Method for determining the relative unpaired glycan content

Abstract

A method is provided for determining the relative unpaired glycan content of an IgG antibody composition. In an exemplary embodiment, the method comprises (b) treating the IgG antibody composition with two enzymes to form a mixture of Fab fragments and Fc fragments, wherein one enzyme cleaves the antibody heavy chain at the N-terminal site of the hinge region disulfide bond and the other enzyme cleaves the β1,4 bond between core GlcNAc residues to form Fc fragments, each containing a pair of coreglycan structures; (b) separating the Fab fragments from the Fc fragments; and (c) quantifying the abundance of (i) paired non-fucosylated Fc fragments, (ii) unpaired non-fucosylated Fc fragments, (iii) paired high-mannose Fc fragments, and / or (iv) unpaired high-mannose Fc fragments to determine the relative unpaired non-fucosylated (AF) glycan content and the relative unpaired high-mannose (HM) glycan content.

Description

Technical Field

[0001] Benefit under 35 U.S.C.§119(e) of U.S. Provisional Patent Application No. 63 / 576,448, filed Apr. 20, 2023, is claimed herein, and its disclosure is incorporated herein by reference.

Background Art

[0002] Glycosylation plays a role in multiple cellular functions, such as protein folding, quality control, molecular transport and sorting, and interaction with receptors on the cell surface. The glycan structure of protein drugs affects their therapeutic effect and influences their biological activity, pharmacokinetics, immunogenicity, solubility, and in vivo clearance. The profile of Fc glycoforms is a product quality attribute of recombinant antibodies as it directly affects the clinical efficacy and pharmacokinetics of antibodies. See, e.g., Reusch and Tejada, Glycobiology 25(12):1325-1334(2015); and Boune et al., Antibodies (Basel) 9(2):22(2020).

[0003] Given their importance, the glycan structure bound to recombinant protein-drugs is often monitored during development and manufacturing (Li et al., Front Immunol 8:1554 (2017)). Traditionally, glycans are monitored using release glycan assays, in which the glycans are cleaved from the protein backbone, chromatographically isolated, and identified. Techniques used for the analysis of immunoglobulin glycosylation are outlined in de Haan et al., Glycobiology 30(4):226-240 (2020). The glycan population of IgG therapeutics is inherently heterogeneous and complex because glycosylation requires the coordinated function of multiple metabolic enzymes, and is complicated by the binding of sugar isomers, occupation of glycosylation sites, and external arm glycosylation, which may include one or more of fucose, galactose, bisected GlcNAc, and sialic acid. Further diversification of the antibody glycan population is attributed to the asymmetry of two N-glycans from any heavy chain, and the involvement of random pairs of two different heavy chain glycans.

[0004] Even when successful, current methods used for glycan analysis ignore the fact that many therapeutic protein molecules, including fusion proteins with IgG and IgG Fc regions, contain two glycosylated Fc chains. Since currently commonly used methods involve cleaving glycans from the protein backbone, such methods cannot provide knowledge about the paired states of glycan species within individual protein molecules. For example, while current methods can be used to determine the total abundance of non-fucosylated glycans in a given antibody composition sample, they do not tell us the level of antibodies containing entirely non-fucosylated heavy chain pairs versus antibodies containing only hemi-non-fucosylated heavy chain pairs. Such information about the paired states of proteins has been suggested to be an important feature of antibody compositions affecting their biological activity levels (see, for example, International Publication No. 2022 / 081824). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2022 / 081824 Brochure [Non-patent literature]

[0006] [Non-Patent Document 1] Reusch and Tejada, Glycobiology 25(12):1325-1334(2015) [Non-Patent Document 2] Boune et al., Antibodies (Basel) 9(2):22(2020) [Non-Patent Document 3] Li et al., Front Immunol 8:1554(2017) [Non-Patent Document 4] de Haan et al., Glycobiology 30(4):226-240(2020) [Overview of the project] [Problems that the invention aims to solve]

[0007] Glycan pair analysis is difficult due to the size of the molecules and the highly heterogeneous glycan population. Specifically, non-fucosylated glycan pair analysis is further complicated by the lower abundance of non-fucosylated glycans and high-mannose glycans compared to other glycans. Therefore, determining the abundance of non-fucosylated glycan pairs and revealing the relative abundance of completely non-fucosylated or hemi-non-fucosylated pairs is complex and time-consuming.

[0008] Therefore, the biopharmaceutical industry needs a simple, efficient, and reliable method for determining the unpaired glycan content of IgG antibody compositions. [Means for solving the problem]

[0009] This specification presents data demonstrating the feasibility and advantages of an improved method for determining the relative unpaired glycan content of an IgG antibody composition. The method comprises simplified sample preparation and analysis thereof, ultimately providing a simpler, faster, and more accurate method for determining the relative unpaired glycan content of an IgG antibody composition.

[0010] Accordingly, the present disclosure provides a method for determining the relative unpaired glycan content of an IgG antibody composition. In an exemplary embodiment, the method includes (a) treating an IgG antibody composition with two enzymes to form a mixture of Fab fragments and Fc fragments, wherein one enzyme cleaves the antibody heavy chain at the N-terminal site of the hinge region disulfide bond and the other enzyme cleaves the β1,4 bond between core GlcNAc residues to form Fc fragments, each containing a pair of coreglycan structures; (b) separating the Fab fragments from the Fc fragments; and (c) quantifying the abundance of (i) paired non-fucosylated Fc fragments, (ii) unpaired non-fucosylated Fc fragments, (iii) paired high-mannose Fc fragments, and / or (iv) unpaired high-mannose Fc fragments to determine the relative unpaired non-fucosylated (AF) glycan content and the relative unpaired high-mannose (HM) glycan content of the IgG antibody composition.

[0011] This disclosure also provides a method for preparing an IgG antibody composition having ADCC activity within a target range, for example, a predetermined target range. In an exemplary embodiment, the method includes determining the relative unpaired glycan content of a sample of an IgG antibody composition according to one of the methods of this disclosure for determining the relative unpaired glycan content. The relative unpaired AF glycan content and / or relative unpaired HM glycan content of the IgG antibody composition may be compared to a target range for unpaired AF glycan content and / or relative unpaired HM glycan content. For example, the target range may be a specification or reference level of a reference IgG antibody composition. Alternatively, for example, the target range for unpaired AF glycan content and / or relative unpaired HM glycan content may be a target range for unpaired AF glycan content and / or relative unpaired HM glycan content that statistically correlates with the target range of ADCC activity level of a reference IgG antibody composition.

[0012] Further methods for analyzing IgG antibody compositions are provided. In exemplary embodiments, the method includes determining the relative unpaired glycan content of a sample of an IgG antibody composition according to one of the methods of the present disclosure for determining the relative unpaired glycan content. In various embodiments, the method includes comparing the relative unpaired AF glycan content and / or relative unpaired HM glycan content of the IgG antibody composition with the relative unpaired AF glycan content and / or relative unpaired HM glycan content of a reference product. The method may be carried out in manufacturing, for example, to determine whether many IgG antibody compositions meet specifications.

[0013] A method for monitoring the production of an IgG antibody composition is further provided. In an exemplary embodiment, the method includes determining the relative unpaired glycan content of an IgG antibody composition according to one of the methods of the present disclosure for determining the relative unpaired glycan content of a first sample obtained at a first time point and a second sample obtained at a second time point different from the first time point. In an exemplary embodiment, the method includes comparing the relative unpaired AF glycan content and / or relative unpaired HM glycan content of the first sample with the relative unpaired AF glycan content and / or relative unpaired HM glycan content of the second sample.

[0014] Methods for generating antibody compositions are also provided in this disclosure. In exemplary embodiments, the method is to determine the relative unpaired glycan content of an antibody composition, determined according to any one of the methods of the disclosure for determining the relative unpaired glycan content, and if it is determined that the relative unpaired glycan content is outside a predetermined target range, the method further includes modifying one or more conditions of a cell culture to obtain a modified cell culture and determining the relative unpaired glycan content. In various examples, the method includes repeating the steps of the method until the relative unpaired glycan content falls within a predetermined target range.

[0015] Further exemplary embodiments and aspects of this disclosure are provided below. [Brief explanation of the drawing]

[0016] [Figure 1A] This is an illustrative diagram of a glycan structure. [Figure 1B] This is an illustrative diagram of an example group of glycans. [Figure 2] This diagram shows the salvage and de novo pathways of fucose metabolism. [Figure 3A] Figure 3A is a typical glycan map chromatogram obtained by HILIC (full-scale view; y-axis maximum approximately 440.00 EU). Figure 3B is a typical glycan map chromatogram obtained by HILIC (enlarged-scale view; y-axis maximum approximately 44.00 EU). [Figure 3B] FIG. 3A is a representative glycan map chromatogram (full scale view; y-axis maximum of about 440.00 EU) obtained by HILIC. FIG. 3B is a representative glycan map chromatogram (expanded scale view; y-axis maximum of about 44.00 EU) obtained by HILIC. [Figure 4A] FIG. 4A is a series of diagrams illustrating antibodies having unpaired or paired non-fucosylated glycans and unpaired or paired high-mannose glycans. FIG. 4B is a diagram providing an exemplary flowchart useful for assigning nomenclature to glycan pairs containing canonical glycans. As shown in the exemplary flowchart of Box 1, if the glycan pair contains 0 non-fucosylated glycans (e.g., both glycans are fucosylated), the glycan pair is "paired fucosylated". If both glycans of the glycan pair are non-fucosylated, the glycan pair is "paired non-fucosylated". If only one glycan is non-fucosylated, the glycan pair is "unpaired non-fucosylated". As shown in the exemplary flowchart of Box 2, if the glycan pair contains 0 high-mannose glycans, Box 1 is used to determine the glycan pair classification. If both glycans contain high-mannose and are non-fucosylated, the glycan pair is "paired high-mannose". If only one glycan of the glycan pair contains high-mannose and is non-fucosylated and the other glycan lacks high-mannose and is fucosylated, the glycan pair is "unpaired high-mannose". If only one glycan of the glycan pair contains high-mannose and the other chain is non-fucosylated, the glycan pair is "paired non-fucosylated". From the perspective of high-mannose, this glycan pair is "unpaired high-mannose". Further information useful for assigning nomenclature to glycan pairs is provided herein. See, for example, Example 1. [Figure 4B]Figure 4A is a series of diagrams illustrating antibodies having unpaired or paired non-fucosylated glycans and unpaired or paired high-mannose glycans. Figure 4B is a diagram providing an exemplary flowchart useful for assigning nomenclature to glycan pairs that include canonical glycans. As shown in the exemplary flowchart of Box 1, if the glycan pair includes 0 non-fucosylated glycans (e.g., both glycans are fucosylated), the glycan pair is "paired fucosylated". If both glycans of the glycan pair are non-fucosylated, the glycan pair is "paired non-fucosylated". If only one glycan is non-fucosylated, the glycan pair is "unpaired non-fucosylated". As shown in the exemplary flowchart of Box 2, if the glycan pair includes 0 high-mannose glycans, Box 1 is used to determine the glycan pair classification. If both glycans include high-mannose and are non-fucosylated, the glycan pair is "paired high-mannose". If only one glycan of the glycan pair includes high-mannose and is non-fucosylated, and the other glycan lacks high-mannose and is fucosylated, the glycan pair is "unpaired high-mannose". If only one glycan of the glycan pair includes high-mannose and the other chain is non-fucosylated, the glycan pair is "paired non-fucosylated". From the perspective of high-mannose, this glycan pair is "unpaired high-mannose". Further information useful for assigning nomenclature to glycan pairs is provided herein. See, for example, Example 1. [Figure 5] It is an explanatory diagram of enzyme digestion. [Figure 6A] Figure 6A is a diagram showing exemplary chromatographic peaks obtained from a 95-minute HILIC-MS separation in which the Fab fragment is separated from the glycosylated Fc fragment. The inset shows an enlarged view of the glycosylated Fc fragment peak. Figure 6B is a diagram showing a further enlarged view of the glycosylated Fc fragment peak. The identity of the glyco-pairs of each peak is recorded. Those labeled in bold indicate the relevant glyco-pairs. [Figure 6B]Figure 6A shows exemplary chromatographic peaks obtained from 95-minute HILIC-MS separation, where the Fab fragment is separated from the glycosylated Fc fragment. The inset shows a magnified view of the glycosylated Fc fragment peak. Figure 6B shows a further magnified view of the glycosylated Fc fragment peak. The identity of the glycopairs for each peak is recorded. Those labeled in bold indicate the associated glycopairs. [Figure 7] Examples of unpaired non-fucosylated Fc fragments, paired non-fucosylated Fc fragments, unpaired high-mannose Fc fragments, and paired high-mannose Fc fragments after enzyme digestion are shown. [Figure 8A] Figure 8A shows a simplified chromatographic separation of the Fab fragment from the Fc fragment, extraction of a single Fc fragment chromatographic peak for mass spectrometry, and an example of the mass spectrum of the Fc fragment chromatographic peak. Figure 8B shows the deconvoluted mass spectrum (left) and a magnified view thereof (right). The peaks in the deconvoluted mass spectrum are compared with a database for peak identification. [Figure 8B] Figure 8A shows a simplified chromatographic separation of the Fab fragment from the Fc fragment, extraction of a single Fc fragment chromatographic peak for mass spectrometry, and an example of the mass spectrum of the Fc fragment chromatographic peak. Figure 8B shows the deconvoluted mass spectrum (left) and a magnified view thereof (right). The peaks in the deconvoluted mass spectrum are compared with a database for peak identification. [Figure 9A] Each figure shows the deconvoluted spectrum of the panel sample described in Example 2, indicating the peak of the relevant glycoform species. [Figure 9B] Each figure shows the deconvoluted spectrum of the panel sample described in Example 2, indicating the peak of the relevant glycoform species. [Figure 9C] Each figure shows the deconvoluted spectrum of the panel sample described in Example 2, indicating the peak of the relevant glycoform species. [Figure 10]This is a schematic diagram of the cell line ADCC assay. [Figure 11A] This is a leverage plot of relative ADCC activity levels (%) measured by a cell line ADCC assay, plotted as a function of measured released non-fucosylated glycans (%). The best-fit line is the diagonal solid line in the center of the shaded area. p<0.0001. [Figure 11B] This is a leverage plot of relative ADCC activity levels (%) measured by a cell line ADCC assay, plotted as a function of measured released high mannose glycan (%). The best-fit line is the diagonal solid line in the center of the shaded area. p=0.2786. [Figure 11C] This graph plots the actual ADCC activity level (%) measured by cell line ADCC assays against the predicted ADCC activity level (%) calculated using Equation 8. [Figure 12A] This is a leverage plot of relative ADCC activity levels (%) measured by a cell line ADCC assay, plotted as a function of measured unpaired non-fucosylated glycans (%). The best-fit line is the diagonal solid line in the center of the shaded area. p<0.0001. [Figure 12B] This is a leverage plot of relative ADCC activity levels (%) measured by a cell line ADCC assay, plotted as a function of measured unpaired high mannose glycan (%). The best-fit line is the diagonal solid line in the center of the shaded area. p=0.0012. [Figure 12C] This graph plots the actual ADCC activity level (%) measured by cell line ADCC assays against the predicted ADCC activity level (%) calculated using Equation 5. [Modes for carrying out the invention]

[0017] This disclosure provides a method for determining the relative unpaired glycan content of an IgG antibody composition. In an exemplary embodiment, the method comprises: (b) treating an IgG antibody composition with two enzymes to form a mixture of Fab fragments and Fc fragments, wherein one enzyme cleaves the antibody heavy chain at the N-terminal site of the hinge region disulfide bond and the other enzyme cleaves the β1,4 bond between core GlcNAc residues to form Fc fragments, each containing a pair of coreglycan structures; (b) separating the Fab fragments from the Fc fragments; and (c) quantifying the abundance of (i) paired non-fucosylated Fc fragments, (ii) unpaired non-fucosylated Fc fragments, (iii) paired high-mannose Fc fragments, and / or (iv) unpaired high-mannose Fc fragments to determine the relative unpaired non-fucosylated (AF) glycan content and the relative unpaired high-mannose (HM) glycan content. As will be further discussed below, the method of the present invention can be used for the preparation of IgG compositions having ADCC activity within a target range, the analysis of IgG antibody compositions, the monitoring of the production of IgG antibody compositions, and / or the production of antibody compositions.

[0018] Glycosylation, glycans, and methods for measuring glycans Many secreted proteins undergo posttranslational glycosylation, a process in which a sugar moiety (e.g., glycans, sugars) is covalently bonded to specific amino acids in the protein. In eukaryotic cells, two types of glycosylation occur: (1) N-linked glycosylation, in which glycans are linked to asparagine in the recognition sequence Asn-X-Thr / Ser (where "X" is any amino acid other than proline), and (2) O-linked glycosylation, in which glycans are linked to serine or threonine. Regardless of the type of glycosylation (N-linked or O-linked), the glycan structures that can be bonded to each site (O or N) are wide-ranging, resulting in minute heterogeneity in protein glycoforms.

[0019] All N-glycans have 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) type consisting of two N-acetylglucosamine (GalNAc) moieties and at least five (e.g., 5, 6, 7, 8, or 9) mannose (Man) residues; (B) complex type containing three or more GlcNAc moieties and any number of other sugar types; or (C) hybrid type containing Man residues on one branch and GlcNAc at the base of the complex branch. Figure 1A (adapted from Stanley et al., Chapter 8: N-Glycans, Essentials of Glycobiology, 2nd ed., Cold Spring Harbor Laboratory Press; 2009) shows the three types of N-glycans.

