Methods for the quantification of glycoproteins

Abstract

The present invention relates to a method for the quantification of a glycoprotein in a sample, comprising quantifying said glycoprotein by comparison to an internal standard, said internal standard comprising a variant form of said glycoprotein, said variant form containing only a core Asn-linked GlcNAc moiety.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to methods for the quantification of glycoproteins in a sample and materials for use in such methods. [Background technology]

[0002] Background of the Invention Recombinant proteins are increasingly being developed for use, for example, as drug products. Analytical tools such as liquid chromatography (LC) and capillary electrophoresis (CE) have been successfully used to separate and quantify size and charge variants of intact monoclonal antibody variants (Staub et al., J Pharm Biomed Anal 55, 810-822, 2011). In addition, immunoassays such as enzyme-linked immunosorbent assays (ELISAs), which utilize specific antibodies against target proteins in human serum or plasma, have also been performed for quantification purposes (Mushens et al., J. Immunol. Methods, 162 (1), 1993). LC-mass spectrometry (MS) is a commonly used method for protein analysis and quantification. In the "bottom-up" MS approach, enzymatic digestion of a target protein generates signature peptides, which are then quantified in the presence of stable isotope-labeled (SIL) peptides as internal standards (IS) (US20120264155A1). Several drawbacks in peptide-level quantification have led to increased interest in intact protein quantification (Jin et al., Bioanalysis 10(11), 851-862, 2018). Selecting the most appropriate signature peptide and optimizing the enzymatic digestion process make assay development complex and time-consuming. Additionally, enzymatic protein digestion increases spectral complexity, making the analysis more susceptible to interferences. However, a major limitation of peptide-level quantification is that information about the entire protein molecule is lost during digestion, meaning that any given peptide cannot truly represent the entire protein. Macchi et al. describe an MS-based method for quantifying intact bispecific antibodies and their variants (Macchi et al., Anal Chem 87:10475-82 (2015)). However, LC-MS-based quantification of intact antibody mixtures presents challenges related to size, structural complexity, and heterogeneity in both LC and MS.Therefore, the use of intact surrogate protein species as IS remains limited.

[0003] Immunoglobulins are glycoproteins. IgG1, IgG2, and IgG4 have a single conserved Asn-linked glycosylation site in the CH2 region (position Asn297 in IgG1), thus carrying two glycans per immunoglobulin molecule. Other immunoglobulins are more heavily glycosylated (see, for example, Maverakis et al. (2015) J Autoimmun, 57:1-13). Due to the variable addition of fucose, galactose, and sialic acid residues to the core complex-type biantennary heptasaccharide (GlcNAc2Man3GlcNAc2), IgG-Fc Asn-linked glycans are highly heterogeneous post-translationally modified. Glycans play important roles in protein conformation, stability, and biological function (Costa et al., Crit Rev Biotechnol 34(4): 281-99 (2014)). Fc glycan heterogeneity varies depending on the species and expression system. Mammalian cell lines are commonly used to produce recombinant glycoproteins, with Chinese hamster ovary (CHO)-based expression systems being the most commonly used. Recombinant biopharmaceuticals are also produced in other expression systems, such as yeast, plants, or insects (Lalonde et al., J Biotechnol 251:128-40 (2017)). However, these latter organisms produce glycan structures that differ from those produced in mammalian cells due to differences in enzymatic mechanisms.

[0004] Deglycosylation of glycoprotein samples can be achieved by various glycosidases, such as peptide:N-glycosidase F (PNGase F), each with its own unique glycan cleavage pattern (Seki et al., (2019) J. Biol. Chem. 294(45): 17143-54). LC-MS analysis after deglycosylation has been applied to characterize the N-glyco-occupancy status of therapeutic monoclonal antibodies (Liu et al., Anal Biochem 509 p142-45, 2016). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] US20120264155A1 [Non-patent literature]

[0006] [Non-Patent Document 1] Staub et al, J Pharm Biomed Anal 55, 810-822, 2011 [Non-patent document 2] Mushens et al, J. Immunol. Methods, 162 (1), 1993 [Non-patent document 3] Jin et al, Bioanalysis 10(11), 851-862, 2018 [Non-patent document 4] Macchi et al., Anal Chem 87:10475-82 (2015) [Non-Patent Document 5] Maverakis et al. (2015) J Autoimmun, 57:1-13 [Non-patent document 6] Costa et al., Crit Rev Biotechnol 34(4): 281-99 (2014) [Non-Patent Document 7] Lalonde et al., J Biotechnol 251:128-40 (2017) [Non-patent document 8] Seki et al., (2019) J. Biol. Chem. 294(45): 17143-54 [Non-Patent Document 9] Liu et al, Anal Biochem 509 p142-45, 2016 Summary of the Invention

[0007] There remains a need for improved methods for accurately quantifying proteins in a sample, particularly in a mixture of proteins.

[0008] The present invention provides a method for the quantification of glycoproteins that uses internal standards with biophysical properties that are highly similar to the individual protein sample components in a mixture, thereby allowing accurate quantification of the composition of such protein samples.

[0009] The internal standard used in the method of the invention is a variant form of the glycoprotein(s) to be quantified, which contains only the core GlcNAc moiety rather than the complete glycan structure. The method of the invention exploits the resulting difference in mass between the glycoprotein in the sample and the variant form of the internal standard to quantify the protein in the sample relative to the internal standard.

[0010] Thus, in a first aspect, the present invention relates to a method for the quantification of one or more glycoproteins in a sample, each glycoprotein to be quantified comprising one or more Asn-linked glycans, said method comprising the steps of: a. providing a sample containing the one or more glycoproteins to be quantified; b. adding an internal standard to the sample, wherein the internal standard comprises a variant form of each of the one or more glycoproteins to be quantified, the variant form being a form that contains only a core Asn-linked GlcNAc moiety; c. quantitating said one or more glycoproteins by comparison with said internal standard; Includes.

[0011] In a further aspect, the present invention relates to a method for the quantification of one or more glycoproteins in a sample, wherein each glycoprotein to be quantified comprises one or more Asn-linked glycans, said method comprising the steps of: a. preparing an internal standard by treating said one or more glycoproteins with one or more enzymes to obtain variant forms of each of said one or more glycoproteins, wherein said variant forms contain only core Asn-linked GlcNAc moieties; b. providing a sample containing the one or more glycoproteins to be quantified; c. adding the internal standard to the sample; d. quantitating said one or more glycoproteins by comparison to said internal standard; Includes.

