 to 8, comprising modifying one or more conditions of the cell culture to primarily alter AF glycan content to achieve the target range of TAF glycan content.

10. The method of any one of claims 5 to 9, further comprising selecting the antibody composition for downstream processing if the TAF glycan content is within a target range.

11. The method of any one of claims 5 to 10, wherein the TAF glycan content is determined before harvesting.

12. The method of any one of claims 5 to 11, wherein the method is an in-process QC assay.

13. 1. A method for monitoring the product quality of an antibody composition, wherein the antibody-dependent cell-mediated cytotoxicity (ADCC) activity level of the antibody composition is a fundamental measure of the product quality of the antibody composition, the method comprising: i. determining the total nonfucosylated (TAF) glycan content of a first sample of the antibody composition obtained at a first time point; ii. Determining the total nonfucosylated (TAF) glycan content of a second sample of said antibody composition obtained at a second time point different from said first time point. and determining that the product quality of the antibody composition is acceptable and / or achieves an ADCC activity level standard when the TAF glycan content is within a target range; The target range of TAF glycan content is based on (1) a target range of ADCC activity levels of a reference antibody, and (2) a first model correlating the ADCC activity level of the antibody composition with the TAF glycan content of the antibody composition; the ADCC activity level predicted by the first model is 95% to 105% of the ADCC activity level predicted by the second model, wherein the second model correlates the ADCC activity level of the antibody composition with each of the high mannose (HM) glycan content and the non-fucosylated (AF) glycan content of the antibody composition; A method wherein an assay that directly measures the ADCC activity of said antibody composition is not performed on said antibody composition.

14. 14. The method of claim 13, wherein the TAF glycan content is determined after collection.

15. 15. The method of claim 13 or 14, wherein the first sample and / or the second sample are obtained from a manufacturing lot.

16. The method of any one of claims 13 to 15, wherein the method is a lot release assay.

17. 17. The method of any one of claims 1 to 16, wherein the assay for directly measuring ADCC activity is a cell-based assay that measures the release of a detectable agent upon lysis of antigen-expressing cells containing the detectable agent by effector cells that bind to an antibody, wherein the antibody binds to both the antigen-expressing cells and the effector cells.

18. The method of any one of claims 1 to 17, wherein the step of determining the TAF glycan content is the only step required to determine the product quality with respect to the ADCC activity level criterion.

19. The method of any one of claims 1 to 18, wherein the method is a quality control (QC) assay.

20. The method of any one of claims 1 to 19, wherein the reference antibody is rituximab.

21. The method of any one of claims 1 to 19, wherein the reference antibody is infliximab.

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