[0020] N-linked glycans found in IgG molecules typically contain one or more monosaccharides, including galactose (Gal), N-glucose (Glc), N-acetylglucosamine (GlcNAc), glucosamine (GlcN), mannose (Man), and fucose (Fuc). Exemplary glycans, their identity, and group classification are shown in Figure 1B.

[0021] N-linked glycosylation is initiated in the endoplasmic reticulum (ER), and as a result of a complex series of reactions, a coreglycan structure is linked, which is basically made up of two GlcNAc residues and three Man residues. The glycan complex formed in the ER is modified by enzymatic action in the Golgi apparatus. If the sugar is relatively difficult for the enzyme to access, it generally remains in its original HM form. If the enzyme can access the sugar, many of the Man residues are cleaved, and the sugar is further modified, resulting in a complex N-glycan structure. 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 glycans (Hanrue Imai-Nishiya (2007), BMC Biotechnology, 7:84). Fucose metabolism is achieved via the salvage pathway or de novo pathway, as shown in Figure 2. In the salvage pathway, free L-fucose is converted to GDP-fucose, whereas in the de novo pathway, GDP-fucose is synthesized via three reactions catalyzed by GMD and FX. GDP-fucose is then transported from the cytoplasm to the Golgi lumen by GDP-Fuc transferase and transferred to receptor oligosaccharides and proteins. The other reaction product, GDP, is converted to guanosine 5-monophosphate (GMP) and inorganic phosphate (Pi) by nucleotide diphosphatases in the lumen. The former is effluxed into the cytoplasm (via a countertransport system coupled to GDP-fucose transport), while the latter is thought to leave the Golgi lumen via the Golgi anion channel GOLAC. See, for example, Nordeen et al. 2000; Hirschberg et al. 2001.

[0022] Therefore, the sugar composition and structural configuration of glycan structures vary, in particular, depending on the glycosylation mechanism in the ER and Golgi apparatus, the accessibility of enzymes in that mechanism to the glycan structure, the order of action of each enzyme, and the stage at which proteins are released from the glycosylation mechanism.

[0023] Various methods can be used to evaluate glycans present in glycoprotein-containing compositions, or to determine, detect, or measure the glycoform profile (e.g., glycoprofile) of a specific sample containing glycoproteins. Suitable methods include, but are not limited to, cation MALDI-TOF analysis, anion 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, as well as combinations thereof. For example, see 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, pages 124-133; Ruhaak LR, Analytical Bioanal Chem, 2010, Vol.397:3457-3481 and Geoffrey, RG Get. al. Analytical Biochemistry 1996, Vol.240, pages 210-226. Furthermore, a suitable method for evaluating glycans present in glycoprotein-containing compositions may include enzymatic cleavage of glycans bound to glycoproteins. The cleaved or released glycans are subsequently separated by hydrophilic interaction liquid chromatography (HILIC) to create a chromatogram with several peaks. Each peak in the chromatogram represents the mean distribution (amount or abundance) of a different glycan. Two representative HILIC chromatograms containing peaks for different glycans are provided in Figures 3A and 3B. For these purposes, peak area % = peak area / total peak area × 100%. Therefore, the level of a particular glycan (or group of glycans) is reported as a percentage. For example, if an antibody composition is characterized as having a 30% Man6 level, it means that 30% of all glycans cleaved from the antibody in the composition are Man6.As will be described in more detail herein, such methods for removing glycans from glycoproteins identify the distribution of glycan content in the glycoprotein, but it should be noted that they do not provide information about paired and / or unpaired glycans.

[0024] This disclosure refers to high-mannose glycans and non-fucosylated glycans in antibody compositions (see, for example, Figure 1B). As used herein, the terms “high-mannose glycan” or “HM glycan” encompass glycans containing 5, 6, 7, 8, or 9 mannose residues, abbreviated as Man5 or M5, Man6 or M6, Man7 or M7, Man8 or M8, and Man9 or M9, respectively. In various embodiments, the level of HM glycan is obtained by summing Man5%, Man6%, Man7%, Man8%, and Man9%. As used herein, the terms “non-fucosylated glycan” or “AF glycan” refer to glycans lacking core fucose, for example, α1,6-linked fucose on GlcNAc residues included in the amide bond with Asn at the N-glycosylation site. Non-fucosylated glycans include, but are not limited to, A1G0, A1G1, A2G0, A2G1(a and b), A2G2, A1G1M4, and A1G1M5. High-mannose glycans also lack core fucose (and therefore constitute a subset of non-fucosylated glycans), but it should be noted that high-mannose glycans have specific characteristics that may be referred to as a separate glycan group. Therefore, unless otherwise specified, high mannose is understood to represent a separate characteristic and can be classified separately from non-fucosylated glycans or as an additional characteristic of non-fucosylated glycans. See, for example, Reusch and Tejada, Glycobiology 25(12):1325-1334 (2015). In various embodiments, the level of non-fucosylated glycans is obtained by summing A1G0%, A2G0%, A2G1a%, A2G1b%, A2G2%, A1G1M5%, and A1G1a%.

[0025] The glycan levels (e.g., amounts, abundances) (e.g., HM glycan %) may be determined (e.g., measured) by any of the various methods known in the Art for evaluating 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 glycoproteins. The glycan levels (e.g., amounts, abundances) (e.g., HM glycan %) of an antibody composition may be determined by measuring the levels (e.g., amounts, abundances) of such glycans in a sample of the antibody composition by a chromatography-based method, e.g., HILIC, and the glycan levels (e.g., amounts, abundances) may be expressed in %, as described herein. The glycan levels of an antibody composition may be expressed as a percentage of all glycans cleaved from the antibody in the composition. The glycan levels (e.g., amounts, abundances) (e.g., HM glycan %) may be determined (e.g., measured) by measuring the levels of such glycans in a sample of the antibody composition. Samples of antibody compositions may be taken, and the levels of glycans (e.g., quantity, abundance) (e.g., HM glycan %) for each sample may be determined (e.g., measured). The percentages of HM glycan and / or AF glycan can be determined.

[0026] Method for determining unpaired glycans in glycan pairs and antibody compositions In the exemplary embodiments of this disclosure, an antibody composition comprises an antibody, each comprising two heavy chains and two light chains. In exemplary embodiments, each antibody in the antibody composition comprises a fragment crystallizable (Fc) region and two Fab regions. The Fc region of the antibody comprises two identical protein fragments derived from the constant domain of the antibody heavy chain, each protein fragment being glycosylated and containing, for example, a glycan. Thus, in exemplary embodiments, the Fc region of the antibody comprises a pair of glycans, or a glycan pair (also referred to herein as a "glyco-pair"). In various embodiments, the two glycans in a glycan pair are structurally distinct from each other, or the glycans in a glycan pair are structurally identical. Glycan pairs can be classified based on the presence or absence of glycans in glycan pairs containing core fucose, and the presence or absence of glycans in glycan pairs containing high mannose. Figure 4A is a series of diagrams illustrating an unpaired non-fucosylated antibody, a paired non-fucosylated antibody, an unpaired high-mannose antibody, and a paired high-mannose antibody. Figure 4B provides an exemplary flowchart useful for assigning nomenclature to glycan pairs containing canonical glycans. Exemplary paired high-mannose glycans include (a) a glycan having two identical high-mannose glycans (e.g., high-mannoses of the same structure), e.g., Man5, Man6, Man7, Man8, or Man9, or (b) a glycan having two non-identical high-mannose glycans (e.g., high-mannose glycans of different structures), where each high-mannose glycan contains Man5, Man6, Man7, Man8, or Man9 (e.g., Man5 as one glycan and Man6, Man7, Man8, or Man9 as the other glycans, or Man6 as one glycan and Man7, Man8, or Man9 as the other glycans, or Man7 as one glycan and Man8, or Man9 as the other glycans, or Man8 as one glycan and Man9 as the other glycans). Examples of unpaired high-mannose glycans include glycans having Man5, Man6, Man7, Man8, or Man9 as one glycan and a fucosylated or non-fucosylated glycan as the other glycan.Examples of unpaired non-fucosylated glycan pairs include, for example, A1G0, A2G0, A2G1a, A2G1b, or A2G2 as one glycan and a fucosylated glycan as the other glycan. Examples of paired non-fucosylated glycans include, for example, two identical non-fucosylated glycans (e.g., A1G0 / A1G0) or two non-identical non-fucosylated glycans (e.g., A1G0 / A2G2). Examples of paired non-fucosylated glycans include (i) A1G0 and A2G0, A2G1a, A2G1b, or A2G2 as one glycan, or (ii) A2G0 on one chain and A2G1a, A2G1b, or A2G2 on the other Fc chain, or (iii) A2G1a on one Fc chain and A2G1b or A2G2 on the other Fc chain, or (iv) A2G1b on one Fc chain and A2G2 on the other chain. A paired non-fucosylated glycan may contain high mannose as one glycan and any of A1G0, A2G0, A2G1a, A2G1b, or A2G2 as the other, for example, M5 / A1G0. From the viewpoint of high mannose, if only one glycan of the pair contains high mannose, the paired non-fucosylated glycan may be considered "unpaired high mannose".

[0027] In exemplary examples, glycan pairs are given designations based on the fucosylated / non-fucosylated state of each glycan in the pair. In exemplary embodiments, glycan pairs are given designations based on the high-mannose state of each glycan in the pair. The appropriate designation will be understood in the context of the glycan structure of interest in a particular scenario. In various embodiments, glycan pairs are given two designations, the first designation based on the fucosylated / non-fucosylated state of each glycan in the pair, and the second designation based on the high-mannose state of each glycan in the pair, and optionally, summary designations are assigned that are used to quantify the abundance of Fc fragments containing (i) paired non-fucosylated glycans, (ii) unpaired non-fucosylated glycans, (iii) paired high-mannose glycans, and / or (iv) unpaired high-mannose glycans. Table A below provides exemplary methods for assigning these designations. In various cases, the summary designation is assigned by applying three rules relating to the first designation (designation 1) and the second designation (designation 2), as follows: (1) If designation 1 is a paired fucosylation, the summary designation is a paired fucosylation (regardless of designation 2); (2) If designation 2 is not an HM, the summary designation is the same as designation 1; and (3) If designation 2 is a paired HM or an unpaired HM and designation 1 is not a paired fucosylation, the summary designation is the same as designation 2 (e.g., scenarios 4 and 10), or a combination of designation 1 and designation 2 (e.g., scenarios 5, 8, and 9).

[0028] [Table 1]

[0029] In various cases, an antibody or its Fc region containing glycan pairs may be described in relation to its glycan pair classification. Therefore, an antibody or its Fc region may be described as "paired non-fucosylated," "unpaired non-fucosylated," "paired fucosylated," "paired high-mannose," or "unpaired high-mannose," depending on its glycan pair classification. Similarly, an antibody or its Fc region may be described as containing paired non-fucosylated glycans (or paired non-fucosylated glycan pairs), unpaired non-fucosylated glycans (or unpaired non-fucosylated glycan pairs), paired high-mannose glycans (paired high-mannose glycan pairs), and / or unpaired high-mannose glycans (unpaired high-mannose glycan pairs), depending on its glycan pair classification. As will be discussed later, antibodies can be cleaved into fragments. The Fc fragment contains the Fc region of the antibody and can be described as "paired non-fucosylated," "unpaired non-fucosylated," "paired fucosylated," "paired high-mannose," or "unpaired high-mannose" depending on the glycan pairing classification of the Fc region.

[0030] Antibody compositions can be characterized with respect to their paired glycan content and / or unpaired glycan content. For example, an antibody composition can be characterized with respect to its paired non-fucosylated glycan content and / or unpaired non-fucosylated glycan content and / or paired high-mannose content and / or unpaired high-mannose content. The abundances described herein (e.g., paired non-fucosylated glycan content and / or unpaired non-fucosylated glycan content and / or paired high-mannose content and / or unpaired high-mannose content) can be referred to as relative or absolute abundances. In exemplary cases, the absolute content of a glycan can be expressed in terms of a unit that measures the level of the glycan itself, e.g., mass per volume unit, moles, mass or molar units, arbitrary units, area under the curve, or the intensity of a mass spectral peak, e.g., a deconvoluted mass spectral peak (as can be determined, e.g., from a chromatograph). In exemplary embodiments, the antibody composition is characterized with respect to the relative abundance of its unpaired glycans, meaning that the amount of unpaired glycans is expressed as an amount relative to the sum of paired and unpaired glycans in the antibody composition. In exemplary embodiments, the antibody composition is characterized with respect to the relative abundance of its unpaired non-fucosylated glycans. In exemplary embodiments, the antibody composition is characterized with respect to the relative abundance of unpaired high-mannose glycans.

[0031] "Relative unpaired non-fucosylated glycan content," "relative relative abundance of unpaired non-fucosylated glycans," and "relative unpaired non-fucosylated glycan %" are all synonymous with "relative relative abundance of unpaired non-fucosylated glycans," and "relative relative abundance of unpaired non-fucosylated glycans" are calculated by dividing the percentage of unpaired non-fucosylated glycans by the sum of the percentage of unpaired non-fucosylated glycans and the percentage of non-fucosylated glycans paired with them, and then multiplying by 100%.

[0032] "Relative unpaired high mannose glycan content," "relative relative abundance of unpaired high mannose glycan pairs," and "relative unpaired high mannose glycan %" are all synonymous with "relative relative abundance of unpaired high mannose glycan," and "relative relative abundance of unpaired high mannose glycan" are calculated by dividing the percentage of unpaired high mannose glycan by the sum of the percentage of unpaired high mannose glycan and the percentage of high mannose glycan paired with it, and multiplying by 100%.

[0033] The term "relative abundance of paired non-fucosylated glycans" is synonymous with "content of paired non-fucosylated glycans" and "relative abundance of paired non-fucosylated glycans." The "percentage of paired non-fucosylated glycans" is calculated by dividing the percentage of paired non-fucosylated glycans by the sum of the percentage of unpaired non-fucosylated glycans and the percentage of paired non-fucosylated glycans, and then multiplying by 100%.

[0034] The term "relative abundance of paired high-mannose glycans" is synonymous with "content of paired high-mannose glycans," and the "relative abundance of paired high-mannose glycans" and the "relative percentage of paired high-mannose glycans" are calculated by dividing the percentage of paired high-mannose glycans by the sum of the percentage of unpaired high-mannose glycans and the percentage of paired high-mannose glycans, and multiplying by 100%.

[0035] In various aspects of this disclosure, the sum of the unpaired relative % of non-fucosylated glycans and the paired relative % of non-fucosylated glycans equals 100%. Therefore, in various aspects, if the paired relative % of non-fucosylated glycans is known, the unpaired relative % of non-fucosylated glycans can be determined (e.g., calculated) by subtracting the paired relative % of non-fucosylated glycans from 100%. In various aspects of this disclosure, if the unpaired relative % of non-fucosylated glycans is known, the paired relative % of non-fucosylated glycans can also be determined (e.g., calculated) by subtracting the unpaired relative % of non-fucosylated glycans from 100%. In various aspects of this disclosure, the sum of the unpaired relative % of high mannose glycans and the paired relative % of high mannose glycans equals 100%. Therefore, in various cases, if the paired relative percentage of high mannose glycans is known, the unpaired relative percentage of high mannose glycans can be determined (e.g., calculated) by subtracting the paired relative percentage of high mannose glycans from 100%.

[0036] Method for determining the relative unpaired glycan content This disclosure provides a method for determining the relative unpaired glycan content of an IgG antibody composition. In an exemplary embodiment, the method includes (a) treating an IgG antibody composition with two enzymes to form a mixture of Fab fragments and Fc fragments, wherein one enzyme cleaves the antibody heavy chain at the N-terminal site of the hinge region disulfide bond and the other enzyme cleaves the β1,4 bond between core GlcNAc residues to form Fc fragments, each containing a pair of coreglycan structures; (b) separating the Fab fragments from the Fc fragments; and (c) quantifying the amount of Fc fragments containing (i) paired non-fucosylated glycans, (ii) unpaired non-fucosylated glycans, (iii) paired high-mannose glycans, and / or (iv) unpaired high-mannose glycans to determine the relative unpaired non-fucosylated (AF) glycan content and the relative unpaired high-mannose (HM) glycan content. In (c), the abundances of (i) paired non-fucosylated Fc fragments, (ii) unpaired non-fucosylated Fc fragments, (iii) paired high-mannose Fc fragments, and / or (iv) unpaired high-mannose Fc fragments are quantified. In exemplary embodiments, the relative abundances of (i) paired non-fucosylated Fc fragments, (ii) unpaired non-fucosylated Fc fragments, (iii) paired high-mannose Fc fragments, and / or (iv) unpaired high-mannose Fc fragments are quantified. In exemplary embodiments, the relative abundances of unpaired non-fucosylated glycans and unpaired high-mannose glycans are determined. In exemplary embodiments, one enzyme and the other enzyme are incubated simultaneously with the IgG antibody composition. For example, the IgG antibody composition may be incubated with one enzyme (which cleaves the antibody heavy chain at the N-terminus), and then the other enzyme (which cleaves the β1,4 bond between core GlcNAc residues) may be added. In exemplary embodiments, one enzyme and the other enzyme are incubated sequentially. For example, an IgG antibody composition may be incubated with one enzyme (which cleaves the antibody heavy chain at the N-terminus), and after the incubation is complete, the IgG antibody composition may be incubated with the other enzyme (which cleaves the β1,4 bond between core GlcNAc residues).