[0012] In yet a further aspect, the present invention relates to a composition comprising a known quantity of a variant form of a glycoprotein, said variant form containing only a core Asn-linked GlcNAc moiety, for use as an internal standard for quantitating said glycoprotein in a sample. [The present invention 1001] 1. A method for the quantification of one or more glycoproteins in a sample, comprising: each glycoprotein to be quantified contains one or more Asn-linked glycans; The method comprises the steps of: a. providing a sample containing the one or more glycoproteins to be quantified; b. adding an internal standard to the sample, the internal standard comprising a variant form of each of the one or more glycoproteins to be quantified, the variant forms containing only core Asn-linked GlcNAc moieties, each core Asn-linked GlcNAc moiety being an N-acetylglycosamine moiety attached directly to an Asn acceptor residue in the polypeptide chain of the variant, and no fucose moiety being linked to the core GlcNAc moiety; and c. Quantifying said one or more glycoproteins by comparison with said internal standard. [The present invention 1002] prior to step c., treating the sample with an enzyme; the enzyme is capable of completely removing Asn-linked glycans from the one or more glycoproteins, but is unable to remove core Asn-linked GlcNAc moieties from the variant forms; The method of the present invention 1001. [The present invention 1003] The method of claim 1002, wherein the enzyme treatment is carried out prior to step b. [The present invention 1004] 1003. The method of claim 1003, wherein the enzyme treatment is carried out after the addition of an internal standard in step b. [The present invention 1005] The method of any one of claims 1002 to 1004, wherein the enzyme is PNGase F (EC 3.5.1.52). [The present invention 1006] The method of claim 1001, further comprising, prior to step b., treating the sample with an enzyme capable of completely removing Asn-linked glycans from said one or more glycoproteins, and optionally then removing or inactivating said enzyme. [The present invention 1007] Any of the aforementioned methods of the present invention, wherein the quantification in step c. is carried out using mass spectrometry. [The present invention 1008] Any of the aforementioned methods of the present invention, wherein the quantification in step c. is performed using a combination of mass spectrometry and non-mass-based separation techniques. [The present invention 1009] Any of the methods of the present invention, wherein the quantification in step c. is carried out using liquid chromatography-mass spectrometry (LC-MS), for example, reverse-phase based LC-MS. [The present invention 1010] Any of the aforementioned methods of the invention which do not include the step of proteolytically digesting or otherwise fragmenting the amino acid chains of the glycoprotein to be quantified. [The present invention 1011] Any of the methods of the invention, wherein both the glycoprotein(s) to be quantified and the variant form(s) thereof used as internal standards are used in their full-length, i.e., form having the full-length amino acid chain, or almost full-length, for example a form comprising 50% or more, such as 75% or more, for example 90% or more, for example 95% or more of their full-length amino acid sequence. [The present invention 1012] Any of the aforementioned methods of the invention, wherein the one or more glycoproteins to be quantified are antibodies. [The present invention 1013] Any of the methods of the invention described above comprising the quantification of two or more, such as two, three, four, five or more, different antibodies. [The present invention 1014] The method of claim 1013, wherein said two or more antibodies are IgG antibodies, for example full-length IgG antibodies. [The present invention 1015] Any of the aforementioned methods of the invention, wherein at least one of the one or more glycoproteins to be quantified contains fucose in a core Asn-linked GlcNAc moiety. [The present invention 1016] Any of the aforementioned methods of the present invention, wherein the sample is a cell culture sample. [The present invention 1017] Any of the aforementioned methods of the invention, wherein the sample comprises purified, recombinantly produced glycoprotein. [The present invention 1018] 1016 or 1017, wherein steps b. and c. of said method are carried out in an automated manner, preferably steps a., b., and c. are carried out in an automated manner. [The present invention 1019] The method of any of claims 1001 to 1015, wherein the sample is a blood sample, a plasma sample, or a serum sample. [The present invention 1020] 1. A process for monitoring the production of a glycoprotein in a cell culture, comprising: Culturing a host cell that produces the glycoprotein; Carrying out the method of the present invention 1016 or 1018 The process comprising: [The present invention 1021] 1. A process for quality control of purified recombinantly produced glycoproteins, comprising: Carrying out the method of the present invention 1017 or 1018 The process comprising: [The present invention 1022] 1. A composition comprising a known number of variant forms of a glycoprotein, the variant form contains only core Asn-linked GlcNAc moieties, each core Asn-linked GlcNAc moiety being an N-acetylglycosamine moiety attached directly to an Asn acceptor residue in the polypeptide chain of the variant, and no fucose moieties are linked to the core GlcNAc moiety; said composition for use as an internal standard for the quantification of said glycoprotein in a sample. [The present invention 1023] 1. A method for the quantification of one or more glycoproteins in a sample, comprising: each glycoprotein to be quantified contains one or more Asn-linked glycans; The method comprises the steps of: a. preparing an internal standard by treating the one or more glycoproteins with one or more enzymes to obtain variant forms of each of the one or more glycoproteins, wherein the variant forms contain only core Asn-linked GlcNAc moieties, each core Asn-linked GlcNAc moiety being an N-acetylglycosamine moiety attached directly to an Asn acceptor residue in the polypeptide chain of the variant, and no fucose moiety being linked to the core GlcNAc moiety; b. providing a sample containing the one or more glycoproteins to be quantified; c. adding the internal standard to the sample; and d. Quantifying said one or more glycoproteins by comparison with said internal standard. [The present invention 1024] The method of claim 1023, wherein said treatment with one or more enzymes in step a. comprises treatment with an endo-β-N-acetylglucosaminidase, such as EndoS2 or EndoS. [The present invention 1025] Any of the methods of claims 1023 to 1024, wherein the treatment with one or more enzymes in step a. comprises treatment with an endo-β-N-acetylglucosaminidase, such as EndoS2 or EndoS, and a fucosidase capable of hydrolyzing the alpha-1,6 linkage from an Asn-linked fucose-alpha-1,6-GlcNAc moiety. [The present invention 1026] The method of any of claims 1023-1025, including one or more of the additional features of claims 1002-1019. [Brief explanation of the drawings]

[0013] [Figure 1]A schematic overview of a possible application of the described method is shown. In this example, a mixture of five IgG antibodies can be quantified using five variant antibodies that serve as internal standards for the five antibodies to be quantified. These internal standards are identical to the fully deglycosylated antibodies to be quantified, except for the remaining core GlcNAc moiety on the internal standards to distinguish them from the antibodies to be quantified. By applying time-resolved deconvolution (TRD), in which observed mass (x-axis) is plotted against elution time (y-axis), a two-dimensional view can be generated, allowing for the identification of individual antibody samples and internal standards. In the third dimension, peak intensities are preserved in such a way that peak volumes can be estimated for further quantitative calculations. [Figure 2A] Figure 2 shows that enzymatic deglycosylation of IgG antibodies with EndoS2 results in sample heterogeneity caused by the presence of defucosylated species. (A) Enzymatic deglycosylation of IgG1-CD19-21D4-E345K with EndoS2 results in efficient removal of glycans down to the core Asn-linked GlcNAc group. However, the presence of defucosylated IgG1-CD19-21D4-E345K results in sample heterogeneity, where some antibodies contain two heavy chains with GlcNAc-fucose moieties, some contain one heavy chain with a GlcNAc-fucose moiety and one heavy chain with a GlcNAc moiety without fucose attached to it, and some contain two heavy chains with GlcNAc moieties without fucose attached to it. (B) Enzymatic deglycosylation of IgG1-CD19-21D4-E345K with PNGase F results in a homogenous population of fully deglycosylated IgG1-CD19-21D4-E345K. [Figure 2B] See legend to Figure 2A. [Figure 3]These figures show that enzymatic treatment of EndoS2-treated IgG antibodies with α-L-fucosidase from L. casei efficiently removed remaining fucose groups, resulting in reduced mass heterogeneity. In panel A, IgG1-CD19-21D4-E345K was treated with EndoS2 alone, resulting in a large proportion of IgG-(GlcNAc-Fuc)2 as well as some IgG-(GlcNAc-Fuc) and IgG-(GlcNAc). In panel B, IgG1-CD19-21D4-E345K was treated with both EndoS2 and α-L-fucosidase, resulting in a single peak of IgG-(GlcNAc). [Figure 4] This shows that PNGase F from a PNGase F-treated sample does not remove residual GlcNAc moieties from IgG antibodies treated with EndoS2 and α-L-fucosidase. (A) PNGase F from the PNGase F-treated sample IgG1-CD19-21D4-E345K does not act on the internal standard IgG1-CD19-21D4-E345K-(GlcNAc)2 treated with EndoS2 and α-L-fucosidase. (B) PNGase F from sample IgG1-CD19-21D4-E345K treated with PNGase F does not act on the internal standard IgG1-CD19-21D4-E345K-(GlcNAc)2 treated with EndoS2 and α-L-fucosidase or on untreated IgG1-b12, whereas EndoS2 and α-L-fucosidase from the internal standard act on untreated IgG1-b12. [Figure 5-1]Figure 5 shows MS and LC analyses of an antibody mixture consisting of deglycosylated human IgG1-CD19-21D4-E345K, IgG1-CD22-huRFB4, IgG1-7D8, IgG1-CD37-37-3, and IgG1-CD52-Campath-E345K antibodies and their corresponding internal standards carrying an Asn-linked GlcNAc moiety on each heavy chain. (A) MS spectra showing overlapping antibody mass peaks and matching internal standards processed by conventional deconvolution mass spectrometry. (B) Elution times of the antibodies and variant antibody internal standards listed in (A) as determined by LC analysis. (C) Time-resolved deconvoluted mass spectra of the MS and LC data of the antibody sample mixtures and variant internal standards listed in (A), with the mass (Da) of each sample or standard mixture on the x-axis and the elution time (min) on the y-axis. The boxes indicate the boundaries of the peak volume integrals. [Figure 5-2] See description of Figure 5-1. [Figure 6-1] Figure 6 shows the sample / IS ratio plotted against the known antibody concentration (µg / mL) for each antibody tested. Results are shown for (A) IgG1-CD19-21D4-E345K, (B) IgG1-CD22-huRFB4, (C) IgG1-7D8, (D) IgG1-CD37-37-3, and (E) IgG1-CD52-Campath-E345K. Figure 6F-J shows the antibody concentration (µg / mL) determined using time-resolved deconvolution MS plotted against the expected known antibody concentration (µg / mL). Results are shown for (F) IgG1-CD19-21D4-E345K, (G) IgG1-CD22-huRFB4, (H) IgG1-7D8, (I) IgG1-CD37-37-3, and (J) IgG1-CD52-Campath-E345K. [Figure 6-2] See description of Figure 6-1. DETAILED DESCRIPTION OF THE INVENTION