[0037] In various aspects of the method of determining relative unpaired glycan content, one enzyme that cleaves the antibody heavy chain at the N-terminal site of the hinge region disulfide bond is a cysteine ​​protease. In various cases, the enzyme is IgdE, a member of the IgdE protease family, optionally expressed by the genus Streptococcus. In various cases, the enzyme is structurally identical or very similar to the IgdE protease expressed by Streptococcus agalactiae. In various aspects, the enzyme is structurally identical or very similar to the enzyme expressed by anaerobic bacteria of the genus Porphyromonas. In various cases, the enzyme is structurally identical or very similar to the enzyme expressed by Porphyromonas gingivalis. In various cases, the site is located between Thr and His or between Lys and Thr in the sequence KTHTCPP (SEQ ID NO: 1) of the IgG1 antibody heavy chain. In an exemplary embodiment, the method includes treating the IgG antibody composition with a cysteine ​​protease for at least 8 hours or at least 12 hours, for example, at least 9 hours, at least 10 hours, at least 11 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours.

[0038] In various embodiments, the other enzyme that cleaves the β1,4 bond between core GlcNAc residues is an IgG-specific enzyme. In exemplary embodiments, the IgG-specific enzyme is an endoglycosidase, such as endoglycosidase D, endoglycosidase F, endoglycosidase F1, endoglycosidase F2, endoglycosidase H, or EndoS or EndoS2 endoglycosidase. In exemplary cases, the endoglycosidase is endoglycosidase S. In exemplary embodiments, the method involves treating the IgG antibody composition with the IgG-specific enzyme for less than 1 hour, for example, less than 55 minutes, less than 50 minutes, less than 45 minutes, less than 40 minutes, less than 35 minutes, or less than 30 minutes. In various embodiments, the method involves treating the IgG antibody composition with the IgG-specific enzyme for about 30 minutes. In various embodiments, other enzymes are added to the IgG composition during incubation with enzymes from the IgdE protease family (e.g., during the last 30 minutes to 1 hour of incubation, if necessary). In various embodiments, other enzymes are added to the IgG composition after incubation with enzymes from the IgdE protease family is complete.

[0039] In exemplary embodiments, the method includes separating the Fab fragment from the Fc fragment of the IgG antibody of the composition. Optionally, the method includes separating the Fab fragment from the Fc fragment by chromatography. Chromatography may be any suitable type capable of separating the Fab fragment from the Fc fragment, but is not limited to affinity chromatography (e.g., protein A chromatography), anion or cation exchange chromatography, liquid chromatography (LC, e.g., reversed-phase LC, HILIC), etc. Optionally, the chromatography is reversed-phase liquid chromatography. In various embodiments, the spectral peak of the Fc fragment elutes as one chromatographic peak selected for analysis of mass spectrometry data. In exemplary embodiments, the method includes performing mass spectrometry data analysis of the Fc fragment to obtain one or more mass spectral peaks. In various embodiments, the method includes deconvolving the mass spectral peak to obtain a deconvoluted mass spectral peak. Appropriate methods for deconvolving mass spectral peaks are known in the art. See Gadgil et al., J.Am.Soc.Mass Spectrom.17,867(2006); Murray et al., Pure and Applied Chemistry, vol.85, no.7, 2013, pp.1515-1609; Marchetti and Mignerey, Nuclear Instruments and Methods in Physics Research Section A:Accelerators, Spectrometers, Detectors and Associated Equipment 324(1-2):288-296(1993); Xu et al., Rapid Commun Mass Spectrom.2018 May 30;32(10):763-774. In an exemplary example, the method includes identifying glycan pairs by comparing the molecular weight of each deconvolved mass spectral peak with a database of glycan pairs and associated molecular weights.In exemplary cases, the method includes the step of determining the relative unpaired unfucosylated (AF) glycan content and the relative unpaired high mannose (HM) glycan content by quantifying the amount of Fc fragments containing (i) paired nonfucosylated glycans, (ii) unpaired nonfucosylated glycans, (iii) paired high mannose glycans, and / or (iv) unpaired high mannose glycans. In various embodiments, the relative unpaired nonfucosylated glycans can be determined by summing the amount of Fc fragments containing paired nonfucosylated glycans with the amount of Fc fragments containing unpaired nonfucosylated glycans. In various embodiments, the relative unpaired high mannose glycans can be determined by summing the amount of Fc fragments containing paired high mannose glycans with the amount of Fc fragments containing unpaired high mannose glycans. A suitable method for carrying out the method of the present disclosure is described in detail in Example 1. The database in exemplary embodiments includes the glycan pairs and their associated theoretical molecular weights in Table 1 of Example 1. In various embodiments, the isolation and quantification of the methods of the Disclosure may be performed in less than 2 hours, optionally less than 95 minutes, less than 85 minutes, less than 75 minutes, less than 60 minutes, less than 45 minutes, less than 30 minutes, or less than 20 minutes (optionally about 19 minutes, about 18 minutes, about 17 minutes, about 16 minutes, about 15 minutes, about 14 minutes, about 13 minutes, about 12 minutes, about 11 minutes, about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, or about 5 minutes). In exemplary embodiments, the methods of the Disclosure may include isolation and quantification in less than 30 minutes or less than 20 minutes. In various embodiments, the isolation and quantification of the methods of the Disclosure may be performed in about 5 minutes to about 25 minutes, about 5 minutes to about 20 minutes, about 5 minutes to about 15 minutes, about 5 minutes to about 10 minutes, about 10 minutes to about 25 minutes, about 15 minutes to about 25 minutes, or about 20 minutes to about 25 minutes). The method for determining the relative unpaired glycan content described herein requires a shorter time than conventional methods.

[0040] Applications for determining the relative unpaired glycan content. The method of the present invention for determining relative unpaired glycans is useful in the development and / or manufacture of antibody compositions. For example, the method for determining relative unpaired glycans can be used to analyze IgG antibody compositions. Thus, a method for analyzing IgG antibody compositions is provided herein. In exemplary embodiments, the method includes determining the relative unpaired glycan content of a sample of an IgG antibody composition according to one of the methods of the present disclosure for determining relative unpaired glycan content. In various embodiments, the method includes comparing the relative unpaired AF glycan content and / or relative unpaired HM glycan content of an IgG antibody composition with the relative unpaired AF glycan content and / or relative unpaired HM glycan content of a reference product. The method may be carried out in manufacture, for example, to determine whether many IgG antibody compositions meet specifications.

[0041] A method for monitoring the production of an IgG antibody composition is further provided. In an exemplary embodiment, the method includes determining the relative unpaired glycan content of an IgG antibody composition according to one of the methods of the present disclosure for determining the relative unpaired glycan content of a first sample obtained at a first time point and a second sample obtained at a second time point different from the first time point. In an exemplary embodiment, the method includes comparing the relative unpaired AF glycan content and / or relative unpaired HM glycan content of the first sample with the relative unpaired AF glycan content and / or relative unpaired HM glycan content of the second sample.

[0042] In various embodiments, each of the first and second samples is a sample of in-process material. In various examples, 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.

[0043] In various embodiments, the methods of the present disclosure are useful for determining whether an IgG antibody composition meets statistical criteria, including appropriate tolerance levels and / or appropriate rejection levels.

[0044] In various embodiments, methods for analyzing an IgG antibody composition and / or monitoring the production of an IgG antibody composition are performed during the development or production of the antibody composition. Therefore, methods for producing an antibody composition are also provided by this disclosure. In exemplary embodiments, the method comprises (A) determining the relative unpaired glycan content of a sample of an IgG antibody composition, the relative unpaired glycan content of a sample of an antibody composition being determined according to any one of the methods of this disclosure for determining relative unpaired glycan content, the sample being a sample of in-process material, and if it is determined that the relative unpaired glycan content is outside a predetermined target range, the method further comprises (B) modifying one or more conditions of a cell culture to obtain a modified cell culture and determining the relative unpaired glycan content, and optionally repeating (A) and (B) until the relative unpaired glycan content is within a predetermined target range. In various cases, one or more conditions of a cell culture are modified to primarily change the relative unpaired HM glycan content in order to achieve a target range of relative unpaired glycan content. In various embodiments, one or more conditions of the cell culture are modified to primarily alter the relative unpaired AF glycan content in order to achieve a target range of relative unpaired glycan content. Further embodiments of the method for producing the antibody composition are provided below. See, for example, the Method for Producing the Antibody Composition.

[0045] Method for changing ADCC and ADCC activity levels The data presented herein support the idea that the relative unpaired glycan content of an antibody composition is related to the ADCC activity level of the antibody composition, and that the ADCC activity level of an antibody composition can be altered by changing the relative unpaired glycan content of the antibody composition. While not bound by any particular theory, the relative unpaired non-fucosylated glycan content and / or relative unpaired high-mannose glycan content of an antibody composition are related to the ADCC activity level of the antibody composition, and changing the relative unpaired non-fucosylated glycan content and / or relative unpaired high-mannose glycan content of an antibody composition results in a change in the ADCC activity level of the antibody composition. Furthermore, since the rate of change in the relative unpaired glycan content has a greater impact on ADCC than the same rate of change in the relative content of paired glycans, the relative unpaired glycan content (e.g., relative unpaired non-fucosylated glycan content and / or relative unpaired high-mannose glycan content) is considered to have a greater leverage on ADCC than the relative content of paired glycans (e.g., relative content of paired non-fucosylated glycans and / or relative content of paired high-mannose glycans). Accordingly, this specification provides a method for changing the ADCC level of an antibody composition. In an exemplary embodiment, the method includes changing the relative unpaired non-fucosylated glycan content and / or the relative unpaired high-mannose glycan content of the antibody composition.

[0046] The term "ADCC," or "antibody-dependent cell-mediated cytotoxicity," refers to the mechanism by which effector cells of the immune system (e.g., natural killer cells (NK cells), macrophages, neutrophils, eosinophils) actively lyse target cells to which specific antibodies are bound. ADCC is part of an adaptive immune response and occurs when an antigen-specific antibody (1) binds to the membrane surface antigen of a target cell via its antigen-binding domain, and (2) binds to an Fc receptor on the surface of an effector cell via its Fc domain. The binding of the antibody's Fc domain to the Fc receptor causes the effector cell to release cytotoxic factors that lead to the death of the target cell (e.g., via cell lysis or cell degranulation).

[0047] 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, Fcγ receptors are receptors for the Fc region of IgG antibodies, Fcα receptors are receptors for the Fc region of IgA antibodies, and FcEPHRON receptors are receptors for the Fc region of IgE antibodies.

[0048] The term "FcγR" or "Fc-gamma receptor" refers to proteins belonging to the IgG superfamily that are involved in inducing phagocytosis in opsonized cells or microorganisms. See, for example, 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, such as 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.

[0049] The terms "ADCC activity" or "ADCC level" refer to the degree to which ADCC is activated or stimulated. Methods for measuring or determining the ADCC level of antibody compositions (including commercially available assays and kits for measuring or determining ADCC levels) are described in the following publications: Yamashita et al., Scientific Reports 6: article number 19772 (2016), doi: 10.1038 / srep19772; Kantokamalakul 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 51As described in “Cr-release and CD107a assays,” J Immunol Methods 406:1-9 (2014), these are well known in the art; all literature is incorporated herein by reference for all purposes. The terms “ADCC assay” or “FcγR reporter gene assay” refer to assays, kits or methods 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 3 or ADCC reporter assays 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 MT-3 cells as target cells labeled with calcein-AM. An illustration of an exemplary calcein-release assay is provided as Figure 10. In an exemplary embodiment of the calcein release assay, a standard curve is constructed using reference antibodies at various concentrations (Figure 10B).

[0050] In exemplary embodiments, the ADCC level of an antibody composition is determined by a quantitative cell line assay that measures the ability of the antibody composition to dose-dependently mediate cytotoxicity in cells that express the antibody antigen and are involved with the FcγRIIIA receptor on effector cells via the antibody's Fc domain. In various embodiments, this method involves the use of target cells having a detectable label released when the target cells are lysed by 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 embodiments, the amount of detectable label released from the target cells is compared to a baseline. The ADCC level may also be reported as ADCC% relative to control ADCC%. In various embodiments, ADCC% is relative ADCC%, which is, by choice, relative to control ADCC%. In various embodiments, control ADCC% is the ADCC% of the reference antibody. In exemplary cases, the control ADCC% is in the range of about 60% to about 130%. By choice, ADCC% is determined by the assay described in Example 3.

[0051] In exemplary embodiments, a method for changing (increasing or decreasing) the ADCC level of an antibody composition includes changing (increasing or decreasing) the relative unpaired non-fucosylated glycan content and / or the relative unpaired high-mannose glycan content of the antibody composition. In exemplary embodiments, a method disclosed herein for changing the ADCC level of an antibody composition includes increasing the relative unpaired non-fucosylated glycan content to increase the level of ADCC activity. In exemplary embodiments, a method for changing the ADCC level of an antibody composition includes increasing the relative unpaired high-mannose glycan content to increase the level of ADCC activity. In various embodiments, the increase in ADCC activity levels provided by the methods of this disclosure is at least or about 1% to about 20% compared to a control (e.g., at least or about 1%, at least or about 2%, at least or about 3%, at least or about 4%, at least or about 5%, at least or about 6%, at least or about 7%, at least or about 8%, at least or about 9%, at least or about 10%, at least or about 11%, at least or about 12%, at least or about 13%, at least or about 14%, at least or about 15%, at least or about 16%, at least or about 17%, at least or about 18%, at least or about 19%, at least or about 20%). A preferred control may be the same protein or antibody composition without a relative increase in unpaired glycan content. In exemplary embodiments, the increase in ADCC activity levels resulting from the methods of this disclosure is approximately 10% to approximately 100%, optionally approximately 10% to approximately 90%, approximately 10% to approximately 80%, approximately 10% to approximately 70%, approximately 10% to approximately 70%, approximately 10% to approximately 50%, approximately 10% to approximately 40%, approximately 10% to approximately 30%, approximately 10% to approximately 20%, approximately 10% to approximately 15%, approximately 20% to approximately 100%, approximately 30% to approximately 100%, approximately 40% to approximately 100%, approximately 50% to approximately 100%, approximately 60% to approximately 100%, approximately 70% to approximately 100%, approximately 80% to approximately 100%, approximately 90% to approximately 100%, or approximately 95% to approximately 100%. The increase may not necessarily be compared to a control.In exemplary embodiments, the increase in ADCC activity level resulting from the method of this disclosure is greater than 100% compared to a control, for example, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or even 1000%. In exemplary embodiments, the ADCC activity level increases by at least about 1.5 times compared to a control. A preferred control may be the ADCC activity level of the same protein or antibody composition that does not change the relative unpaired glycan content. In exemplary embodiments, the ADCC activity level increases by at least about 2 times compared to a control. In exemplary embodiments, the ADCC activity level increases by at least about 3 times compared to a control. In exemplary embodiments, the ADCC activity level increases by at least about 4 times or about 5 times compared to a control. In various embodiments, the increase in the ADCC activity level of an antibody composition is related to an increase in the relative unpaired glycan content. For example, the increase in the level of ADCC activity of an antibody composition is at least or about X% per approximately 1% increase in relative unpaired glycan content, where X% is at least or about 1% to about 20% increase (e.g., at least or about 1% increase, at least or about 2% increase, at least or about 3% increase, at least or about 4% increase, at least or about 5% increase, at least or about 6% increase, at least or about 7% increase, at least or about 8% increase, at least or about 9% increase, at least or about 10% increase, at least or about 11% increase, at least or about 12% increase, at least or about 13% increase, at least or about 14% increase, at least or about 15% increase, at least or about 16% increase, at least or about 17% increase, at least or about 18% increase, at least or about 19% increase, at least or about 20% increase).For example, X% could be approximately 10% to 100%, and by arbitrary choice, approximately 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 10% to 15%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, or 95% to 100%.

[0052] In various embodiments, a method for modifying the ADCC level of an antibody composition includes reducing the relative unpaired non-fucosylated glycan content to decrease the level of ADCC activity. In various embodiments, a method for modifying the ADCC level of an antibody composition includes reducing the relative unpaired high-mannose glycan content to decrease the level of ADCC activity. In various embodiments, the reduction in ADCC activity levels provided by the methods of this disclosure is at least or about 1% to about 20% compared to a control (e.g., at least or about 1% reduction, at least or about 2% reduction, at least or about 3% reduction, at least or about 4% reduction, at least or about 5% reduction, at least or about 6% reduction, at least or about 7% reduction, at least or about 8% increase, at least or about 9% increase, at least or about 10% increase, at least or about 11% increase, at least or about 12% increase, at least or about 13% increase, at least or about 14% increase, at least or about 15% increase, at least or about 16% increase, at least or about 17% increase, at least or about 18% increase, at least or about 19% increase, at least or about 20% increase). A preferred control may be the same protein or antibody composition with no change in relative unpaired glycan and overall glycan composition content. In exemplary embodiments, the reduction in ADCC activity levels resulting from the methods of this disclosure is approximately 10% to approximately 100%, optionally approximately 10% to approximately 90%, approximately 10% to approximately 80%, approximately 10% to approximately 70%, approximately 10% to approximately 60%, approximately 10% to approximately 50%, approximately 10% to approximately 40%, approximately 10% to approximately 30%, approximately 10% to approximately 20%, approximately 10% to approximately 15%, approximately 20% to approximately 100%, approximately 30% to approximately 100%, approximately 40% to approximately 100%, approximately 50% to approximately 100%, approximately 60% to approximately 100%, approximately 70% to approximately 100%, approximately 80% to approximately 100%, approximately 90% to approximately 100%, or approximately 95% to approximately 100%. The reduction may not necessarily be compared to a control. In exemplary embodiments, the reduction in ADCC activity levels resulting from the method of the present disclosure is greater than 100% compared to the control, for example, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or even 1000%.In exemplary embodiments, the level of ADCC activity is reduced by at least about 1.5 times compared to a control. A preferred control may be the ADCC activity level of the same protein or antibody composition with no change in glycan content. In exemplary embodiments, the level of ADCC activity is reduced by at least about 2 times compared to a control. In exemplary embodiments, the level of ADCC activity is reduced by at least about 3 times compared to a control. In exemplary embodiments, the level of ADCC activity is reduced by at least about 4 times or about 5 times compared to a control. In various embodiments, the reduction in the ADCC activity level of the antibody composition is related to a reduction in the relative unpaired glycan content. For example, the decrease in the level of ADCC activity of an antibody composition is at least or about X% per approximately 1% decrease in relative unpaired glycan content, where X% is at least or about 1% to about 20% decrease (e.g., at least or about 1% decrease, at least or about 2% decrease, at least or about 3% decrease, at least or about 4% decrease, at least or about 5% decrease, at least or about 6% decrease, at least or about 7% decrease, at least or about 8% increase, at least or about 9% increase, at least or about 10% increase, at least or about 11% increase, at least or about 12% increase, at least or about 13% increase, at least or about 14% increase, at least or about 15% increase, at least or about 16% increase, at least or about 17% increase, at least or about 18% increase, at least or about 19% increase, at least or about 20% increase). For example, X% could be approximately 10% to 100%, and by arbitrary choice, approximately 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 10% to 15%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, or 95% to 100%.

[0053] In exemplary embodiments, any changes (increases or decreases) brought about by the methods disclosed herein are compared to a “control.” In exemplary embodiments, the control is the level of ADCC activity when the steps of the method are not performed. In exemplary embodiments, the control is the level of ADCC activity when the relative unpaired non-fucosylated glycan content and / or relative unpaired high-mannose glycan content are not changed (increased or decreased). For example, a preferred control may be the ADCC activity level of the same protein or antibody composition, but without an increase in relative unpaired glycan content (e.g., relative unpaired non-fucosylated glycan content and / or relative unpaired high-mannose glycan content), or a preferred control may be the ADCC activity level of the same protein or antibody composition, but without a decrease in relative unpaired glycan content (e.g., relative unpaired non-fucosylated glycan content and / or relative unpaired high-mannose glycan content). In exemplary cases, the control may be the ADCC activity level of the same protein or antibody composition produced under the same cell culture conditions, except for conditions modified to cause a change in relative unpaired glycan content. In exemplary embodiments, the control may be the ADCC activity level of the same protein or antibody composition produced under a first set of cell culture conditions that result in an ADCC activity level outside the target range. In various embodiments, the control may be the ADCC activity level of the same protein or antibody composition produced under a first set of cell culture conditions that result in a relative unpaired non-fucosylated glycan content and / or a relative unpaired high mannose glycan content that is outside the target range.

[0054] The term "target range" refers to a range of values ​​based on a reference standard or reference product. For example, the target range for ADCC activity levels may be the range of ADCC activity levels indicated by the reference product. Also, for example, the target range for relative unpaired glycan content (e.g., relative unpaired non-fucosylated glycan content and / or relative unpaired high-mannose glycan content) may be the range of relative unpaired glycan content of the reference product (e.g., relative unpaired non-fucosylated glycan content and / or relative unpaired high-mannose glycan content). In various embodiments, the target range is a predetermined target range, meaning that the target range was determined or confirmed at a previous point in time.

[0055] Methods to change the unpaired glycan content In exemplary embodiments, a method for altering (increasing or decreasing) the ADCC level of an antibody composition includes altering (increasing or decreasing) the unpaired non-fucosylated glycan content and / or unpaired high-mannose glycan content of the antibody composition. In exemplary embodiments, a method disclosed herein for altering the ADCC level of an antibody composition includes increasing the unpaired non-fucosylated glycan content and / or unpaired high-mannose content to increase the level of ADCC activity. In various embodiments, a method for altering the ADCC level of an antibody composition includes increasing the unpaired non-fucosylated glycan content and / or unpaired high-mannose content by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, or more. In various embodiments, a method for modifying the ADCC level of an antibody composition includes increasing the unpaired non-fucosylated glycan content and / or unpaired high mannose content by more than 5% or more than 10%, for example, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%. In various embodiments, the method includes increasing the unpaired non-fucosylated glycan content and / or unpaired high mannose content by at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more.

[0056] In exemplary embodiments, methods disclosed herein for modifying the ADCC level of an antibody composition include reducing the level of ADCC activity by decreasing the unpaired non-fucosylated glycan content. In various embodiments, methods for modifying the ADCC level of an antibody composition include reducing the unpaired non-fucosylated glycan content and / or unpaired high mannose content by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, or more. In various embodiments, methods for modifying the ADCC level of an antibody composition include reducing the unpaired non-fucosylated glycan content and / or unpaired high mannose content by more than 5% or more than 10%, for example, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%. In various embodiments, the method includes reducing the unpaired non-fucosylated glycan content and / or unpaired high mannose content by at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more.

[0057] In exemplary embodiments, the increase or decrease in unpaired non-fucosylated glycan content and / or unpaired high-mannose glycan content is compared to a “control.” In exemplary embodiments, the control is the unpaired glycan content of a control protein or antibody composition produced under the same cell culture conditions, except for the conditions resulting in the increase or decrease in unpaired glycan content. In exemplary embodiments, the control may be the unpaired glycan content of the same protein or antibody composition produced under a first set of cell culture conditions that result in ADCC activity levels outside the target range. In various embodiments, the control may be the unpaired glycan content of the same protein or antibody composition produced under a first set of cell culture conditions that result in unpaired non-fucosylated glycan content and / or unpaired high-mannose glycan content that are outside the target range.

[0058] While not bound by any particular theory, conditions that result in a change (increase or decrease) in the non-fucosylated glycan content and / or high mannose content may result in an increase or decrease in the unpaired non-fucosylated glycan content and / or unpaired high mannose content of the antibody composition. In various cases, the unpaired non-fucosylated glycan content and / or unpaired high mannose content may be increased or decreased in accordance with any one of the teachings in International Publication Brochures 2013 / 114164, 2013 / 114245, 2013 / 114167, 2015128793, or 2016 / 089919, 2018 / 170099, or 2019 / 191150 (each of which is incorporated herein by reference). In various cases, the unpaired non-fucosylated glycan content and / or unpaired high mannose content can be increased or decreased by selecting clones that produce antibodies or antibody protein products containing levels of unpaired non-fucosylated glycan content and / or unpaired high mannose content within a target range.

[0059] Method for producing an antibody composition A simple and efficient method for predicting the level of effector function (e.g., ADCC) exhibited by a particular antibody composition based on its given glycoform profile is described herein. Data is provided herein supporting the idea that the ADCC activity level of an antibody composition can be predicted based on the relative unpaired glycan content of the antibody composition. While not bound by any particular theory, the unpaired non-fucosylated glycan content and / or relative unpaired high-mannose glycan content of an antibody composition are predictors of the ADCC activity level of the antibody composition. Such predicted ADCC levels are useful during antibody production when the antibody needs to have an ADCC activity level within a target range. For example, by monitoring the unpaired non-fucosylated glycan content and / or unpaired high-mannose glycan content of an antibody composition, it is possible to predict whether the antibody composition exhibits an ADCC activity level within a target range. If the target range for the ADCC activity level of an antibody composition is known, the target range for the relative unpaired non-fucosylated glycan content and / or relative unpaired high-mannose glycan content can be determined. If the ADCC activity level calculated based on the unpaired non-fucosylated glycan content and / or unpaired high mannose glycan content or unpaired non-fucosylated glycan content and / or unpaired high mannose glycan content falls within the target range, a selection of an antibody composition for further processing, e.g., downstream processing, may occur. In exemplary embodiments, the target range is based on the target range of ADCC activity levels for a model that correlates the ADCC activity level of a reference antibody and an antibody composition with the non-fucosylated glycan content and / or high mannose glycan content of the antibody composition, and optionally a model that correlates the ADCC activity level of an antibody composition with the unpaired non-fucosylated glycan content and / or unpaired high mannose glycan content of the antibody composition.

[0060] Accordingly, this disclosure provides a method for producing an antibody composition. In an exemplary embodiment, the method includes (i) determining the relative unpaired non-fucosylated glycan content and / or relative unpaired high mannose glycan content of a sample of the antibody composition, and (ii) selecting the antibody composition for downstream processing based on the relative unpaired non-fucosylated glycan content and / or relative unpaired high mannose glycan content determined in (i). In an exemplary embodiment, the method for producing an antibody composition includes (i) determining the unpaired non-fucosylated glycan content and / or unpaired high mannose glycan content of the antibody composition, (ii) determining the ADCC level of the antibody composition based on the unpaired non-fucosylated glycan content and / or unpaired high mannose glycan content determined in (i), and (iii) selecting the antibody composition for downstream processing if the ADCC level of the antibody composition determined in (ii) is within the target ADCC range. In exemplary embodiments, a method for producing an antibody composition includes (i) determining the unpaired non-fucosylated glycan content and / or unpaired high-mannose glycan content of a sample of the antibody composition taken from a cell culture containing glycosylated competent cells expressing the antibody of the antibody composition; (ii) optionally modifying the cell culture to adjust the unpaired non-fucosylated glycan content and / or unpaired high-mannose glycan content, and determining the unpaired non-fucosylated glycan content and / or unpaired high-mannose glycan content of a sample of the antibody composition taken from the modified cell culture; and (iii) selecting an antibody composition for downstream processing based on the unpaired non-fucosylated glycan content and / or unpaired high-mannose glycan content.

[0061] Methods for preparing IgG antibody compositions having ADCC activity levels within a target range are also provided herein. In exemplary embodiments, the method includes determining the relative unpaired glycan content of a sample of an IgG antibody composition according to one of the methods of the present disclosure for determining relative unpaired glycan content. The relative unpaired AF glycan content and / or relative unpaired HM glycan content of an IgG antibody composition may be compared to a target range for unpaired AF glycan content and / or relative unpaired HM glycan content. For example, the target range may be a specification or reference level of a reference IgG antibody composition. Alternatively, for example, a predetermined target range for unpaired AF glycan content and / or relative unpaired HM glycan content may be a predetermined target range for unpaired AF glycan content and / or relative unpaired HM glycan content that statistically correlates with a target range for the ADCC activity level of a reference IgG antibody composition.

[0062] Methods for generating antibody compositions are also provided in this disclosure. In exemplary embodiments, the method is to determine the relative unpaired glycan content of an antibody composition, determined according to any one of the methods of the disclosure for determining the relative unpaired glycan content, and if it is determined that the relative unpaired glycan content is outside a predetermined target range, the method further includes modifying one or more conditions of a cell culture to obtain a modified cell culture and determining the relative unpaired glycan content. In various examples, the method includes repeating the steps of the method until the relative unpaired glycan content falls within a predetermined target range.

[0063] In various embodiments, the sample is taken from a cell culture containing glycosylated competent cells expressing the antibody of the antibody composition. Optionally, the method further includes modifying one or more conditions of the cell culture to alter the relative unpaired nonfucosylated glycan content and / or the relative unpaired high mannose glycan content of the antibody composition, and determining the relative unpaired nonfucosylated glycan content and / or the relative unpaired high mannose glycan content of a sample of the antibody composition taken from the modified cell culture. In exemplary embodiments, the method includes repeating the alterations until the relative unpaired nonfucosylated glycan content and / or the relative unpaired high mannose glycan content falls within a target range. In various embodiments, the relative unpaired nonfucosylated glycan content and / or the relative unpaired high mannose glycan content are determined in real time with respect to the production of the antibody composition. As used herein, “real time” means a determination made during the progress of the production process without interrupting the process. The production of therapeutic proteins will be understood to involve live cells and sensitive materials that cannot be retained indefinitely while assays and determinations are being performed. In the numerical example in question, "real-time" determination may be made within 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 10 minutes, 5 minutes, 1 minute, 30 seconds, 20 seconds, 10 seconds, 5 seconds, 1 second, or 0.1 seconds (while the manufacturing process is in progress) after the measurement is taken. In various cases, if the relative unpaired non-fucosylated glycan content and / or relative unpaired high mannose glycan content are within the target range, the method includes selecting an antibody composition for downstream processing. In various embodiments, the relative unpaired non-fucosylated glycan content and / or relative unpaired high mannose glycan content correlate with the ADCC activity level of the antibody composition. Optionally, the method further comprises determining the ADCC activity level of the antibody composition based on the relative unpaired non-fucosylated glycan content and / or relative unpaired high-mannose glycan content determined in (i). Optionally, if the ADCC activity level is within the target range, the method comprises selecting the antibody composition for downstream processing.

[0064] In various embodiments, a method for producing an antibody composition includes changing the ADCC level of the antibody composition in accordance with the method for changing the ADCC level of the antibody composition described herein.

[0065] In various cases, a method for producing an antibody composition includes determining the relative unpaired glycan content of the antibody composition according to one of the methods of this disclosure for determining the relative unpaired glycan content of the antibody composition.

[0066] Downstream processing The relative percentages of unpaired high-mannose glycans and / or unpaired non-fucosylated glycans are determined (e.g., measured) to provide more information about the antibody-dependent cell-mediated cytotoxicity (ADCC) percentage of the antibody composition. The determination (e.g., measurement) can be performed at any stage of manufacturing. In particular, the measurement may be performed before or after sampling, or before or during any stage of downstream processing. Exemplary downstream processing includes any chromatographic unit operation, including capture chromatography, intermediate chromatography, and / or polish chromatography unit operations; virus inactivation and neutralization; virus filtration; and / or final formulation. In various embodiments, the relative percentages of unpaired high-mannose glycans and / or unpaired non-fucosylated glycans are determined (e.g., measured) in real time, near real time, and / or retrospectively. Monitoring and measurement can be performed using known techniques and commercially available instruments.

[0067] In various embodiments of this disclosure, the determination (e.g., measurement) of unpaired high mannose glycans % and / or unpaired non-fucosylated glycans % is performed before recovery. As used herein, the term “recover” means that the cell culture medium containing the recombinant protein of interest is collected and separated from at least the cells of the cell culture. Recovery can be performed continuously. In some embodiments, recovery is performed using centrifugation and may further include precipitation, filtration, etc. In various embodiments, the determination is performed before recovery. In various embodiments, the determination is performed before chromatography, optionally before protein A chromatography. In some embodiments, the determination (e.g., measurement) of unpaired high mannose glycans relative % and / or unpaired non-fucosylated glycans relative % is performed at least 3 days, at least 4 days, or at least 5 days before recovery. Optionally, the determination (e.g., measurement) of unpaired high mannose glycans relative % and / or unpaired non-fucosylated glycans relative % is performed in real time with respect to antibody production.

[0068] In various embodiments of this disclosure, the determination (e.g., measurement) of the relative percentage of unpaired high mannose glycans and / or unpaired non-fucosylated glycans is performed after recovery. In various embodiments, the determination is performed after chromatography (optionally, protein A chromatography). In various embodiments, the determination is performed after recovery and chromatography, and optionally, after protein A chromatography.

[0069] With respect to the methods disclosed herein, the antibody composition is selected in various embodiments for further processing, e.g., downstream processing, and this selection is based on specific parameters, e.g., ADCC%, unpaired high mannose glycan relative%, and / or unpaired non-fucosylated glycan relative%, respectively. In various cases, the methods disclosed herein include using the antibody composition in further processing, e.g., downstream processing, based on specific parameters, e.g., ADCC%, unpaired high mannose glycan relative%, and / or unpaired non-fucosylated glycan relative%, respectively. In various cases, the methods disclosed herein include performing further processing, e.g., downstream processing, using an antibody composition based on specific parameters, e.g., ADCC%, unpaired high mannose glycan relative%, and / or unpaired non-fucosylated glycan relative%, respectively.

[0070] In exemplary cases, downstream processing includes or consists of any processing that occurs after (or downstream of) the processing in which the unpaired relative percentages of high mannose glycans and / or unpaired relative percentages of non-fucosylated glycans are determined (e.g., measured). For example, if the unpaired relative percentages of high mannose glycans and / or unpaired relative percentages of non-fucosylated glycans are determined (e.g., measured) at recovery, downstream processing is any processing that takes place after (or downstream of) recovery, and in various embodiments includes: dilution, packing, filtration, formulation, further chromatography, viral filtration, viral inactivation, or a combination thereof. For example, if the relative percentage of unpaired high mannose glycans and / or unpaired non-fucosylated glycans is determined (e.g., measured) after chromatography, e.g., protein A chromatography, then downstream processing includes or consists of any processing performed after (or downstream of) chromatography, and in various embodiments, downstream processing includes: dilution, packing, filtration, formulation, further chromatography, viral filtration, viral inactivation, or a combination thereof. In exemplary cases, further chromatography is ion exchange chromatography (e.g., cation exchange chromatography or anion exchange chromatography).

[0071] Chromatographic stages / types used in downstream processes include capture chromatography or affinity chromatography, which are used to separate recombinant products from other proteins, aggregates, DNA, viruses, and other such impurities. In an exemplary case, the initial chromatography is performed using protein A (e.g., protein A bound to a resin). In various embodiments, intermediate and polish chromatography further purifies the recombinant protein to remove bulk contaminants, exogenous viruses, trace impurities, aggregates, isoforms, etc. Chromatography can be performed in a binding and elution mode, where the recombinant protein of interest is bound to the chromatography medium and impurities flow through, or in a flow-through mode, where impurities are bound 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 multi-mode chromatography (MM), hydroxyapatite chromatography (HA); reversed-phase chromatography and gel filtration.