[0014] Detailed Description of the Invention definition As used herein, the term "glycoprotein" refers to a protein containing one or more glycans (i.e., oligosaccharides or carbohydrate side chains) covalently attached to the polypeptide chain. The process of attaching glycans to a polypeptide is known as glycosylation. One of the most common types of glycosylation is Asn-linked glycosylation, in which a glycan is attached to the amide nitrogen of an asparagine residue in the polypeptide.

[0015] The term "internal standard" has its ordinary meaning in the art and refers to a known quantity of a substance, herein a variant glycoprotein, that is added to a sample containing an analyte to be quantified, e.g., a glycoprotein, which can then be used to quantify the analyte by determining the ratio of the analyte signal to the internal standard signal.

[0016] The term "immunoglobulin" refers to a class of structurally closely related glycoproteins consisting of two pairs of polypeptide chains: a pair of low-molecular-weight light (L) chains and a pair of heavy (H) chains, all four of which may be interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain typically consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region typically consists of three domains, CH1, CH2, and CH3. The heavy chains are interconnected via disulfide bonds at the so-called "hinge region." Each light chain typically consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability (or hypervariable regions whose sequences can be hypervariable and / or in the form of structurally defined loops), also called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged in the following order from amino to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Unless otherwise stated or contradicted by the context, CDR sequences herein are identified using DomainGapAlign according to the IMGT rules (Lefranc MP., Nucleic Acids Research 1999;27:209-212 and Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38, D301-307 (2010); internet http address (www.imgt.org / ) See also:

[0017] Reference to amino acid positions in the Fc region / Fc domain in the present invention follows EU numbering (Edelman et al., Proc Natl Acad Sci U.S.A. 1969 May;63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest. 5th Edition - 1991 NIH Publication No. 91-3242).

[0018] The Fc region of an immunoglobulin is typically defined as the fragment of an antibody that would be generated after digestion of the antibody with papain, encompassing the two CH2-CH3 regions and connecting regions, such as the hinge region, of the immunoglobulin. The constant domains of the antibody heavy chain define the antibody isotype, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE. The Fc region mediates antibody effector functions through cell surface receptors called Fc receptors and proteins of the complement system.

[0019] As used herein, the term "glycosylation" refers to a post-translational modification involving the selective attachment of glycans to polypeptides, such as antibodies, to stabilize the structure of the polypeptide or provide function to the polypeptide. Glycan structures vary between organisms, but generally contain two or more GlcNAc moieties, multiple mannose moieties, and optionally additional carbohydrate moieties, such as galactose moieties, fucose moieties, and sialic acid moieties. The most predominant types of glycosylation, N-linked and O-linked glycosylation, occur in the endoplasmic reticulum and Golgi apparatus of eukaryotic cells, respectively. N-linked (or Asn-linked) glycosylation refers to the process of linking carbohydrate structures to the side chain nitrogen atom of asparagine (Asn) residues in polypeptides, while O-linked glycosylation refers to the process of linking carbohydrate structures to the side chain oxygen atom of serine (Ser) or threonine (Thr) residues in polypeptides. Immunoglobulins, such as IgG molecules, have a single conserved Asn-linked glycosylation site at amino acid position Asn297 in the CH2 domain. As used herein, the term "deglycosylation" refers to the removal, e.g., enzymatic removal, of glycans from a polypeptide, e.g., an antibody.

[0020] As used herein, the term "core GlcNAc moiety" or "core Asn-linked GlcNAc moiety" refers to an N-acetylglycosamine moiety, also known as GlcNAc, that is attached directly (and therefore not via another moiety) to an Asn acceptor residue in a polypeptide chain. In other words, it is the GlcNAc moiety that is most proximal to the polypeptide chain.

[0021] As used herein, the term "fucose" or "fucosylation" refers to a fucose moiety that is part of the glycan structure of a glycoprotein. The fucose moiety can be present in various locations in complex glycan structures. In this context, fucose moieties linked to a core GlcNAc moiety are of particular interest.

[0022] As used herein, the term "complete removal" or variations thereof, in the context of removing Asn-linked glycans from glycoproteins, refers to the removal of the entire glycan structure from the Asn acceptor site in the polypeptide chain, including the core GlcNAc moiety, whereby the Asn acceptor residue is converted to Asp.

[0023] The term "hinge region" as used herein is intended to refer to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216 to 230 according to EU numbering.

[0024] As used herein, the term "CH2 region" or "CH2 domain" is intended to refer to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to EU numbering. However, the CH2 region may also be of any of the other subtypes described herein.

[0025] As used herein, the term "CH3 region" or "CH3 domain" is intended to refer to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to EU numbering. However, the CH3 region may also be of any of the other subtypes described herein.

[0026] As used herein, the term "isotype" refers to the immunoglobulin class encoded by the heavy chain constant region genes (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE, or IgM, or any allotype thereof, e.g., IgG1m(za) and IgG1m(f)). Furthermore, each heavy chain isotype can be combined with either a kappa (κ) or lambda (λ) light chain.

[0027] The term "antibody" (Ab), in the context of the present invention, refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either, capable of specifically binding to an antigen. The variable regions of the heavy and light chains of an immunoglobulin molecule contain binding domains that interact with antigens. As described above, the constant or "Fc" region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, such as C1q, the first component of the classical pathway of complement activation. An antibody can also be a multispecific antibody, e.g., a bispecific antibody, or similar molecule. The term "bispecific antibody" refers to an antibody having specificity for at least two different, typically non-overlapping, epitopes. Such epitopes can be on the same or different targets. When the epitopes are on different targets, such targets can be on the same cell or different cells or cell types. As indicated above, unless otherwise stated or clearly contradicted by the context, the term antibody as used herein encompasses antibody fragments that retain the ability to specifically bind to an antigen. Such fragments can be provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant expression. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0028] The term "full length antibody," as used herein, refers to an antibody that contains all heavy and light chain constant and variable domains that correspond to those normally found in a wild-type antibody of that isotype. A "full length amino acid sequence" refers to the entire amino acid sequence of a protein as it is present in the sample to be quantified, e.g., the entire amino acid sequence of the heavy chain of an antibody in the sample.

[0029] As used herein, the terms "antigen-binding region," "antigen binding region," "binding region," or "antigen-binding domain" refer to the region of an antibody capable of binding to an antigen. This binding region is typically defined by the VH and VL domains of an antibody, which can be further subdivided into regions of hypervariability (or hypervariable regions whose sequences can be in the form of hypervariable and / or structurally defined loops), also called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). An antigen can be any molecule, such as a polypeptide present on a cell, bacterium, or virion.

[0030] As used herein, the term "target" or "target antigen" refers to a molecule to which the antigen-binding region of an antibody binds. A target includes any antigen against which an antibody is directed. The terms "antigen" and "target" may be used interchangeably with respect to an antibody and may apply to the same meaning and purpose for any aspect or embodiment of the present invention.

[0031] A "variant form" is a glycoprotein molecule that contains one or more modifications, e.g., amino acid substitutions, compared to the "parent" glycoprotein. Exemplary parent antibody formats include, but are not limited to, wild-type antibodies, full-length antibodies or Fc-containing antibody fragments, bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, or any combination thereof. Modifications can include modifications of glycans or of amino acid sequences. In the context of the present invention, amino acid substitutions in variants include the following: Original amino acid - position - substituted amino acid is shown as:

[0032] Three-letter or one-letter codes are used, including the codes Xaa and X to denote amino acid residues. Thus, the designation "E345R" or "Glu345Arg" means that the variant contains a substitution of glutamic acid with arginine at the variant amino acid position corresponding to the amino acid at position 345 of the parent antibody.

[0033] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which an expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, CHO cells, HEK 293 cells, PER.C6, NS0 cells, and lymphocytic cells, as well as other eukaryotic hosts such as plant cells and fungi.

[0034] As used herein, the term "plasma half-life" refers to the time it takes for the concentration of a polypeptide in blood plasma to decrease to half of its initial concentration during elimination (after the distribution phase). For antibodies, the distribution phase is typically considered to be 1 to 3 days. During this period, there is an approximately 50% decrease in blood plasma concentration due to redistribution between plasma tissues.

[0035] As used herein, the term "antibody-drug conjugate" refers to an antibody or Fc-containing polypeptide having specificity for at least one type of malignant cell, a drug, and a linker that connects the drug to, for example, the antibody. The linker is cleavable or non-cleavable in the presence of malignant cells, and the antibody-drug conjugate kills the malignant cells.

[0036] Further aspects and embodiments of the present invention As described above, in a first aspect, the present invention relates to a method for the quantification of one or more glycoproteins in a sample, wherein each glycoprotein to be quantified comprises one or more Asn-linked glycans, said method comprising the steps of: a. providing a sample containing the one or more glycoproteins to be quantified; b. adding an internal standard to the sample, wherein the internal standard comprises a variant form of each of the one or more glycoproteins to be quantified, the variant form being a form that contains only a core Asn-linked GlcNAc moiety; c. quantitating said one or more glycoproteins by comparison with said internal standard; Includes.

[0037] In one embodiment, the method of the present invention does not include a step of proteolytically digesting or otherwise fragmenting the polypeptide chain of the glycoprotein to be quantified.

[0038] In one embodiment, prior to step c., the method comprises treating the sample with an enzyme that is capable of completely removing Asn-linked glycans from said one or more glycoproteins but is not capable of removing core Asn-linked GlcNAc moieties from said variant forms, i.e., the enzyme is capable of completely removing Asn-linked glycans extending from a core GlcNAc moiety but is not capable of removing Asn-linked glycans that do not have any extension from a core GlcNAc moiety, such as Asn-linked glycans consisting solely of an optionally fucosylated core GlcNAc moiety.

[0039] As shown in the Examples herein, PNGase F is an example of an enzyme that can completely remove Asn-linked glycans from glycoproteins. That is, this enzyme removes the entire Asn-linked glycan from glycoproteins, including those containing the core GlcNAc moiety (thereby converting Asn to Asp). However, it is also shown in the Examples herein that PNGase F does not remove the core GlcNAc moiety from glycoprotein variants that only have the core Asn-linked GlcNAc moiety. Therefore, treatment of a sample with an enzyme having the properties of PNGase F results in complete deglycosylation of glycoproteins in the sample, but not of an internal standard that retains the core Asn-linked GlcNAc. The methods of the present invention exploit the resulting difference in mass for quantitation of proteins in a sample relative to an internal standard. Figure 1 illustrates a (non-limiting) embodiment of the method of the present invention.

[0040] In one embodiment, treatment with an enzyme capable of completely removing Asn-linked glycans from the one or more glycoproteins but not removing core Asn-linked GlcNAc moieties from variant forms occurs prior to step b. In another embodiment, the enzyme treatment occurs after addition of an internal standard in step b. In one embodiment, the enzyme is peptide:N-glycosidase F, otherwise known as PNGase F (EC 3.5.1.52) (Norris et al., Structure 2(11): p1049-59 (1994)).

[0041] In another embodiment, the method comprises, prior to step b., treating the sample with an enzyme capable of completely removing Asn-linked glycans from said one or more glycoproteins, and optionally then removing or inactivating said enzyme prior to step b. For example, if an enzyme is used to deglycosylate the glycoprotein to be quantified that can completely remove Asn-linked glycans from the glycoprotein but also remove core Asn-linked GlcNAc from variant forms, it may be beneficial or even necessary to remove or inactivate said enzyme prior to addition of the variant forms in order to preserve the mass difference between the variant forms and the deglycosylated protein to be quantified.

[0042] As described, the method of the present invention uses a variant form of the glycoprotein to be quantified as an internal standard. Preferably, for each glycoprotein to be quantified, the corresponding variant form in the internal standard is a homogenous preparation, i.e., for each glycoprotein, all variant molecules are identical in mass.

[0043] The variant forms used are those containing only core Asn-linked GlcNAc moieties. In particular, each core Asn-linked GlcNAc moiety can consist of a single N-acetylglucosamine moiety attached directly to an Asn acceptor residue in the polypeptide chain of said variant form of each of said one or more glycoproteins. In a preferred embodiment, no fucose moiety is linked to said core GlcNAc moiety.

[0044] In another embodiment, the core GlcNAc moiety is fucosylated, preferably 100% fucosylated, i.e., all molecules of the variant form used as an internal standard are fucosylated.

[0045] In preferred embodiments, the variant form used as an internal standard is greater than 95% glycosylated, i.e., greater than 95% of the Asn acceptor sites out of the total number of acceptor sites of all variant molecules in the composition have an attached GlcNAc. In further embodiments, the variant form is greater than 98%, such as greater than 99%, e.g., 100% glycosylated.

[0046] As described, quantification of the one or more glycoproteins is performed by comparison with the internal standard. This overcomes the problem that external calibration curves, consisting of samples measured as separate measurements, are affected by experimental conditions, such as MS instrument conditions, spray conditions, sample recovery, carryover, etc., when used exclusively. To compensate for any possible variations during the entire sample preparation and analysis process, both the internal standard and the sample analyte are analyzed simultaneously. The amount of internal standard added should be an appropriate ratio to the analyte after considering the dynamic range. As long as the concentrations of the internal standard and sample analyte(s) fall within a linear dynamic range, no calibration curve is required. However, the use of an external calibration curve in conjunction with an internal standard is common practice and provides the best results. This is because the use of an external standard (ES) is expected to be free of interference from, for example, endogenous proteins and / or sample matrix, and the internal standard simultaneously corrects for any bias in instrument stability during analysis. To obtain a calibration curve, the MS peak intensity ratio (ES / IS) is calculated (Y value) and plotted against the known ES concentration (X value). Upon sample analysis (spiked with IS), the MS peak intensity ratio (Y value) is calculated (unknown / IS), and the sample concentration can be estimated from the calibration curve by interpolation.

[0047] In one embodiment, the quantification in step c. is performed using mass spectrometry. In a further embodiment, the quantification in step c. is performed using a combination of mass spectrometry and a non-mass-based separation technique. Such a combination of different techniques may be particularly useful when the sample has a complex composition and / or multiple glycoproteins are to be quantified. A non-mass-based separation technique may allow the separation of two or more proteins with highly similar masses, thereby allowing for separate quantification. For example, in one embodiment, a sample may contain two or more glycoproteins with a mass difference of less than 75 Da, e.g., less than 60 Da, e.g., less than 50 Da, after the addition of an internal standard. In one embodiment, the quantification in step c. is performed using liquid chromatography-mass spectrometry (LC-MS), e.g., a reverse-phase based LC-MS.