[0072] In various embodiments, downstream processing includes virus inactivation. Enveloped viruses have a capsid surrounded by a lipoprotein membrane or "envelope" and are therefore susceptible to inactivation. In various cases, virus inactivation methods include thermal inactivation / pasteurization, pH inactivation, UV and gamma irradiation, use of high-intensity broad-spectrum white light, addition of chemical inactivators, and treatment with surfactants and solvents / surfactants.

[0073] In various embodiments, downstream processing includes viral filtration. In various embodiments, viral filtration includes removing non-enveloped viruses. In various embodiments, viral filtration includes the use of microfilters or nanofilters.

[0074] In various embodiments, downstream processing includes formulation, which is carried out in one or more steps. In various embodiments, after the completion of chromatography, the purified recombinant protein is exchanged for formulation buffer. In exemplary embodiments, buffer exchange is performed using ultrafiltration and diafiltration (UF / DF). In exemplary embodiments, the recombinant protein is exchanged for the desired formulation buffer using diafiltration and concentrated to the desired final formulation concentration using ultrafiltration. In various embodiments, further stability-enhancing excipients are added following UF / DF formulation.

[0075] Additional steps The methods disclosed herein include additional steps in various embodiments. For example, in some embodiments, the method includes one or more upstream or downstream steps involved in the production, purification, and formulation of an antibody composition. Optionally, the downstream steps are any of the downstream process steps described herein or known in the art. See, for example, Downstream Processing. In exemplary embodiments, the method includes steps for producing host cells expressing recombinant glycosylated proteins (e.g., antibodies). In some embodiments, the host cells are prokaryotic host cells, e.g., E. coli or Bacillus subtilis, or in some embodiments, eukaryotic host cells, e.g., yeast cells, filamentous fungal cells, protozoan cells, insect cells, or mammalian cells (e.g., CHO cells). Such host cells are described in the art. For example, see Kunert et al., Appl. Microbiol Biotechnol. 100:3451-61 (2016) and refer to “cell” in this specification. For example, the method involves, in some examples, introducing a recombinant glycosylated protein or a vector containing a nucleic acid comprising a nucleotide sequence encoding its polypeptide chain into a host cell.

[0076] In exemplary embodiments, the method includes the step of maintaining cells, for example, glycosylated competent cells, in a cell culture. Thus, the method may include the step of performing any one or more of the steps described in the section on maintaining cells in cell cultures herein.

[0077] In exemplary embodiments, the method disclosed herein includes a step for isolating and / or purifying antibodies from a culture. In exemplary embodiments, the method includes, but is not limited to, one or more chromatographic steps, including, for example, affinity chromatography (e.g., protein A affinity chromatography), ion exchange chromatography, and / or hydrophobic interaction chromatography. In exemplary embodiments, the method includes a step for generating crystalline biomolecules from a solution containing recombinant glycosylated proteins.

[0078] The methods of this disclosure, in various embodiments, include one or more steps for preparing a composition, and in some embodiments, include one or more steps for preparing a composition comprising a pharmaceutical composition comprising a purified recombinant glycosylated protein. Such compositions are described herein.

[0079] Cell maintenance in cell cultures Regarding a method for producing the antibody composition of this disclosure, the antibody composition may be produced by maintaining cells in a cell culture. The cell culture may 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, osmotic pressure, etc. In an exemplary embodiment, the cell culture before inoculation is shaken in a CO2 incubator with 5% CO2 under standard humidification conditions (e.g., 70 rpm). In an exemplary embodiment, about 10 in 1.5 L of medium 6 The cell culture is seeded at a seeding density of individual cells / mL.

[0080] In exemplary embodiments, the methods of the present disclosure include maintaining glycosylation-eligible cells in cell culture medium at a pH of approximately 6.85 to approximately 7.05, for example, in various embodiments, approximately 6.85, approximately 6.86, approximately 6.87, approximately 6.88, approximately 6.89, approximately 6.90, approximately 6.91, approximately 6.92, approximately 6.93, approximately 6.94, approximately 6.95, approximately 6.96, approximately 6.97, approximately 6.98, approximately 6.99, approximately 7.00, approximately 7.01, approximately 7.02, approximately 7.03, approximately 7.04, or approximately 7.05.

[0081] In exemplary embodiments, the method includes maintaining the cell culture at a temperature of 30°C to 40°C. In typical embodiments, the temperature is approximately 32°C to 38°C or approximately 35°C to 38°C.

[0082] In exemplary embodiments, the method includes maintaining an osmotic pressure of approximately 200 mOsm / kg to approximately 500 mOsm / kg. In exemplary embodiments, the method includes maintaining an osmotic pressure of approximately 225 mOsm / kg to approximately 400 mOsm / kg or approximately 225 mOsm / kg to approximately 375 mOsm / kg. In exemplary embodiments, the method includes maintaining an osmotic pressure of approximately 225 mOsm / kg to approximately 350 mOsm / kg. In various embodiments, the osmotic pressure (mOsm / kg) is maintained at approximately 200, 225, approximately 250, approximately 275, approximately 300, approximately 325, approximately 350, approximately 375, approximately 400, approximately 425, approximately 450, approximately 475, or approximately 500.

[0083] In exemplary embodiments, the method includes maintaining the dissolved oxygen (DO) level of the cell culture at approximately 20% to approximately 60% oxygen saturation during the initial cell culture period. In typical examples, the method includes maintaining the DO level of the cell culture at approximately 30% to approximately 50% (e.g., approximately 35% to approximately 45%) oxygen saturation during the initial cell culture period. In typical examples, the method includes maintaining the DO level of the cell culture at approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, or approximately 60% oxygen saturation during the initial cell culture period. In exemplary embodiments, the DO level is approximately 35 mmHg to approximately 85 mmHg, or approximately 40 mmHg to approximately 80 mmHg, or approximately 45 mmHg to approximately 75 mmHg.

[0084] The cell culture is maintained in any one or more culture media. In an exemplary embodiment, the cell culture is maintained in a medium suitable for cell growth and / or provided with one or more feed media according to any appropriate supply schedule. In an exemplary embodiment, the method includes maintaining the cell culture in a medium containing glucose, fucose, lactate, ammonia, glutamine and / or glutamate. In an exemplary embodiment, the method includes maintaining the cell culture in a medium containing manganese at a concentration of about 1 μM or less during the initial cell culture period. In an exemplary embodiment, the method includes maintaining the cell culture in a medium containing about 0.25 μM to about 1 μM manganese. In an exemplary embodiment, the method includes maintaining the cell culture in a medium containing a negligible amount of manganese. In an exemplary embodiment, the method includes maintaining the cell culture in a medium containing copper at a concentration of about 50 ppb or less during the initial cell culture period. In an exemplary embodiment, the method includes maintaining the cell culture in a medium containing copper at a concentration of about 40 ppb or less during the initial cell culture period. In exemplary embodiments, the method includes maintaining the cell culture in a medium containing copper at a concentration of approximately 30 ppb or less during the initial cell culture period. In exemplary embodiments, the method includes maintaining the cell culture in a medium containing copper at a concentration of approximately 20 ppb or less during the initial cell culture period. In exemplary embodiments, the medium contains copper at a concentration of approximately 5 ppb or more, or approximately 10 ppb or more. In exemplary embodiments, the cell culture medium contains mannose. In exemplary embodiments, the cell culture medium does not contain mannose.

[0085] In typical embodiments, the type of cell culture is either fed-batch or continuous perfusion. However, the methods of this disclosure are advantageously not limited to any particular type of cell culture.

[0086] Cells maintained in cell culture may be glycosylated competent cells. In exemplary embodiments, glycosylated competent cells are eukaryotic cells, including, but not limited to, yeast cells, filamentous fungal cells, protist cells, algal cells, insect cells, or mammalian cells. Such host cells are described in the art. See, for example, Kunert et al., Appl. Microbiol Biotechnol. 100:3451-61 (2016). In exemplary embodiments, eukaryotic cells are mammalian cells. In exemplary embodiments, mammalian cells are non-human mammalian cells. In some embodiments, the cells include 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 cancer cells (e.g., HeLa), human 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 These include 3T3, mouse fibroblasts L929, mouse neuroblastoma cells N2a, human breast cancer cells MCF-7, retinoblastoma cells Y79, human retinoblastoma cells SO-Rb50, human liver cancer cells Hep G2, mouse B myeloma cells J558L, or neonatal hamster kidney (BHK) cells (Gaillet et al. 2007; Khan, Adv Pharm Bull 3(2):257-263(2013)).

[0087] Cells that are not competent for glycosylation can also be transformed into glycosylation-competent cells, for example, by introducing genes that encode the relevant enzymes required for glycosylation. Exemplary enzymes include, but are not limited to, oligosaccharide transferases, glycosidases, glucosidase I, glucosidase II, calnexin / calreticulin, glycosyltransferases, mannosidases, GlcNAc transferases, galactosyltransferases, and sialyltransferases.

[0088] In typical embodiments, glycosylation-eligible cells are not genetically modified to alter the activity of enzymes in the de novo or salvage pathways. These two pathways of fucose metabolism are shown in Figure 2. In typical embodiments, glycosylation-eligible cells are not genetically modified to alter the activity of one or more of the following: fucosyltransferases (FUTs, e.g., FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9), fucose kinases, GDP-fucose pyrophosphorylase, GDP-D-mannose-4,6-dehydratase (GMD), and GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase (FX).

[0089] In a typical embodiment, glycosylation-eligible cells are not genetically modified to knock out the gene encoding FX. In typical embodiments, glycosylation-eligible cells are not genetically modified to alter the activity of β(1,4)-N-acetylglucosaminyltransferase III (GNTIII) or GDP-6-deoxy-D-lyxo-4-hexose reductase (RMD). In exemplary embodiments, glycosylation-eligible cells are not genetically modified to overexpress GNTIII or RMD.

[0090] Antibodies and their fragments As used herein, the term “antibody” refers to a conventional immunoglobulin-type protein comprising 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-type” structure of two identical pairs of polypeptide chains, each pair having one “light” chain (typically with a molecular weight of about 25 kDa) and one “heavy” chain (typically with a molecular weight of about 50–70 kDa). Antibodies have one variable region and one constant region. In the IgG structure, the variable region is generally about 100–110 or more amino acids and includes three complementarity-determining regions (CDRs), which are primarily involved in antigen recognition and substantially different among other antibodies that bind to different antigens. See, for example, Janeway et al., “Structure of the Antibody Molecule and the Immunoglobulin Genes”, Immunobiology: The Immune System in Health and Disease, 4th ed. Elsevier Science Ltd. / Garland Publishing, (1999)).

[0091] In short, in the antibody backbone, CDRs are embedded within the framework of the variable regions of the heavy and light chains, where they constitute a region that plays a major role in antigen binding and recognition. The variable region contains at least three heavy or light chain CDRs (see also 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; Chothia et al., 1989, Nature 342:877-883), which are located within the framework region (referred to as framework regions 1-4, FR1, FR2, FR3, and FR4, by Kabat et al., 1991; see also Chothia and Lesk, 1987, ibid.).

[0092] Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses (e.g., IgM1 and IgM2, but not limited to these). The light chain constant region may be, for example, a kappa or lambda light chain constant region, e.g., a human kappa or human lambda light chain constant region. The heavy chain constant region may be, for example, an alpha, delta, epsilon, gamma, or mu heavy chain constant region, e.g., a human alpha, human delta, human epsilon, human gamma, or human mu heavy chain constant region. In various embodiments, the antibody is IgG containing one of IgG1, IgG2, IgG3, or IgG4. In various examples, the antibody is IgG1.

[0093] In various embodiments, antibodies can be monoclonal or polyclonal antibodies. Illustrative examples include mammalian antibodies such as mouse antibodies, rat antibodies, rabbit antibodies, goat antibodies, horse antibodies, chicken antibodies, hamster antibodies, pig antibodies, and human antibodies. In certain embodiments, recombinant glycosylated proteins are monoclonal human antibodies.

[0094] In various embodiments, antibodies are chimeric antibodies or humanized antibodies. 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 generally, a sequence of amino acid sequences from at least two species. The term “humanized,” when used in reference to antibodies, refers to an antibody having at least a CDR region of a non-human source that has been manipulated to have a structure and immune function more similar to a true human antibody than the antibody of its original source. For example, humanization may include transplanting a CDR from a non-human antibody, such as a mouse antibody, into a human antibody. Humanization may also include the selection of amino acid substitutions to make the non-human sequence look more like a human sequence.

[0095] Antibodies, in various embodiments, are cleaved into fragments by enzymes such as papain and pepsin. Papain cleaves the antibody to produce two Fab fragments and a single Fc fragment. Pepsin cleaves the antibody to produce an F(ab')2 fragment and a pFc' fragment. As described herein, a method for determining the unpaired glycan content comprises two enzyme digestions, one of which is used to produce a mixture of Fab and Fc fragments.

[0096] Advantageously, this method is not limited to the antigen specificity of the antibody. Therefore, the antibody has some binding specificity to substantially any antigen. In exemplary embodiments, the antibody binds to a hormone, growth factor, cytokine, cell surface receptor or any ligand thereof. In exemplary embodiments, the antibody binds to a protein expressed on the cell surface of an immune cell. In exemplary embodiments, the antibody binds to 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, CD61 , 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, CD 98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CDw108, CD109, CD114, CD115, CD116, CD117, CD118, CD1 19, CD120a, CD120b, CD121a, CDw121b, CD122, CD123, CD124, CD125, CD126, CD127, CDw128, CD129, CD130, CDw131, CD132, CD134, CD135 It binds to a cluster of differentiation molecules selected from the group consisting of 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.

[0097] In exemplary embodiments, the antibody is specified in U.S. Patent Publication No. 7947809 and U.S. Patent Application Publication No. 20090041784 (glucagon receptor), U.S. Patent No. 7939070, U.S. Patent No. 7833527, U.S. Patent No. 7767206 and U.S. Patent No. 7786284 (IL-17 receptor A), U.S. Patent No. 7872106 and U.S. Patent No. 7592429 (sclerostin), U.S. Patent No. 7871611, U.S. Patent No. 7815907, U.S. Patent No. 7037498, U.S. Patent No. 7,700,742 and U.S. Patent Publication No. 20100255538 (IGF-1 receptor), U.S. Patent No. 7,868,140 (B7RP1), U.S. Patent No. 7,807,159 and U.S. Patent Publication No. 20110091455 (myostatin), U.S. Patent No. 7,736,644, U.S. Patent No. 7,628,986, U.S. Patent No. 7,524,496 and U.S. Patent Publication No. 20100111979 (deletion variant of epidermal growth factor receptor), U.S. Patent No. 7,728,110 (SA RSV), U.S. Patent No. 7718776 and U.S. Patent Publication No. 20100209435 (OPGL), U.S. Patent No. 7658924 and U.S. Patent No. 7521053 (Angiopoietin-2), U.S. Patent No. 7601818, U.S. Patent No. 7795413, U.S. Patent Publication No. 20090155274, U.S. Patent Publication No. 20110040076 (NGF), U.S. Patent No. 7579186 (TGF-βII receptor), U.S. Patent No. 7541438 (Connective Tissue Growth Factor), U.S. Patent No. 7438910 (IL1-R1), U.S. Patent No. 7423128 (Properdin), U.S. Patent No. 7411057, U.S. Patent No. 7824679, U.S. Patent No. 7109003, U.S. Patent No. 6682736, U.S. Patent No. 7132281, and U.S. Patent No. 7807797 (CTLA-4), U.S. Patent No. 7084257, U.S. Patent No. 7790859, U.S. Patent No. 7335743, U.S. Patent No. 7084257,and U.S. Patent Publication No. 20110045537 (Interferon-Gamma), U.S. Patent No. 7932372 (MAdCAM), U.S. Patent No. 7906625, U.S. Patent Publication No. 20080292639, and U.S. Patent Publication No. 20110044986 (Amyloid), U.S. Patent No. 7815907 and U.S. Patent No. 7700742 (Insulin-like Growth Factor I), U.S. Patent No. 7566772 and and U.S. Patent No. 7,964,193 (Interleukin-1β), U.S. Patent No. 7,563,442, U.S. Patent No. 7,288,251, U.S. Patent No. 7,338,660, U.S. Patent No. 7,626,012, U.S. Patent No. 7,618,633, and U.S. Patent Publication No. 201,000,98694 (CD40), U.S. Patent No. 7,498,420 (c-Met), U.S. Patent No. 7,326,414, U.S. Patent No. 7,592,430, and U.S. Patent No. 7728113 (M-CSF), U.S. Patent No. 6924360, U.S. Patent No. 7067131, and U.S. Patent No. 7090844 (MUC18), U.S. Patent No. 6235883, U.S. Patent No. 7807798, and U.S. Patent Publication No. 20100305307 (Epidermal Growth Factor Receptor), U.S. Patent No. 6716587, U.S. Patent No. 7872113, and U.S. Patent No. 7465450 U.S. Patent No. 7,186809, U.S. Patent No. 7,317090, and U.S. Patent No. 7,638606 (Interleukin-4 Receptor), U.S. Patent Publication No. 20110135657 (BETA-KLOTHO), U.S. Patent Nos. 7,887799 and 7,879323 (Fibroblast Growth Factor-like Polypeptides), U.S. Patent No. 7,867494 (IgE), U.S. Patent Publication No. 20100254975 (Alpha-4 Beta-7), U.S. Patent Publication No. 20100197005 and U.S. Patent No. 7537762 (Activin-like receptor kinase 1), U.S. Patent No. 7585500 and U.S. Patent Publication No. 20100047253 (IL-13), U.S. Patent Publication No. 20090263383 and U.S. Patent No. 7449555 (CD148),U.S. Patent Publication No. 20090234106 (Activin A), U.S. Patent Publication No. 20090226447 (Angiopoietin-1 and Angiopoietin-2), U.S. Patent Publication No. 20090191212 (Angiopoietin-2), U.S. Patent Publication No. 20090155164 (C-FMS), U.S. Patent No. 7537762 (Activin receptor-like kinase-1), U.S. Patent No. 7371381 (Galanin), U.S. Patent Publication No. 20070196376 (Insulin-like Growth factors), U.S. Patent No. 7,267,960 and U.S. Patent No. 7,741,115 (LDCAM), U.S. Patent No. 7,265,212 (CD45RB), U.S. Patent No. 7,709,611, U.S. Patent Publication No. 20060127393 and U.S. Patent Publication No. 20100040619 (DKK1), U.S. Patent No. 7,807,795, U.S. Patent Publication No. 20030103978 and U.S. Patent No. 7,923,008 (Osteoprotegain), U.S. Patent Publication No. 20090208489 U.S. Patent Publication No. 20080286284 (PSMA), U.S. Patent No. 7888482, U.S. Patent Publication No. 20110165171, and U.S. Patent Publication No. 20110059063 (PAR2), U.S. Patent Publication No. 20110150888 (HEPCIDIN), U.S. Patent No. 7939640 (B7L-1), U.S. Patent No. 7915391 (C-Kit), U.S. Patent No. 7807796, U.S. Patent No. 7193058, and U.S. Patent No. 742 U.S. Patent No. 7669 (ULBP), U.S. Patent No. 7786271, U.S. Patent No. 7304144, and U.S. Patent Publication No. 20090238823 (TSLP), U.S. Patent No. 7767793 (SIGIRR), U.S. Patent No. 7705130 (HER-3), U.S. Patent No. 7704501 (Ataxin-1-like polypeptide), U.S. Patent No. 7695948 and U.S. Patent No. 7199224 (TNFα-converting enzyme), U.S. Patent Publication No. 20090234106 (Activin A),U.S. Patent Application Publication No. 20090214559 and U.S. Patent No. 7438910 (IL1-R1), U.S. Patent No. 7579186 (TGFβ Type II receptor), U.S. Patent No. 7569387 (TNF receptor-like molecule), U.S. Patent No. 7541438 (Connective tissue growth factor), U.S. Patent No. 7521048 (TRAIL receptor-2), U.S. Patent No. 6319499, U.S. Patent No. 7081523, and U.S. Patent Publication No. 20080182976 (Erythropoietin receptor), U.S. Patent Publication No. 20080166352 and U.S. Patent No. 7435796 (B7RP1), U.S. Patent No. 7423128 (Properdin), U.S. Patent No. 7422742 and U.S. Patent No. 7141653 (Interleukin-5), U.S. Patent No. 6740522 and U.S. Patent No. 7411050 (RANKL), U.S. Patent No. 7378091 (Carbohydrate anhydride IX (CA IX) Tumor antigens), U.S. Patent No. 7318925 and U.S. Patent No. 7288253 (Palatinoid hormones), U.S. Patent No. 7285269 (TNF), U.S. Patent No. 6692740 and U.S. Patent No. 7270817 (ACPL), U.S. Patent No. 7202343 (Unicellular chemoattractant protein-1), U.S. Patent No. 7144731 (SCF), U.S. Patent No. 6355779 and U.S. Patent No. 7138500 (4-1BB), U.S. Patent No. 7135174 (PDGFD), U.S. Patent No. 6630143 and U.S. Patent No. 7045128 (Flt-3 ligand), U.S. Patent No. 6,849,450 (metalloproteinase 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 Publication No. 2011,002,7287 (proprotein converter subtilisin type 9 (PCSK9)), U.S. Patent Publication No. 2011,001,4201 (IL-18 receptor),and one of those described in U.S. Patent Application Publication No. 20090155164 (C-FMS). The above patents and published patent applications are incorporated herein by reference in their entirety for the purpose of disclosing variable domain polypeptides, nucleic acids encoding variable domains, host cells, vectors, methods for producing such variable domain encoding polypeptides, pharmaceutical compositions, and methods for treating diseases associated with individual targets of variable domain-containing antigen-binding proteins or antibodies.