[0048] The glycoprotein to be quantified can be any glycoprotein containing one or more Asn-linked glycans, including native glycoproteins, recombinant glycoproteins, therapeutic glycoproteins, diagnostic glycoproteins, enzymes, etc. In one embodiment, the glycoprotein to be quantified contains only Asn-X-Thr glycosylation sites and does not contain any Asn-X-Ser sites. In another embodiment, the glycoprotein to be quantified is greater than 95% glycosylated, i.e., greater than 95% of the Asn acceptor sites out of the total number of acceptor sites of all molecules in the composition have an attached glycan. In a further embodiment, the glycoprotein to be quantified is greater than 98%, e.g., greater than 99%, e.g., 100% glycosylated.

[0049] In some embodiments, two or more, e.g., two, three, four, five, or more, different glycoproteins will be quantified, preferably simultaneously. In particular, the provided sample may contain 2-10 different antibodies, e.g., 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 different antibodies. In certain embodiments of the invention, where a sample comprises two or more antibodies to be quantified, the least abundant of said two or more distinct antibodies is present in an amount that is at least 1% (w / w), 2% (w / w), 3% (w / w), 4% (w / w), 5% (w / w), 6% (w / w), 7% (w / w), 8% (w / w), 9% (w / w), or 10% (w / w) of the amount of the most abundant of said two or more distinct antibodies. In particular, two or more antibodies are ... It may be present in an amount such that it is 1, 3:4-5:1, 1:5-4:1, 1:5-3:1, 1:5-2:1, 1:5-1:1, 1:5-1:2, 1:5-1:3, 1:5-1:4, 1:4-4:1, 1:4-3:1, 1:4-2:1, 1:4-1:1, 1:4-1:2, 1:4-1:3, 1:3-4:1, 1:3-3:1, 1:3-2:1, 1:3-1:1, 1:3-1:2, 1:2-4:1, 1:2-3:1, 1:2-2:1, 1:2-1:1, 1:1-4:1, 1:1-3:1, or for example 1:1-2:1.

[0050] The native glycoprotein to be quantified can be of any origin, including, for example, animal origin, e.g., mammalian origin, or other eukaryotic origin, e.g., fungal or yeast origin. Similarly, recombinantly produced glycoproteins can be produced in any type of host cell capable of Asn-linked glycosylation, including animal host cells, e.g., mammalian host cells, e.g., CHO cells, or human cells, e.g., HEK cells, or fungal or yeast cells.

[0051] In one embodiment, the one or more glycoproteins to be quantified are antibodies. In a further embodiment, the method comprises quantification of two or more, e.g., two, three, four, five, or more, different antibodies. In yet a further embodiment, the two or more different antibodies are IgG antibodies, e.g., full-length IgG antibodies. "Different," in this context, can be a difference in amino acid sequence and / or any post-translational modification other than Asn-linked glycosylation, such as conjugation, terminal clipping, or other amino acid residue modification. In one embodiment, the difference is a difference in a post-translational modification other than Asn-linked glycosylation. In one embodiment, the method comprises simultaneous quantification of an antibody-drug conjugate and an unconjugated antibody in the same sample.

[0052] In a further embodiment, the sample containing one or more glycoproteins to be quantified is a sample obtained from one of the steps of methods described in the art for generating bispecific antibodies in vitro by half-molecule exchange (see, e.g., Labrijn et al. (2013) PNAS 110:5145 and WO2011131746). In some of these methods, parent homodimeric antibodies are mixed and subjected to controlled reducing conditions in vitro, which separate the antibodies into half molecules and allow reassembly and reoxidation to form the bispecific antibody. The quantification methods of the present invention can be used to monitor the progress of bispecific antibody formation and / or assay purity by simultaneous quantification of the parent homodimeric antibodies and the resulting bispecific antibody.

[0053] In a further embodiment, the sample containing one or more glycoproteins to be quantified is a sample obtained from one of the steps of the method described in WO2019243626 (Genmab).

[0054] WO2019243626 describes a method for generating an output mixture of two or more different antibodies that have differences in their amino acid sequences, which differences allow for separation of the antibodies by chromatography; - two or more different antibodies are present in the output mixture in a desired or predetermined concentration ratio or essentially in a desired or predetermined concentration ratio; - the method comprises the steps of: a. providing an input mixture in which two or more different antibodies are absent or essentially absent in a desired or predetermined concentration ratio; b. separating the two or more antibodies by chromatography; c. recovering the two or more antibodies in the amounts required to provide an output mixture. Includes.

[0055] The term "output mixture," as used herein, is intended to refer to an antibody mixture in which two or more different antibodies are present in a desired or predetermined concentration ratio. The term "input mixture," as used in the context of an "output mixture," is intended to refer to an antibody mixture in which at least two of two or more different antibodies are present in a concentration ratio that is not the desired or predetermined concentration ratio and / or is present in a concentration ratio that is not within an acceptable deviation from the desired or predetermined concentration ratio.

[0056] Thus, the methods of the invention can be used, for example, to quantify antibodies in an input mixture or an output mixture.

[0057] The sample provided in the methods of the present invention can be any type of sample containing glycoproteins. In one embodiment, the sample is a cell culture sample, i.e., a sample taken from or derived from a culture of cells, e.g., recombinant host cells, that produce the glycoprotein(s) to be quantified. Thus, the methods of the present invention can be used, for example, to monitor glycoprotein production, including monitoring the quantities and / or ratios of the glycoproteins, e.g., antibodies, produced when multiple glycoproteins are produced in cell culture.

[0058] Thus, in a further aspect, the present invention relates to a process for monitoring the production of a glycoprotein in a cell culture, said process comprising culturing a host cell producing said glycoprotein and carrying out a method according to the invention as described herein. Depending on the outcome of the quantification, cell culture growth conditions can be adjusted to regulate glycoprotein production, or the ratio of glycoproteins if multiple glycoproteins are produced.

[0059] The methods of the invention can also be used for quality control (GC) of glycoprotein preparations or batches after purification and / or polishing. Thus, in one embodiment, the sample comprises a purified glycoprotein, e.g., a purified, recombinantly produced glycoprotein.

[0060] Thus, in a further aspect, the present invention relates to a process for the quality control of purified, recombinantly produced glycoproteins, said process comprising carrying out a method according to the invention as described herein.

[0061] The method according to the present invention can be carried out in an automated manner, i.e., without human intervention in one or all steps of the method. In one embodiment, steps b. and c. of the method are carried out in an automated manner. In a further embodiment, steps a., b., and c. are carried out in an automated manner. The method can provide quantitative results in a sufficiently short time frame, for example, 4 hours or less, to allow adjustment of the ongoing cell culture process.

[0062] In another embodiment, the sample is or is derived from a bodily fluid sample, e.g., a blood, plasma, or serum sample. Thus, the methods of the invention can be used to monitor the fate, e.g., plasma half-life, of a glycoprotein(s) in a subject, such as a human subject.

[0063] The method of the present invention is suitable for quantifying glycoproteins over a wide range of concentrations. In one embodiment, the concentration of each glycoprotein to be quantified in the sample is greater than 0.001 g / L, e.g., greater than 0.005 g / L, e.g., greater than 0.01 g / L. In another embodiment, the concentration of each glycoprotein to be quantified in the sample is less than 1 g / L, e.g., less than 0.25 g / L, e.g., less than 0.1 g / L.

[0064] As stated, in a further aspect, the present invention relates to a method for the quantification of one or more glycoproteins in a sample, each glycoprotein to be quantified comprising one or more Asn-linked glycans, said method comprising the steps of: a. preparing an internal standard by treating said one or more glycoproteins with one or more enzymes to obtain variant forms of each of said one or more glycoproteins, wherein said variant forms contain only core Asn-linked GlcNAc moieties; b. providing a sample containing the one or more glycoproteins to be quantified; c. adding the internal standard to the sample; d. quantitating said one or more glycoproteins by comparison to said internal standard; Includes.