[0098] In exemplary embodiments, the antibodies include muromonab-CD3 (a product marketed under the trade name Orthoclone Okt3®), absiximab (a product marketed under the trade name Reopro®), rituximab (a product marketed under the trade names MabThera® and Rituxan®), basiliximab (a product marketed under the trade name Simulect®), daclizumab (a product marketed under the trade name Zenapax®), palivizumab (a product marketed under the trade name Synagis®), infliximab (a product marketed under the trade name Remicade®), Trastuzumab (product sold under the brand name Herceptin®), alemtuzumab (products sold under the brand names MabCampath® and Campath-1H®), adalimumab (product sold under the brand name Humira®), tositumomab-I131 (product sold under the brand name Bexxar®), ephalizumab (product sold under the brand name Raptiva®), cetuximab (product sold under the brand name Erbitux®), ibritumomab tiuxeta (Products sold under the brand name Zevalin®), omalizumab (products sold under the brand name Xolair®), ocrelizumab (products sold under the brand name Ocrevus®), bevacizumab (products sold under the brand name Avastin®), natalizumab (products sold under the brand name Tysabri®), ranibizumab (products sold under the brand name Lucentis®), panitumumab (products sold under the brand name Vectibix®), eculizumab (products sold under the brand name Products sold under the brand name Soliris (registered trademark), certolizumab pegol (product sold under the brand name Cimzia (registered trademark)), golimumab (product sold under the brand name Simponi (registered trademark)), canakinumab (product sold under the brand name Ilaris (registered trademark)), catumakisomab (product sold under the brand name Removab (registered trademark)), ustekinumab (product sold under the brand name Stellara (registered trademark)), tocilizumab (products sold under the brand names RoActemra (registered trademark) and Actemra (registered trademark)),Ofatumumab (product marketed under the trade name Arzerra®), denosumab (product marketed under the trade name Prolia®), belimumab (product marketed under the trade name Benlysta®), laxibakumab, ipilimumab (product marketed under the trade name Yervoy®), and pertuzumab (product marketed under the trademark name Perjeta Antibody, such as daclizumab).

[0099] In exemplary embodiments, the antibody binds to a tumor-associated antigen and is an anti-cancer antibody. Examples of suitable anti-cancer antibodies include, but are not limited to, anti-BAFF antibodies such as belimumab; anti-CD20 antibodies such as rituximab; anti-CD22 antibodies such as epratuzumab; anti-CD25 antibodies such as daclizumab; anti-CD30 antibodies such as iratumumab, anti-CD33 antibodies such as gemtuzumab, anti-CD52 antibodies such as alemtuzumab; anti-CD152 antibodies such as ipilimumab; anti-EGFR antibodies such as cetuximab; anti-HER2 antibodies such as trastuzumab and pertuzumab; anti-IL6 antibodies such as siltuximab; and anti-VEGF antibodies such as bevacizumab; and anti-IL6 receptor antibodies such as tocilizumab.

[0100] antibody composition The methods of this disclosure relate to antibody compositions. In various embodiments, the composition comprises only one type of antibody. In various examples, the composition comprises antibodies, and each antibody in the composition comprises the same or substantially identical amino acid sequence. In various embodiments, the composition comprises antibodies in which each antibody in the composition comprises an amino acid sequence that is at least 90% identical to the amino acid sequences of all other antibodies in the composition. In various embodiments, the composition comprises antibodies in which each antibody in 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 sequences of all other antibodies in the composition. In various embodiments, the composition comprises antibodies in which each antibody in the composition comprises the same or essentially identical amino acid sequence (e.g., at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequences of all other antibodies in the composition), but the glycan pair classifications of the antibodies in the composition may differ from one another.

[0101] In various embodiments, the composition contains only one type of antibody. In various examples, the composition contains antibodies, and each antibody in the antibody composition contains the same or substantially identical amino acid sequence. In various embodiments, the antibody composition contains antibodies, and each antibody in the antibody composition contains an amino acid sequence that is at least 90% identical to the amino acid sequences of all other antibodies in the antibody composition. In various embodiments, the antibody composition contains antibodies, and each antibody in the antibody composition contains 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 sequences of all other antibodies in the antibody composition. In various embodiments, the antibody composition contains antibodies in which each antibody in the antibody composition contains the same or essentially identical amino acid sequence (e.g., at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequences of all other antibodies in the antibody composition), but the glycan pair classifications of the antibodies in the antibody composition may differ from one another. In exemplary embodiments, the antibody composition contains a heterogeneous mixture of antibodies having different glycan pair classifications. In various examples, an antibody composition may be characterized with respect to its unpaired AF glycan content, unpaired HM glycan content, paired AF glycan content, and / or paired HM glycan content. In various examples, an antibody composition may be characterized with respect to its relative abundance of unpaired AF glycan and / or relative abundance of unpaired HM glycan.

[0102] The following embodiments are provided merely to illustrate the present invention and are not intended to limit the scope of the invention. [Examples]

[0103] Example 1 This example describes an exemplary simplified method for determining the paired glycan content and unpaired glycan content of an antibody composition.

[0104] In a previous study, the paired unpaired and high-mannose glycan content and the unpaired unpaired and high-mannose glycan content were determined for two different antibody compositions, and a statistical relationship was established that correlated the unpaired glycan content of the antibody composition with ADCC activity and FcγRIIIa binding. In this previous study, the method used to measure the level of unpaired or paired glycopairs consisted of three steps: an antibody cleavage step, a chromatographic separation step, and a detection step based on mass spectrometry (MS). Briefly, samples of antibody compositions containing antibody A or antibody B were treated with the IgdE (FabALACTICA® or FL, manufactured by Genovis Inc., Cambridge, MA) enzyme, which cleaves the antibody heavy chain above the hinge to produce Fab antibody fragments and Fc antibody fragments. The FL enzyme is IgG1 heavy chain sequence [ka] The second and third amino acids were cleaved. Subsequently, the resulting Fab fragment and glycosylated Fc fragment were separated and characterized by hydrophilic interaction liquid chromatography (HILIC) using water / organic compound and MS-based detection with ion-pair reagents as solvents. HILIC separation was performed over 95 minutes on a Waters Acquity UPLC Glycoprotein Amide column using a mobile phase composition of 20:80 (v / v) of water and acetonitrile containing 0.1% TFA. After 1 minute, a linear gradient of 0.44 ml / min was applied for the next 74 minutes to separate glycopairs based on their interaction with the stationary phase. MS detection was performed using an Agilent 6545XT QToF MS with an Agilent Jet Stream (AJS) electrospray ionization source (ESI) with the following settings: capillary voltage -4500V; dry gas -11mL / min; nebulizer pressure -25psi and gas temperature -340℃; mass range 1000~3000m / z. Deconvolution was performed using Bioconfirm B.09.00 (Agilent) with S / N 30 and output mass range set to 40~60kDa.

[0105] The above method successfully measured the content of unpaired and paired non-fucosylated and high-mannose glycans to establish a statistically significant correlation with biological function. However, subsequent studies were conducted to improve the efficiency of the method for determining the levels of unpaired and paired non-fucosylated and high-mannose glycans. Glycan pair analysis is complex due to the high degree of heterogeneity of the glycopair population, and HILIC separation aims to reduce this complexity by introducing high-resolution separation before MS analysis. Glycan pairs are separated into a glycopair map of approximately 15 chromatographic peaks (Figure 6A). However, HILIC-MS glycan pair analysis remains complex for several reasons. Firstly, paired fucosylated glycoparticles (Fc,A2G0F / A2G0F, Fc,A2G0F / A2G1F, and Fc,A2G1F / A2G1F) that do not significantly affect ADCC are the dominant species in the analysis and elute across the entire chromatographic region of the glycosylated Fc fragment chromatogram (Figure 6A-6B). Secondly, many of the important chromatographic peaks are not sufficiently resolved: major non-fucosylated and high-mannose glycoparticles co-elute with the major paired fucosylated glycoparticles and / or are poorly separated. As a result, it is difficult to establish a correct window for extracting the mass spectrum of each chromatographic peak, and a defective extraction window can artificially inflate or contract the peak area of ​​important non-fucosylated and high-mannose glycoparticles, reducing the accuracy of the analysis. Thirdly, structural isomers and glycopairs with partially reduced disulfide bonds (Rd, +2Da) yield multiple chromatographic peaks for species classified as the same glycopair. Finally, important non-fucosylated and high-mannose glycopairs (shown in bold in Figure 6B) are present in very low abundances, often accounting for less than 5% of the population. In summary, these challenges complicate data analysis and quantification, requiring careful consideration by highly skilled analysts and ultimately increasing the time required for these methods as a whole.

[0106] The new method was designed to involve the preparation, separation, measurement, and analysis of a simplified sample containing components essential for the quantification of non-fucosylated high-mannose glycans (paired or unpaired), without the presence of large amounts of non-essential components. For example, the previous method identified and quantified all types of glycopairs, including high-mannose and all fucosylated and non-fucosylated glycopairs, but this level of resolution was unnecessary for determining the levels of unpaired and paired non-fucosylated and high-mannose glycopairs. Furthermore, the separation prior to MS-based detection was designed so that glycosylated Fc fragments eluted under a single chromatographic peak in the total ion chromatogram chromatographically separated from the Fab region. In the previous HILIC-MS method, the low chromatographic resolution of important fucosylated and high-mannose glycopairs from abundant non-fucosylated glycopairs makes it difficult to determine the correct mass spectral extraction window for accurate quantification. To simplify analysis in the new method, all glycosylated Fc fragments are intentionally co-eluted under a single chromatographic peak in the total ion chromatogram. The mass spectrum is extracted from the entire chromatographic peak of the Fc fragment and deconvolved. The deconvoluted mass spectral peak in the single deconvoluted mass spectrum is utilized for the identification and quantification of glycopairs. Using this approach, Fc fragments are separated by their difference (delta) in molecular weight (MW). By intentionally removing the chromatographic separation of glycosylated Fc fragments, data analysis is simplified, and subsequently, quantitative accuracy and inter-sample comparability are improved. Furthermore, sample throughput is dramatically increased. An automated identification system for faster identification and quantification of paired and unpaired non-fucosylated and high-mannose glycopairs has also been incorporated into the new method.

[0107] The new method involved two-enzyme digestion of the antibody composition before chromatographic separation and MS-based detection. This two-enzyme digestion is shown in Figure 5. Briefly, the antibody composition sample was treated with the enzyme IgdE (FabALACTICA® or FL, manufactured by Genovis Inc., Cambridge, MA), which cleaves the IgG1 antibody heavy chain above the hinge to produce Fab antibody fragments and Fc antibody fragments. The FL enzyme is used to digest the IgG1 heavy chain sequence. [ka] The second and third amino acids are cleaved. Digestion was carried out overnight at 37°C. A second digestion using EndoS (IgGZERO®, Genovis Inc.), an IgG-specific endoglycosidase S enzyme, was performed for 30 minutes at 37°C. EndoS is an IgG-specific endoglycosidase that acts on complex N-glycans at the Fc-glycosylation site of IgG. This enzyme hydrolyzes the β1,4 bond between core GlcNAc residues in the N-glycan, leaving the innermost GlcNAc on the Fc (with or without fucose) intact. EndoS has limited activity against high-mannose and hybrid glycans, which is used to identify and quantify high-mannose glycopairs, and then to identify and quantify the relative abundance % of paired and unpaired high-mannose.

[0108] These two enzymatic digestions resulted in a simplified population of glycopairs that could be classified into one of two classes of non-fucosylated glycopairs: (1) paired non-fucosylated (both heavy chains of the Fc fragment were non-fucosylated) and (2) unpaired non-fucosylated (only one heavy chain of the Fc fragment was non-fucosylated, while the other heavy chain was fucosylated) (Figure 7). Based on the assumption that both non-fucosylated glycans and high-mannose glycans are highly potent, pairs containing combinations of high-mannose and non-fucosylated glycans were classified as non-fucosylated pairs and unpaired high-mannose pairs, as needed, according to Table A.

[0109] EndoS exhibits limited activity towards high-mannose and hybrid glycans, which was used to identify high-mannose glycopairs and quantify high-mannose glycan pairs. Individual Fc high-mannose glycopairs could be classified into one of two high-mannose classes: (1) paired high-mannose (both heavy chains of the Fc fragment contained high-mannose glycans) and (2) unpaired high-mannose (only one heavy chain of the Fc fragment contained high-mannose, while the other heavy chain was fucosylated or unfucosylated) (Figure 7). A summary is provided in Table A and according to the three rules described herein.

[0110] After enzyme digestion, the obtained Fc and Fab fragments were chromatographically separated using high-speed reversed-phase liquid chromatography (RP-LC) separation with MS detection. This separation yielded a single chromatographic peak containing both the Fab fragment and the Fc fragment, which was sufficiently separated from the processing enzyme. The diagrams of the chromatographic separation and MS analysis steps are shown in Figures 8A-8B.

[0111] The apparatus used to carry out the steps of the method has the following capabilities:

[0112] [Table 2]

[0113] The autosampler temperature was maintained at 4°C, aliquots were injected, and separation was performed at 60°C using a 2.1 ID × 50 mm RP analysis column. Mobile phase A was 0.1% (v / v) trifluoroacetic acid in water, and mobile phase B was 0.1% (v / v) trifluoroacetic acid in acetonitrile. An example of the mobile phase gradient is shown below.

[0114] [Table 3]

[0115] MS acquisition was performed using an Agilent 6545 XT Q-TOF with an AJS ESI source in positive ion mode. Source conditions were as follows: gas temperature -325°C, dry and sheath gas flow rate -12 L / min, sheath gas temperature -300°C, nebulizer -40 psi, capillary voltage -5000 V, fragmenter voltage -280 V, and skimmer -120 V. Spectra were acquired in the m / z range 1200 to 3200.