[0065] The method may include, mutatis mutandis, one or more of the further features described above for the method of the first aspect of the invention.

[0066] In one embodiment, the step of preparing an internal standard comprises treatment with endo-β-N-acetylglucosaminidase (ENGase EC 3.2.1.96) (Fairbanks (2017) Chem Soc Rev46:5128), such as EndoS2 or EndoS.

[0067] In a preferred embodiment, the variant form is one that contains only a core Asn-linked GlcNAc moiety, without any fucose moieties linked to the core GlcNAc moiety.

[0068] In one embodiment, the treatment with one or more enzymes in step a. comprises treatment with an endo-β-N-acetylglucosaminidase and a fucosidase capable of hydrolyzing the alpha-1,6-linkage from an Asn-linked fucose-alpha-1,6-GlcNAc moiety, such as fucosidase 29A, also known as LcFuc29A (GH29), EC number 3.2.1.51, from Lactobacillus casei or Bacteroides fragilis (Tsai et al. 2017 ACS Chem. Biol 12:63). In a further embodiment, the step of preparing an internal standard comprises treatment with EndoS2 or EndoS (preferably EndoS2), followed by treatment with a fucosidase.

[0069] In one embodiment of the above-described method of the present invention, at least one of the one or more glycoproteins to be quantified contains fucose at some of its core Asn-linked GlcNAc moieties, i.e., at least some of its core Asn-linked GlcNAc moieties.

[0070] In one embodiment, the internal standard used in the methods of the present invention is not isotopically labeled.

[0071] In one embodiment, both the glycoprotein to be quantified and its variant form used as an internal standard are used in their full length, i.e., in a form having the full length amino acid chain, or in a form that is almost full length, e.g., comprising 50% or more, such as 75% or more, for example 90% or more, for example 95% or more of their full length amino acid sequence, e.g., comprising 50% or more, such as 75% or more, for example 90% or more, for example 95% or more of the heavy chain antibody sequence.

[0072] In a further aspect, the present invention relates to a composition comprising a known quantity of variant forms of a glycoprotein, said variant forms containing only core Asn-linked GlcNAc moieties, for use as an internal standard for quantitating said glycoprotein in a sample. Preferably, said variant forms contain only core Asn-linked GlcNAc moieties, and no fucose moieties are linked to said core GlcNAc moieties.

[0073] Additional Items of the Disclosure 1. 1. A method for the quantification of one or more glycoproteins in a sample, comprising: each glycoprotein to be quantified contains one or more Asn-linked glycans; The method comprises the steps of: a. providing a sample containing the one or more glycoproteins to be quantified; b. adding an internal standard to the sample, wherein the internal standard comprises a variant form of each of the one or more glycoproteins to be quantified, the variant form being a form that contains only a core Asn-linked GlcNAc moiety; and c. Quantifying said one or more glycoproteins by comparison with said internal standard.

[0074] 2. 2. The method according to item 1, wherein said variant form is a form containing only a core Asn-linked GlcNAc moiety, and no fucose moiety is linked to said core GlcNAc moiety.

[0075] 3. prior to step c., treating the sample with an enzyme; the enzyme is capable of completely removing Asn-linked glycans from the one or more glycoproteins, but is unable to remove core Asn-linked GlcNAc moieties from the variant forms; Follow steps 1 or 2.

[0076] 4. The method according to item 3, wherein the enzyme treatment is carried out prior to step b.

[0077] 5. The method according to item 3, wherein the enzyme treatment is carried out after the addition of an internal standard in step b.

[0078] 6. 6. The method according to any one of items 3 to 5, wherein the enzyme is PNGase F (EC 3.5.1.52).

[0079] 7. 3. The method according to item 1 or 2, comprising, prior to step b., treating the sample with an enzyme capable of completely removing Asn-linked glycans from said one or more glycoproteins, and optionally subsequently removing or inactivating said enzyme.

[0080] 8. The method according to any one of the preceding items, wherein the quantification in step c. is performed using mass spectrometry.

[0081] 9. The method according to any one of the preceding items, wherein the quantification in step c. is performed using a combination of mass spectrometry and non-mass-based separation techniques.

[0082] 10. The method according to any one of the preceding items, wherein the quantification in step c. is carried out using liquid chromatography-mass spectrometry (LC-MS), for example, reverse-phase based LC-MS.

[0083] 11. The method according to any one of the preceding items, which does not include the step of proteolytically digesting or otherwise fragmenting the amino acid chains of the glycoprotein to be quantified.

[0084] 12. 10. The method according to any one of the preceding items, wherein both the glycoprotein(s) to be quantified and the variant form(s) thereof used for internal standard are used in their full length, i.e. in a form having the full length amino acid chain, or almost full length, for example in a form comprising 50% or more, such as 75% or more, for example 90% or more, for example 95% or more of their full length amino acid sequence.

[0085] 13. 10. The method according to any one of the preceding items, wherein the one or more glycoproteins to be quantified are antibodies.

[0086] 14. The method according to any one of the preceding items, comprising quantitation of two or more, e.g., two, three, four, five, or more, different antibodies.

[0087] 15. 15. The method according to item 14, wherein the two or more antibodies are IgG antibodies, such as full-length IgG antibodies.

[0088] 16. The method according to any one of the preceding items, wherein at least one of the one or more glycoproteins to be quantified contains fucose in a core Asn-linked GlcNAc moiety.

[0089] 17. The method according to any one of the preceding items, wherein the sample is a cell culture sample.

[0090] 18. The method according to any one of the preceding items, wherein the sample comprises purified, recombinantly produced glycoprotein.

[0091] 19. 19. The method according to item 17 or 18, wherein steps b. and c. of the method are carried out in an automated manner, preferably steps a., b., and c. are carried out in an automated manner.

[0092] 20. 17. The method according to any one of items 1 to 16, wherein the sample is a blood sample, a plasma sample, or a serum sample.

[0093] twenty one. 1. A process for monitoring the production of a glycoprotein in a cell culture, comprising: Culturing a host cell that produces the glycoprotein; Follow the procedure in accordance with item 17 or 19. The process comprising:

[0094] twenty two. 1. A process for quality control of purified recombinantly produced glycoproteins, comprising: Follow the procedure in accordance with item 18 or 19 The process comprising:

[0095] twenty three. 1. A composition comprising a known number of variant forms of a glycoprotein, The composition, wherein the variant form is a form containing only a core Asn-linked GlcNAc moiety for use as an internal standard for quantitating the glycoprotein in a sample.

[0096] twenty four. 1. A method for the quantification of one or more glycoproteins in a sample, comprising: each glycoprotein to be quantified contains one or more Asn-linked glycans; The method comprises the steps of: a. preparing an internal standard by treating said one or more glycoproteins with one or more enzymes to obtain variant forms of each of said one or more glycoproteins, wherein said variant forms contain only core Asn-linked GlcNAc moieties; b. providing a sample containing the one or more glycoproteins to be quantified; c. adding the internal standard to the sample; and d. Quantifying said one or more glycoproteins by comparison with said internal standard.

[0097] twenty five. 25. The method according to item 24, wherein the treatment with one or more enzymes in step a. comprises treatment with an endo-β-N-acetylglucosaminidase, such as EndoS2 or EndoS.

[0098] 26. 26. The method according to item 24 or 25, wherein the variant form is a form containing only a core Asn-linked GlcNAc moiety, and no fucose moiety is linked to the core GlcNAc moiety.

[0099] 27. 26. The method according to any of items 24 to 25, wherein the treatment with one or more enzymes in step a. comprises treatment with an endo-β-N-acetylglucosaminidase, such as EndoS2 or EndoS, and a fucosidase capable of hydrolyzing alpha-1,6 linkages from Asn-linked fucose-alpha-1,6-GlcNAc moieties.

[0100] 28. The method according to any one of items 24 to 27, including one or more of the further features of items 2 to 20.

[0101] array TIFF0007778705000001.tif211153TIFF0007778705000002.tif218153TIFF0007778705000003.tif126153

[0102] This invention is further illustrated by the following examples, which should not be construed as further limiting. [Example]

[0103] Example 1: Expression and antibody production of human IgG1-CD19-21D4, human IgG1-CD22-huRFB4, human IgG1-7D8, human IgG1-CD37-37-3, and human IgG1-CD52-Campath, and variants For antibody expression of the isolated immunoglobulin proteins, variable heavy (VH) and variable light (VL) chain sequences were prepared by gene synthesis (GeneArt Gene Synthesis; ThermoFisher Scientific, Germany) and cloned into the pcDNA3.3 expression vector (ThermoFisher Scientific, US) containing the IgG1m(f) allotype heavy chain (HC; SEQ ID NO: 1) and light chain (LC; SEQ ID NO: 3) constant regions. The heavy chain constant region amino acid sequences used are shown in the sequence reference table below.

[0104] The desired mutations were introduced either by gene synthesis or site-directed mutagenesis. The antibodies mentioned in this application are the previously described IgG1-CD19-21D4 (WO2007002223; VH:SEQ ID NO 4; VL:SEQ ID NO 8), IgG1-CD22-huRFB4 (humanized variant of the murine antibody published in Weber et al., J Immunol Res 2015, 561814 (2015)), IgG1-7D8 (WO2004 / 035607; VH:SEQ ID NO 16; VL:SEQ ID NO 20), IgG1-CD37-37-3 (WO2011 / 112978; VH:SEQ ID NO 23; VL:SEQ ID NO 27), and IgG1-CD52-Campath (Crowe et al., Immunology 87(1):105-110). (1992); VH: SEQ ID NO 30; VL: SEQ ID NO 34). The sequences are also provided herein. The IgG1-CD19-21D4 and IgG1-CD52-Campath antibodies used in the examples have a mutation at amino acid position E345, E345K (SEQ ID NO: 2), which enhances Fc:Fc interactions between the individual antibodies. Because these antibody variants are monomeric in solution, the presence of these mutations does not affect the methods described herein.

[0105] Sequences were either subcloned from the pcDNA 3.3 expression vector into the in-house developed expression vector pGENpr6DGV (IgG1-CD19-21D4-E345K, IgG1-CD22-huRFB4, IgG1-CD37-37-3, and IgG1-CD52-Campath-E345K) or cloned into the vector pCon7D8-DGV (for IgG1-7D8), and both ORFs were expressed from the same vector. These expression vectors contained both the antibody open reading frame controlled by an upstream CMV promoter and a downstream TK polyA transcription termination signal, and a glutamine synthetase selectable marker expressed under the control of an SV40 promoter fragment and SV40 polyA transcription termination signal. The vectors were transfected into cells of the CHO-K1 cell line (ECACC cat. nr. 85051005), adapted for suspension growth in a chemically defined medium, or into cells of the CHO-K1SV cell line (ECACC cat. nr. 85051005), adapted for suspension growth in a chemically defined medium, at 1 μg / 1.0E+06 cells by nucleofection (Lonza Nucleofector 2b) using the Amaxa Solution V kit essentially according to the manufacturer's instructions, program T020 (Lonza). Cells containing the expression vectors were grown in 96-well plates in CD-CHO medium (Life Technologies / Thermo Scientific) containing GS EM supplement (Sigma) under MSX selection (Sigma) for 4 weeks, after which a panel of parental cell cultures demonstrating growth and IgG expression were expanded to larger volumes. The top producing clones were tested for IgG expression on the Ambr15 platform (TAP Biosystems), after which the best producing parental cells were selected for inoculation of 500 mL into a 3 L bioreactor to supply the IgG material. The cell culture was harvested after 14 days, and the IgG-containing supernatant was collected by filtration.

[0106] The antibody was purified by Protein A affinity chromatography. The cell culture supernatant was filtered through a 0.2 μM dead-end filter and then loaded onto an appropriately sized MabSelect SuRe column (GE Healthcare). The antibody was eluted with 0.1 M citric acid-NaOH, pH 3. The eluate was immediately neutralized with 2 M Tris-HCl, pH 9, and dialyzed overnight against 12.6 mM sodium phosphate, 140 mM NaCl, pH 7.4 (GE Healthcare). After dialysis, the sample was filter-sterilized through a 0.2 μM dead-end filter. The concentration of the purified IgG was determined by absorbance at 280 nm. The purified protein was analyzed by CE-SDS, HP-SEC, and mass spectrometry.

[0107] Example 2: Enzymatic deglycosylation of IgG antibodies with EndoS2 reveals sample heterogeneity caused by the presence of defucosylated species, while PNGase F completely deglycosylates IgG antibodies For absolute quantification of IgG antibody clones in complex protein mixtures, mass spectrometry can be used in combination with IgG antibody internal standards. These internal standards should have similar biophysical properties (e.g., similar ionization efficiency and physicochemical properties) to those of the sample analytes, but should be distinguishable from them. The Asn-linked glycosylation site, Asn297, contains structurally diverse Asn-linked glycan branches, all of which contain two N-acetylglucosamine (GlcNAc) groups at their base. The endoglycosidase EndoS2 hydrolyzes the linkage between these two GlcNAc groups, leaving only the Asn-linked core GlcNAc moiety most proximal to the polypeptide chain. Here, we tested whether enzymatic treatment with EndoS2 would enable the generation of internal standard antibodies that retain a small, uniform mass tag.

[0108] To hydrolyze the linkage between the two GlcNAc residues at the base of the N-glycan core of the Fc-glycan, 50 μL of 200 μg / mL antibody IgG1-CD19-21D4-E345K was treated with 1 μL of EndoS2 (40 U / μL; Genovis, cat. no. A0-GL1-020), an endoglycosidase from the M49 serogroup A Streptococcus strain, for 1 hour at 37° C. Alternatively, for complete deglycosylation, 50 μL of 200 μg / mL IgG1-CD19-21D4-E345K was treated with 1 μL of peptide-N-glycosidase F (PNGase F; 2.5 U / mL; PROzyme, cat. no. GKE-5006D) overnight at 37° C.

[0109] Samples were analyzed by liquid chromatography-mass spectrometry (LC-MS) using a Dionex Ultimate 3000 UHPLC system (Thermo Scientific) coupled online to an Orbitrap Q-Exactive Plus mass spectrometer (Thermo Scientific). Samples were injected (5 μL) onto a BioResolve reversed-phase monoclonal antibody column (1.0 mm × 5 cm, 2.7 μm particle size with 450 Å pore size; Waters, cat. no. 186009015). Mobile phase A contained 0.1% formic acid (v / v) in water (BioSolve, cat. no. 0023244101BS), and mobile phase B contained 0.1% (v / v) formic acid in acetonitrile (BioSolve, cat. no. 0001934101BS). Proteins were eluted at 200 μL / min using the following linear gradient: 25% B at 0 min, 30% B at 2 min, 40% B at 12 min, 80% B at 13 min, 80% B at 18 min, 25% B at 19 min, and 25% B at 25 min. Complete MS spectra were acquired at a resolution setting of 17,500 and a 5 × 10 6 Spectra were acquired with an automatic gain control (AGC) target value of 0.01 and a maximum injection time of 200 ms. Each spectrum consisted of 10 microscans.

[0110] The raw data were analyzed using Genedata Expressionist Refiner MS (version 13.0) software. The retention time (RT) range was restricted to 1-22 min, and the m / z range was restricted to 1400-4000 Da. Spectra were deconvoluted. Harmonic suppression deconvolution of intact protein activity (Mode: Fully automated; Equalization: Standard; Multiple retention times per mass: Yes; Steps: 1.0 Da) was used with curvature-based peak detection (Smoothing: 7 scans; Central calculation: Weighted by intensity (0% threshold); Boundary determination: Inflection point) and the following advanced settings: Minimum peak intensity: 1%; Minimum search mass: 5.0 kDa; Maximum search mass: 500.0 kDa; Relative mass window: 2%; Maximum mass window: 1.0 kDa; Mass deconvolution: Relative mass window 10%, Maximum mass window 5.0 kDa). The deconvoluted spectra were then averaged using the arithmetic mean method. Here, peaks in the deconvoluted spectra were detected using a spectral peak detection activity (smoothing: 3 points; peak detection: ascending-based; median calculation: local maxima; boundary determination: inflection points). Peaks with intensities less than 1% of the maximum intensity were excluded.