[0116] Next, chromatographic peaks containing the Fc fragment were selected for data analysis, and the mass spectrum of one chromatographic peak containing the Fc fragment was extracted and deconvolved (Figures 8A-8B). Deconvolution was performed using Byos (Protein Metrics Inc, Cupertino, CA), with an input m / z range of 1300-3200 m / z and an output range of 30-70 kDa. The intensity of each deconvolved mass spectral peak of the antibody composition was used for quantification.

[0117] EndoS-treated glycopairs were identified by matching the molecular weight (MW) of the deconvoluted mass spectral peaks of the antibody composition against a custom database of theoretical MWs (Table 1). The glycan MW used in this approach is the N-bond-associated glycan MW (natural glycan MW-H2O). The database consists of theoretical MWs of two EndoS-treated glycans (glycan pairs), where complex glycans were hydrolyzed to either GlcNac or GlucNac+Fuc, while high-mannose and hybrid glycans remained intact. The database consists of approximately 40 individual EndoS glycan pair theoretical MWs classified as (1) non-fucosylated pairs, (2) non-fucosylated unpairs, (3) high-mannose pairs, and (4) high-mannose unpairs (Table 1). Glycan pairs hydrolyzed by EndoS are shown in italics in the following text.

[0118] To facilitate the automated identification of EndoS-treated glycopairs (defined as an intact Fc and two glycan portions), the amino acid sequence of FL-treated Fc was first loaded into the protein analysis software to generate the theoretical mass spectral peak (MW) of Fc. Furthermore, the software was instructed to assume that all possible disulfide bonds remained intact and both C-terminal lysines were clipped. Secondly, the EndoS glycan pair theoretical MW database was loaded into the protein analysis software as a list of custom modifications. Following this user input, the protein analysis software then automatically determined a list of theoretical MWs for EndoS glycopairs (see Table 1). If an MW matched a theoretical MW in the custom database for EndoS glycopairs, the software automatically identified the deconvoluted mass spectral peak.

[0119] [Table 4]

[0120] [Table 5]

[0121] The most abundant EndoS glycopair is Fc,GlucNac+fucose / GlucNac+fucose, which contains a fucosylated glycan on each heavy chain, and is therefore not applicable to determining non-fucosylated or high-mannose glycan pairs. In previous studies using HILIC-MS, the highly abundant and unapplicable paired fucosylated glycopair elutes across the entire chromatogram, and the presence of the target species is often masked when glycosylated Fc is eluted (Figure 6B). In this study, EndoS treatment simplifies the highly abundant paired fucosylated glycopair to a single species Fc,GlucNac+fucose / GlucNac+fucose. This single deconvoluted mass peak is well separated from the target species by delta molecular weight, facilitating streamlined data interpretation (Figure 8B).

[0122] The abundance of individual EndoS glycopairs was determined from the relative intensity or area of ​​the deconvoluted mass spectral peaks identified as follows: Glyco-to-Strength % = Deconvoluted Peak Intensity / Total Deconvoluted Peak Intensity × 100 [Formula 1]

[0123] The abundance of paired non-fucosylated glycopairs was determined by summing the individual glycopairs classified as paired non-fucosylated glycopairs, and similarly, the abundance of unpaired non-fucosylated glycopairs was determined by summing the individual glycopairs classified as unpaired non-fucosylated glycopairs. Equation 2 was used to calculate the relative abundance % of the unpaired non-fucosylated glycan content in the antibody composition: Unpaired non-fucosylated glycan content % = (Unpaired non-fucosylated glyco [Formula 2]

[0124] The abundance of paired high-mannose glycopairs was determined by summing the individual glycopairs classified as paired high-mannose glycopairs, and similarly, the abundance of unpaired high-mannose glycopairs was determined by summing the individual glycopairs classified as unpaired high-mannose glycopairs. Equation 3 was used to calculate the relative abundance % of the unpaired high-mannose glycan content in the antibody composition: Unpaired high mannose glycan content % = (Unpaired high mannose glycan per unit / Total high mannose glycan per unit (Unpaired + per unit)) × 100 [Formula 3]

[0125] Previous studies have used 95-minute HILIC-MS analysis to separate highly complex glycopair populations to determine the levels of deglycosylation and high-mannose glycan pairs. This approach remains challenging because highly abundant and unapplicable paired fucosylated glycopairs often mask the presence of the desired glycopair as they elute across the entire glycosylated Fc chromatographic region. Important high-mannose and non-fucosylated glycopairs are difficult to quantify accurately because they co-elute and / or shoulder with highly abundant paired fucosylated glycopairs. This example demonstrates the feasibility of a streamlined method that simplifies the glycopair population before analysis by EndoS. Rapid 17-minute chromatographic separation before MS detection simplifies the analysis by obtaining a well-resolved chromatographic peak of one of the Fc fragments for further quantification. The quantification of EndoS glycopairs is achieved using deconvoluted mass spectral peaks, where highly abundant paired fucosylated species are a single deconvoluted mass spectral peak well-separated by MW from important non-fucosylated glycopairs and high-mannose glycopairs. A custom EndoS glycopair MW database facilitates the automated identification and quantification of important non-fucosylated and high-mannose glycopairs. This novel method provides rapid analysis and improved quantification accuracy when determining the relative abundance % of unpaired non-fucosylated glycan content and paired non-fucosylated glycan content, as well as the relative abundance % of unpaired high-mannose glycan content and paired high-mannose glycan content in antibody compositions.

[0126] Example 2 This example describes an exemplary application of a simplified method for determining the relative percentages of paired and unpaired non-fucosylated and high-mannose glycan content in an antibody composition.

[0127] The novel method described in Example 1 was performed on a panel of antibody composition samples, each of which contained a sample of an IgG1 monoclonal antibody or reference product (RP) lot produced by a specific clone. The deconvoluted mass spectra of the selected samples are shown in Figures 9A-9C. Data for paired non-fucosylated glycopairs and unpaired non-fucosylated glycopairs are provided in Tables 2A and 2B, respectively. Data for paired high-mannose glycopairs and unpaired high-mannose glycopairs are provided in Tables 2C and 2D, respectively.

[0128] [Table 6]

[0129] [Table 7]

[0130] [Table 8]

[0131] [Table 9]

[0132] The relative unpaired and paired non-fucosylated glycan content and the relative unpaired and paired high-mannose glycan content were calculated as described in Example 1, and the data are provided in Table 3 below.

[0133] [Table 10]

[0134] Based on this data, the RP range for unpaired non-fucosylated glycan content was 85.4–88.4, and the range for unpaired high mannose glycan content was 67.8–81.2. The clones with the best unpaired glycan profiles were clones 203, 116, 113, 126, and 236. Clones 116 and 126 had unpaired non-fucosylated glycan content within the RP range, while clone 203 had both unpaired non-fucosylated glycan content within the RP range and unpaired high mannose glycan content.

[0135] Example 3 This example describes an exemplary method for measuring ADCC activity levels.

[0136] The ADCC activity level (expressed as a % relative value) for a panel of samples of IgG1 monoclonal antibody compositions is determined using a quantitative cell-based assay that measures the antibody's ability to mediate cytotoxicity in a dose-dependent manner in target cells that stably express the antigen, via the Fc domain of the antibody and its involvement with the FcγRIIIA(158V) receptor on NK92-M1 effector cells. These events activate the effector cells, leading to the destruction of the target cells via exocytosis of the cytolytic granule complex perforin / granzyme. A schematic diagram of the ADCC assay is shown in Figure 10. Briefly, target cells are labeled with calcein-acetoxymethyl (calcein-AM), which readily enters the cells and is subsequently cleaved by intercellular esterases and captured within the cells. When the target cells are lysed, fluorescent calcein is released into the culture medium. The level of calcein released from the lysed target cells is determined by measuring the fluorescence of the reaction supernatant using an Envision (Perkin Elmer) fluorescence plate reader. Each assay is performed three times, and the mean and standard deviation are recorded. The data is fitted to the mean fluorescence value using a constrained 4-parameter fit with SoftMaxPro software, calculated by the EC50 standard / EC50 sample ratio, and reported as the percentage of ADCC activity relative to the reference standard.

[0137] Example 4 HILIC is a quantitative analysis of the N-linked glycan distribution of antibodies and comprises the following three steps: (1) releasing and labeling N-linked glycans from reference and test samples using PNGase F and a fluorophore capable of specifically derivatizing free glycans; (2) loading a sample within an effective linear range onto a 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.

[0138] Standards and test samples are prepared by the following steps: (1) Dilute the sample and control with water; (2) Add PNGase F to the sample and control and incubate to release N-linked glycans; (3) Mix with a fluorophore-labeled solution using a fluorophore such as 2-aminobenzoic acid; (4) Centrifuge to precipitate the protein and remove the supernatant; (5) Dry and reconstitute the labeled glycans in the injection solution.

[0139] The solutions used in this assay are mobile phase A (100 mM ammonium formate, target pH 3.0) and mobile phase B (acetonitrile). The apparatus used to carry out the steps of the method has the following capabilities:

[0140] [Table 11]

[0141] The instrument settings for HPLC using a BEH glycan 1.7 μm column (2.1 mm ID × 150 mm) and 2-aminobenzoic acid fluorophore labeling for hydrophilic interaction analysis are shown below.

[0142] [Table 12]

[0143] An example of a moving phase gradient is shown below.

[0144] [Table 13]

[0145] The results report will be structured in the following format.

[0146] [Table 14]

[0147] Example 5 This example describes the relationship between ADCC activity and the released percentage of high mannose glycan, released percentage of non-fucosylated glycan, unpaired percentage of high mannose glycan, and unpaired percentage of non-fucosylated glycan for an antibody composition.

[0148] The ADCC activity levels of the antibody compositions in the panel were determined by a cell-based ADCC assay. The percentage of released high-mannose glycan and free non-fucosylated glycan was determined by the method described in Example 4. Table 4 shows the ADCC activity levels, released high-mannose glycan percentage, and released non-fucosylated glycan percentage for each antibody composition in the panel.

[0149] [Table 15]

[0150] These data were analyzed using the JMP suite of computer programs (SAS Institute, Cary, NC) for statistical analysis. Figure 11A is the ADCC leverage plot for released non-fucosylated glycans %, and Figure 11B is the leverage plot for released high-mannose glycans %. The best-fit line is the diagonal solid line in the center of the shaded area. As shown in Figures 11A and 11B, released non-fucosylated glycans showed a statistically significant relationship with the ADCC activity level of the antibody composition (p<0.0001), while released high-mannose glycans did not (p=0.2786).

[0151] The relationship between %ADCC and the percentage of released non-fucosylated glycans and the percentage of released high-mannose glycans can be described by formula 4. Predicted %ADCC = -5.8 + (21.7% released non-fucosylated glycans) + (4.3% released high-mannose glycans) [Formula 4]

[0152] The predicted %ADCC value for each sample was calculated by substituting the measured percentages of released non-fucosylated glycans and released high-mannose glycans into Equation 4. The actual ADCC% (as measured in the cell system assay) is plotted against the predicted ADCC% (as calculated by Equation 4), and this plot is provided as Figure 11C. Root mean square error (RMSE), r 2 The statistical parameters, including the p-value, are shown in Figure 11C. These results suggest that Equation 4 accurately predicts actual (measured) ADCC and highlight a statistically significant correlation between the percentage of released non-fucosylated glycans and ADCC (p<0.0001). Higher levels of non-fucosylated glycans result in higher ADCC activity. The effect of high mannose on ADCC was weak and not statistically significant.

[0153] In the approach outlined in Example 4, glycans are released from the Fc of the antibody composition, so glycan pair information is not captured, and it is not possible to determine the utilization of both unpaired and paired non-fucosylated glycans and unpaired and paired high-mannose glycans on ADCC activity. The previous HILIC-MS method outlined in Example 1 showed that an increase in the level of unpaired non-fucosylated glycans has an increased influence on ADCC, presumably because the proportion of non-fucosylated glycans is spread over a wider distribution of molecules in the antibody composition. Paired high-mannose glycans have been shown to have a very low influence on ADCC activity, but the structural reason for this is currently unknown.

[0154] The RP-LC MS approach outlined in Example 1 for measuring the levels of paired and unpaired non-fucosylated and high-mannose glycan content provides a robust yet simplified approach for determining the relationship between ADCC activity and glycan pairs. The overall distribution of total non-fucosylated and total high-mannose glycans determined by the approaches described in Examples 1 and 4 should yield similar results. However, the RP-LC MS method was developed as a targeted workflow focused on the analysis of unpaired non-fucosylated high-mannose glycan content, and complete compositional data are likely to be less precise compared to the well-established released glycan assay described in Example 4. The optimal use of glycan pair data for non-fucosylated and high-mannose subgroups is applied in the form of a correction factor for released non-fucosylated and high-mannose glycan content determined using Example 4 to explain the glycan pair contribution. Using this correction, the relationship between ADCC activity and unpaired non-fucosylated glycans and unpaired high-mannose glycans for antibody compositions can be determined.

[0155] The paired and unpaired non-fucosylated glycan content and the paired and unpaired high-mannose glycan content of the same antibody composition were determined according to the procedure outlined in Example 1. The results are shown in Table 3.

[0156] Using the paired non-fucosylated glycan fraction, the freed non-fucosylated glycan data in Table 4 was corrected to describe the portion of the antibody composition in which non-fucosylated glycans were paired (paired non-fucosylated fraction). Equation 5 can be used to determine the percentage of unpaired non-fucosylated glycans in the entire antibody composition. % of unpaired non-fucosylated glycans = % of free non-fucosylated glycans (1 pair of non-fucosylated fractions) / (1 + 1 pair of non-fucosylated fractions) × 100 [Formula 5]

[0157] Using the relatively paired high-mannose glycan content, corrections were applied to the released high-mannose glycan data in Table 4 to describe the paired high-mannose glycan portion of the antibody composition (paired high-mannose fraction). Equation 6 can be used to determine the unpaired high-mannose glycan percentage of the entire antibody composition. % Unpaired high mannose glycan = % Released high mannose glycan (1 paired high mannose fraction) / (1 + 1 paired high mannose fraction) × 100 [Formula 6]

[0158] The ADCC activity level, the percentage of unpaired non-fucosylated glycans, and the percentage of unpaired high-mannose glycans were calculated as shown in Equations 5 and 6, and the data are shown in Table 5 below.

[0159] [Table 16]

[0160] Next, the data in Table 5 was analyzed using a complete set of JMP computer programs for statistical analysis. Figure 12A is the ADCC leverage plot for % unpaired non-fucosylated glycans, and Figure 12B is the leverage plot for % unpaired high mannose glycans. The best-fit line is the diagonal solid line in the center of the shaded area. As shown in Figures 12A and 12B, both % unpaired non-fucosylated glycans and % unpaired high mannose glycans showed a statistically significant relationship with ADCC activity levels (p<0.0001 for % unpaired non-fucosylated glycans, and p=0.0012 for % unpaired high mannose glycans). These results support the finding that % unpaired high mannose glycans significantly contribute to ADCC activity.

[0161] In summary, these data support the importance of paired states. While the total percentage of released high-mannose glycan content did not appear to have a statistically significant relationship with ADCC activity levels, the percentage of unpaired high-mannose glycan content was demonstrated to be statistically significant in correlation with ADCC activity.

[0162] The relationship between %ADCC, %unpaired non-fucosylated glycans, and %unpaired high-mannose glycans can be described by formula 7. Predicted %ADCC = -27.5 + (29.6% unpaired non-fucosylated glycans) + (14.5% unpaired high-mannose glycans) [Formula 7]

[0163] The measured values ​​of % unpaired high-mannose glycan and % unpaired non-fucosylated glycan were substituted into Equation 7 to calculate the predicted %ADCC value for each sample. The actual %ADCC (measured in the cell system assay) was plotted against the predicted %ADCC (calculated by Equation 7), and the plot is provided as Figure 12C. Root mean square error (RMSE), r 2 The statistical parameters, including the p-value, are shown in Figure 12C.

[0164] These results suggest that Equation 7 accurately predicts the actual (measured) ADCC and highlights a statistically significant direct correlation between % unpaired non-fucosylated glycan, % unpaired high-mannose glycan, and ADCC activity levels (p<0.0001). Higher levels of unpaired non-fucosylated glycan and unpaired high-mannose content result in higher ADCC activity. The influence of unpaired non-fucosylated glycan was stronger than the influence of unpaired high-mannose glycan on ADCC activity (29.6 and 14.5, respectively). By determining the unpaired non-fucosylated glycan and unpaired high-mannose glycan, the ADCC level of the antibody composition can be predicted, and therefore, the quality of the product, e.g., the quality of the antibody composition, can be predicted.

[0165] Using a similar approach, the paired non-fucosylated and paired high-mannose glycan contents can be determined by correcting for the released non-fucosylated and high-mannose glycan contents, respectively. In the current antibody composition, as seen in Table 3, the abundance of paired glycan species is much lower, and therefore a lower impact on ADCC activity is expected. Nevertheless, this approach can be used to further understand the relationship between paired glycan content and ADCC activity. Paired non-fucosylated glycans are expected to have a statistically significant relationship with ADCC. As mentioned above, paired high-mannose glycan contents are expected to have a very low impact on ADCC activity.

[0166] These data support the importance of glycan pair analysis for understanding structure-function relationships.

[0167] Example 6 This example describes a method for determining product quality during the manufacturing of an antibody composition.

[0168] Samples of cell cultures containing antibody-producing clone 203 are collected at different pre-collection times after inoculation and at post-collection times before protein A chromatography. The novel method described in Example 1 is performed on the obtained samples. For each sample, data on paired high-mannose glycan content, unpaired high-mannose glycan content, paired non-fucosylated glycan content, and unpaired non-fucosylated glycan content are recorded. The ranges for unpaired high-mannose glycan content and unpaired non-fucosylated glycan content are predetermined. Samples considered to be outside this range are marked, and troubleshooting for the corresponding manufacturing date is monitored. Cell culture parameters were found to be fluctuating on this day, and efforts were made to prevent further fluctuations. The process of collecting samples at different points before and after collection, and the process of determining the unpaired high-mannose glycan content and unpaired non-fucosylated glycan content of the samples are repeated. Samples are considered to be within the predetermined range.

[0169] Example 7 This example describes a method for determining the product quality of different manufacturing lots of an antibody composition.

[0170] Samples from lots 1-10 of IgG1 monoclonal antibody are obtained after storage at 4°C for 12, 24, and 36 months. The novel method described in Example 1 is performed on the obtained samples. For each sample, data are recorded for paired high-mannose glycan species, unpaired high-mannose glycan pair content, paired non-fucosylated glycan content, and unpaired non-fucosylated glycan pair content. The predicted ADCC activity level for each lot is determined using Formula 7 and the data for % unpaired high-mannose glycan molecules and % unpaired non-fucosylated glycan molecules. Lots within the predetermined ADCC range are determined to be acceptable for administration to patients, while lots outside the range are discarded.

[0171] Example 8 This example describes a simplified method for determining paired and unpaired glycan content for the large-scale preparation of IgG described in Examples 1-7 above.

[0172] Three batches of IgG were prepared in a large bioreactor, and samples were obtained from each batch and used for two-enzyme digestion, essentially as described in Example 1. Briefly, the samples were treated overnight with the enzyme IgdE (FabALACTICA® or FL, manufactured by Genovis Inc., Cambridge, MA), followed by a second digestion with EndoS (IgGZERO®, manufactured by Genovis Inc.) for 30 minutes. After the two-enzyme digestion, the resulting Fc and Fab fragments were separated chromatographically by using high-speed reversed-phase liquid chromatography (RP-LC) separation with MS detection, essentially as described in Example 1. This separation yielded a single chromatographic peak containing the Fab fragment and the Fc fragment well separated from the processing enzymes. The chromatographic peak containing the Fc fragment was then selected for data analysis, and the mass spectrum of the single chromatographic peak containing the Fc fragment was extracted and deconvolved, essentially as described in Example 1. As essentially described in Example 1, EndoS-treated glycoparticle pairs were identified by matching the molecular weight (MW) of the deconvoluted mass spectral peaks of the antibody composition with a custom database of theoretical MWs (Table 1). Data for unpaired non-fucosylated glycoparticle pairs, paired non-fucosylated glycoparticle pairs, unpaired high-mannose glycoparticle pairs, and paired non-fucosylated glycoparticle pairs are shown in Table 6.

[0173] [Table 17]

[0174] The relative unpaired and paired non-fucosylated glycan content and the relative unpaired and paired high-mannose glycan content were calculated as described in Example 1, and the data are provided in Table 7 below.

[0175] [Table 18]

[0176] This example demonstrates that a novel, simplified method for determining relative unpaired glycan content can be implemented using large-scale preparation of IgG antibody compositions.

[0177] Example 9 This example describes a simplified method for determining paired and unpaired glycan content for antibody compositions containing IgG different from that described in Examples 1-8. The IgG in this example targeted a different antigen than that of the IgG in Examples 1-8.

[0178] Samples of antibody compositions containing IgG different from the IgG described in Examples 1-8 were used for two-enzyme digestion, essentially as described in Example 1. Briefly, the samples were treated overnight with the enzyme IgdE (FabALACTICA® or FL, manufactured by Genovis Inc., Cambridge, MA), followed by a second digestion with EndoS (IgGZERO®, manufactured by Genovis Inc.) for 30 minutes. After the two-enzyme digestion, the resulting Fc and Fab fragments were separated chromatographically by using high-speed reversed-phase liquid chromatography (RP-LC) separation with MS detection, essentially as described in Example 1. This separation yielded a single chromatographic peak containing the Fab fragment and the Fc fragment well separated from the processing enzymes. The chromatographic peak containing the Fc fragment was then selected for data analysis, and the mass spectrum of the single chromatographic peak containing the Fc fragment was extracted and deconvolved, essentially as described in Example 1. As essentially described in Example 1, EndoS-treated glycoparticle pairs were identified by matching the molecular weight (MW) of the deconvoluted mass spectral peaks of the antibody composition with a custom database of theoretical MWs (Table 1). Data for unpaired non-fucosylated glycoparticle pairs, paired non-fucosylated glycoparticle pairs, unpaired high-mannose glycoparticle pairs, and paired non-fucosylated glycoparticle pairs are shown in Table 8.

[0179] [Table 19]

[0180] The relative unpaired and paired non-fucosylated glycan content and the relative unpaired and paired high-mannose glycan content were calculated as described in Example 1, and the data are provided in Table 9 below.

[0181] [Table 20]

[0182] This example demonstrates that a novel, simplified method for determining relative unpaired glycan content can be used with IgG antibody compositions other than those described in Examples 1-8.

[0183] Example 10 This example describes a simplified method for determining paired and unpaired glycan content for antibody compositions containing IgG with the same isotype and antigen specificity as the IgG of Examples 1-7, but with a different allotype (e.g., including a modified Fc region sequence compared to the IgG of Examples 1-7).

[0184] Samples of antibody compositions containing different allotypes of IgG (compared to the IgG described in Examples 1-7) were subjected to two-enzyme digestion, essentially as described in Example 1. Briefly, the samples were treated overnight with the enzyme IgdE (FabALACTICA® or FL, manufactured by Genovis Inc., Cambridge, MA), followed by a second digestion with EndoS (IgGZERO®, manufactured by Genovis Inc.) for 30 minutes. After the two-enzyme digestion, the resulting Fc and Fab fragments were separated chromatographically by using high-speed reversed-phase liquid chromatography (RP-LC) separation with MS detection, essentially as described in Example 1. This separation yielded a single chromatographic peak containing the Fab fragment and the Fc fragment well separated from the processing enzymes. The chromatographic peak containing the Fc fragment was then selected for data analysis, and the mass spectrum of the single chromatographic peak containing the Fc fragment was extracted and deconvolved, essentially as described in Example 1. As essentially described in Example 1, EndoS-treated glycopairs were identified by matching the molecular weight (MW) of the deconvoluted mass spectral peaks of the antibody composition with a custom database of theoretical MWs, the custom database used in this example is described in Table 10. Data for unpaired non-fucosylated glycopairs, paired non-fucosylated glycopairs, unpaired high-mannose glycopairs, and paired non-fucosylated glycopairs are shown in Table 11.

[0185] [Table 21]

[0186] [Table 22]

[0187] [Table 23]

[0188] The relative unpaired and paired non-fucosylated glycan content and the relative unpaired and paired high-mannose glycan content were calculated as described in Example 1, and the data are provided in Table 12 below.

[0189] [Table 24]

[0190] Example 11 This example describes a simplified method for determining paired and unpaired glycan content for a panel of antibody composition samples, where each individual sample in the panel contained either an IgG1 monoclonal antibody produced by a specific clone or a sample from a reference product (RP) lot.

[0191] The panel samples were subjected to two-enzyme digestion, essentially as described in Example 1. Briefly, the samples were treated overnight with the enzyme IgdE (FabALACTICA® or FL, manufactured by Genovis Inc., Cambridge, MA), followed by a second digestion with EndoS (IgGZERO®, manufactured by Genovis Inc.) for 30 minutes. After the two-enzyme digestion, the resulting Fc and Fab fragments were separated chromatographically by using high-speed reversed-phase liquid chromatography (RP-LC) separation with MS detection, essentially as described in Example 1. This separation yielded a single chromatographic peak containing both the Fab fragment and the Fc fragment well-isolated from the processing enzymes. The chromatographic peak containing the Fc fragment was then selected for data analysis, and the mass spectrum of the single chromatographic peak containing the Fc fragment was extracted and deconvolved, essentially as described in Example 1. As essentially described in Example 1, EndoS-treated glycoparticle pairs were identified by matching the molecular weight (MW) of the deconvoluted mass spectral peaks of the antibody composition with a custom database of theoretical MWs (Table 10). Data for unpaired non-fucosylated glycoparticle pairs, paired non-fucosylated glycoparticle pairs, unpaired high-mannose glycoparticle pairs, and paired non-fucosylated glycoparticle pairs are shown in Table 13.

[0192] [Table 25]

[0193] The relative unpaired and paired non-fucosylated glycan content and the relative unpaired and paired high-mannose glycan content were calculated as described in Example 1, and the data are provided in Table 14 below.

[0194] [Table 26]

[0195] Example 12 This example describes a simplified method for determining paired and unpaired high-mannose glycan content in an antibody composition containing entirely non-fucosylated IgG.

[0196] A sample of an antibody composition containing completely unfucosylated IgG was used in a two-enzyme digestion essentially as described in Example 1. Briefly, the sample was treated overnight with the enzyme IgdE (FabALACTICA® or FL, manufactured by Genovis Inc., Cambridge, MA), followed by a second digestion with EndoS (IgGZERO®, manufactured by Genovis Inc.) for 30 minutes. After the two-enzyme digestion, the resulting Fc and Fab fragments were separated chromatographically by using high-speed reversed-phase liquid chromatography (RP-LC) separation with MS detection, essentially as described in Example 1. This separation yielded a single chromatographic peak containing the Fab fragment and the Fc fragment well separated from the processing enzymes. The chromatographic peak containing the Fc fragment was then selected for data analysis, and the mass spectrum of the single chromatographic peak containing the Fc fragment was extracted and deconvolved, essentially as described in Example 1. As essentially described in Example 1, EndoS-treated glycoparticle pairs were identified by matching the molecular weight (MW) of the deconvoluted mass spectral peaks of the antibody composition with a custom database of theoretical MWs (Table 15). Data for unpaired high-mannose glycoparticle pairs and paired non-fucosylated glycoparticle pairs are shown in Table 16.

[0197] [Table 27]

[0198] [Table 28] The relative unpaired high mannose glycan content and the paired high mannose glycan content were calculated as described in Example 1, and the data is provided in Table 17 below.

[0199] [Table 29]

[0200] All references cited herein, including publications, patent applications, and patents, are incorporated herein by reference to such an extent that each reference is shown to be incorporated individually and specifically by reference, and that the entirety of these references is described herein.

[0201] The use of the terms “one (a)” and “one (an)” and “it,” and similar reference subjects in relation to the descriptions in this disclosure (particularly in relation to the following claims), should be construed as encompassing both singular and plural unless otherwise indicated herein or unless explicitly contradicted by the context. The terms “comprising,” “having,” “including,” and “containing,” should be construed as non-restrictive terms unless otherwise specified, meaning “including, but not limited to,” including the components of the designation but not excluding other elements.

[0202] Unless otherwise indicated herein, the descriptions of value ranges are intended solely as a convenient way of referring to each individual value and endpoint within the range, incorporating each individual value and endpoint as if they were individually listed herein.

[0203] All methods described herein may be performed in any suitable order, unless otherwise specified herein or unless it is clearly inconsistent with the context. The use of any examples or exemplary language provided herein (e.g., "etc.") is intended solely to clarify the disclosure and does not impose any limitation on the scope of the disclosure unless otherwise claimed. No language herein should be construed as indicating that any unclaimed element is essential for the practice of the disclosure.

[0204] This specification describes preferred embodiments of the Disclosure, for example, the best modes known to the inventors for carrying out the Disclosure. Variations of these preferred embodiments will become apparent to those skilled in the art by reading the above description. The inventors expect that those skilled in the art will adopt such variations as needed, and the inventors intend that the Disclosure will be carried out in forms other than those specifically described herein. Accordingly, this Disclosure includes all variations and equivalents of the subject matter enumerated in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the above elements is incorporated herein in all possible variations unless otherwise indicated herein or unless explicitly contradicted in the context.

Claims

1. a. The IgG antibody composition is treated with two enzymes to form a mixture of Fab fragments and Fc fragments, wherein one enzyme cleaves the antibody heavy chain at the N-terminal site relative to the hinge region disulfide bond, and the other enzyme cleaves the β1,4 bond between core GlcNAc residues, thereby forming Fc fragments, each containing a pair of coreglycan structures. b. Separating the Fab fragment from the Fc fragment, c. i. Paired non-fucosylated Fc fragments, ii. Unpaired, non-fucosylated Fc fragments, iii. Paired high-mannose Fc fragments, and / or iv. Unpaired high mannose Fc fragment The amount of each is quantified to determine the relative unpaired non-fucosylated (AF) glycan content and the relative unpaired high-mannose (HM) glycan content. A method for determining the relative unpaired glycan content of an IgG antibody composition, including [the specified substance].

2. The method according to claim 1, wherein one of the enzymes is a cysteine ​​protease.

3. The method according to claim 2, wherein the cysteine ​​protease cleaves the sequence KTHTCPP (SEQ ID NO: 1) of the IgG1 antibody heavy chain at a site between Thr and His or between Lys and Thr.

4. The method according to claim 2 or 3, comprising treating the IgG antibody composition with the cysteine ​​protease for at least 8 hours or at least 12 hours.

5. The method according to any one of claims 1 to 4, wherein the other enzyme is an IgG-specific enzyme.

6. The method according to claim 5, wherein the IgG-specific enzyme is an endoglycosidase.

7. The method according to claim 6, wherein the endoglycosidase is Endo S endoglycosidase.

8. The method according to any one of claims 5 to 7, comprising treating the mixture with the IgG-specific enzyme for less than one hour.

9. The method according to claim 8, comprising treating the IgG antibody composition with the IgG-specific enzyme for about 30 minutes.

10. The method according to any one of claims 1 to 9, wherein the Fab fragment is separated from the Fc fragment by chromatography.

11. The method according to claim 10, wherein the chromatography is reversed-phase liquid chromatography.

12. The method according to claim 10 or 11, comprising performing mass spectrometry on a chromatographic fraction containing the Fc fragment to obtain one or more mass spectral peaks.

13. The method according to claim 12, comprising deconvolving the mass spectral peak to obtain a deconvolved mass spectral peak.

14. The method according to claim 13, comprising identifying a glycan pair by comparing the molecular weight of each deconvolved mass spectral peak with a database of glycan pairs and associated molecular weights.

15. The method according to any one of claims 1 to 14, wherein (b) and (c) occur in less than two hours.

16. The method according to claim 15, wherein (b) and (c) occur in less than 75 minutes.

17. The method according to claim 16, wherein (b) and (c) occur in less than 60 minutes.

18. Determining the relative unpaired glycan content of a sample of the IgG antibody composition according to the method described in any one of claims 1 to 17, The relative unpaired AF glycan content and / or relative unpaired HM glycan content of the IgG antibody composition are compared with the target range of the relative unpaired AF glycan content and / or relative unpaired HM glycan. A method for preparing an IgG antibody composition having ADCC activity within the target range, including [a specific component].

19. Determining the relative unpaired glycan content of a sample of the IgG antibody composition according to the method described in any one of claims 1 to 17, Optionally, the relative unpaired AF glycan content and / or relative unpaired HM glycan content of the IgG antibody composition are compared with the relative unpaired AF glycan content and / or relative unpaired HM glycan content of the reference product. A method for analyzing an IgG antibody composition containing the following.

20. The method according to claim 18 or 19, wherein the method is part of the process for producing the IgG antibody composition.

21. The method according to any one of claims 18 to 20, wherein the method is performed in real time during the production of the IgG antibody composition.

22. The method according to any one of claims 18 to 21, wherein the sample is a sample of an in-process material.

23. The method according to any one of claims 18 to 22, wherein the relative unpaired glycan content is determined before or after harvesting.

24. The method according to claim 23, wherein the relative unpaired glycan content is determined after sampling.

25. The method according to any one of claims 18 to 25, wherein the sample is obtained from a manufacturing lot.

26. The relative unpaired glycan content of the IgG antibody composition is determined for the first sample obtained at a first time point and the second sample taken at a second time point different from the first time point, according to the method described in any one of claims 1 to 17. Optionally, the relative unpaired AF glycan content and / or relative unpaired HM glycan content of the first sample are compared with the relative unpaired AF glycan content and / or relative unpaired HM glycan content of the second sample. A method for monitoring the production of an IgG antibody composition containing [a specific substance].

27. The method according to claim 26, wherein each of the first sample and the second sample is a sample of an in-process material.

28. The method according to claim 26, wherein the first sample is a sample of in-process material and the second sample is a sample of a manufacturing lot.

29. The method according to claim 26, 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.

30. (A) comprising determining the relative unpaired glycan content of a sample of the IgG antibody composition according to the method of any one of claims 1 to 17, If the sample is an in-process material sample and the relative unpaired glycan content is determined to be outside the target range, (B) modify one or more conditions of the cell culture to obtain a modified cell culture and determine the relative unpaired glycan content, By choice, steps (A) and (B) are repeated until the relative unpaired glycan content falls within the target range. A method for producing an IgG antibody composition further comprising the above.

31. The method according to claim 30, wherein one or more conditions of the cell culture are modified to primarily change the relative unpaired HM glycan content in order to achieve the target range of relative unpaired glycan content.

32. The method according to claim 30, wherein one or more conditions of the cell culture are modified to primarily change the relative unpaired AF glycan content in order to achieve the target range of relative unpaired glycan content.

Images