[0111] Enzymatic treatment of IgG1-CD19-21D4-E345K with EndoS2 resulted in efficient removal of the glycan up to the Asn-linked GlcNAc group, leaving only the innermost GlcNAc residue and a single attached core fucose (IgG-(GlcNAc-Fuc)2; Figure 2). However, the presence of antibody species without core fucosylation (i.e., without a core fucose group attached to the innermost GlcNAc residue via an α-1,6-linkage) in antibody preparations leads to sample heterogeneity: in addition to IgG-(GlcNAc-Fuc)2 antibodies, EndoS2-treated samples also contain a few antibodies containing one heavy chain with an innermost GlcNAc residue linked to core fucose and one heavy chain with an innermost GlcNAc residue without core fucose (-Fuc, or IgG-(GlcNAc-Fuc)), and a few antibodies containing two heavy chains with innermost GlcNAc residues without a core fucose group (-Fuc2 or IgG-(GlcNAc)). In contrast, enzymatic treatment of antibody IgG1-CD19-21D4-E345K with PNGase F (Fig. 2B) resulted in a homogenous population of fully deglycosylated IgG1-CD19-21D4-E345K.

[0112] Example 3: Enzymatic defucosylation of EndoS2-treated IgG antibodies with Lactobacillus casei α-L-fucosidase removes fucose groups from core GlcNAc groups As shown in Example 2, enzymatic deglycosylation of IgG antibodies with EndoS2 results in a heterogeneous population of IgG antibodies with glycan groups consisting of a GlcNAc core group with or without an attached fucose group. As a result, the antibody can contain two heavy chains with an innermost GlcNAc residue linked to a core fucose group (IgG-(GlcNAc-Fuc)2), or alternatively, one or both of the heavy chains can have a core GlcNAc group without an attached fucose group (IgG-(GlcNAc-Fuc) or IgG-(GlcNAc), respectively).

[0113] Here, we tested whether enzymatic defucosylation could be used to eliminate this heterogeneity by removing the remaining fucose groups on IgG-(GlcNAc-Fuc)2 and IgG-(GlcNAc-Fuc), while leaving the GlcNAc groups intact.

[0114] Fifty microliters of 200 μg / mL IgG1-CD19-21D4-E345K was treated for 6 hours at 37°C with 1 μL of endoglycosidase EndoS2 (40 U / μL; Genovis, cat. no. A0-GL1-020) to hydrolyze the linkage between the two GlcNAc residues at the base of the N-glycan core of the Fc-glycan, and 1 μL of Lactobacillus casei fucosidase (fucosidase 29A; 0.5 mg / mL; NZYtech, cat. no. CZ0566) to hydrolyze the α-1,6 linkage of the core fucose group to the innermost GlcNAc residue. Alternatively, 50 μL of 200 μg / mL IgG1-CD19-21D4-E345K was treated for 6 hours at 37°C with 1 μL EndoS2. LC-MS was performed as described in Example 2.

[0115] Enzymatic treatment of EndoS2-treated IgG antibodies with α-L-fucosidase from L. casei efficiently removed the remaining fucose groups from IgG-(GlcNAc-Fuc)2 and IgG-(GlcNAc-Fuc), resulting in IgG antibodies with uniform GlcNAc tags (Figure 3B).

[0116] Example 4: PNGase F from PNGase F-treated samples does not remove remaining GlcNAc moieties from IgG antibodies treated with EndoS2 and α-L-fucosidase As shown in Example 3, enzymatic deglycosylation of IgG antibodies with EndoS2 and α-L-fucosidase leaves small, uniform GlcNAc moieties on both heavy chains. When used as internal standards, these IgG-(GlcNAc)2 antibodies can be added to a complex mixture of antibodies that have been completely deglycosylated by the endoglycosidase PNGase F. To determine whether depletion or inactivation of PNGase F from antibody samples might be required before the IgG-(GlcNAc)2 internal standard could be added, we tested whether PNGase F could remove remaining GlcNAc moieties from IgG-(GlcNAc)2 antibodies.

[0117] Fifty microliters of 400 μg / mL IgG1-CD19-21D4-E345K was treated for 6 hours at 37°C with 2 μL of endoglycosidase EndoS2 (40 U / μL; Genovis, cat. no. A0-GL1-020) to hydrolyze the linkage between the two GlcNAc residues at the base of the N-glycan core of the Fc-glycan, and 2 μL of Lactobacillus casei fucosidase (fucosidase 29A; 0.5 mg / mL; NZYtech, cat. no. CZ0566) to hydrolyze the α-1,6 linkage of the core fucose group to the innermost GlcNAc residue. In parallel, 50 μL of 400 μg / mL IgG1-CD19-21D4-E345K was completely deglycosylated overnight at 37° C. with 2 μL of PNGase F (2.5 U / mL) as described in Example 2. After treatment, 25 μL of each sample was mixed 1:1. LC-MS was performed as described in Example 2.

[0118] Alternatively, 50 μL of 600 μg / mL IgG1-CD19-21D4-E345K was treated with 3 μL EndoS2 and 3 μL α-L-fucosidase for 6 hours at 37°C. In parallel, 50 μL of 600 μg / mL IgG1-CD19-21D4-E345K was completely deglycosylated with 3 μL PNGase F (2.5 U / mL) overnight at 37°C. 25 μL of each sample and 25 μL of 600 μg / mL IgG1-b12 were mixed 1:1:1 and incubated for 3 hours at 37°C. LC-MS was performed as described in Example 2.

[0119] Addition of PNGase F-treated IgG1-CD19-21D4-E345K to EndoS2 and α-L-fucosidase-treated IgG antibody (IgG1-CD19-21D4-E345K-(GlcNAc)2) did not result in removal of the remaining GlcNAc moieties on the EndoS2 and α-L-fucosidase-treated IgG antibody (Figure 4A), indicating that inactivation or removal of PNGase F from the sample analyte is not required.

[0120] Addition of PNGase F-treated IgG1-CD19-21D4-E345K to the internal standard IgG1-CD19-21D4-E345K treated with EndoS2 and α-L-fucosidase and to untreated IgG1-b12 did not result in complete deglycosylation of the internal standard or untreated IgG1-b12. Instead, IgG1-b12 appeared as IgG1-b12-(GlcNAc)2 and IgG1-b12-(GlcNAc)-(GlcNAc-Fuc) variants, suggesting that the enzymatic activity of EndoS2 and α-L-fucosidase from the internal standard dominates over the enzymatic activity of PNGase F from the sample analyte in this mixture.

[0121] Example 5: Analysis of antibody mixtures is facilitated by multidimensional separation methods As described in Examples 2-4, highly homogeneous variant antibody internal standards can be generated by subsequent deglycosylation of antibody samples with EndoS2 endoglycosidase and Lactobacillus casei fucosidase (29A). Use of this method results in whole, intact proteins with only remaining Asn-linked core GlcNAc moieties on both heavy chains. These antibody variants can be used as internal standards for quantification of individual antibodies in antibody mixtures. However, mass spectrometry-based separation and detection of antibody-containing samples is most appropriate when all analyzed antibody samples and their designated internal standards do not have overlapping mass profiles, i.e., (nearly) isobaric masses. Here, we describe how an LC-based separation method can be added to the procedure to enable time-resolved deconvolution of samples containing antibody mixtures with overlapping mass profiles (i.e., (nearly) isobaric masses).

[0122] An antibody mixture was generated by mixing equal concentrations of human antibodies IgG1-CD19-21D4-E345K, IgG1-CD22-huRFB4, IgG1-7D8, IgG1-CD37-37-3, and IgG1-CD52-Campath-E345K at a total final concentration of 8 mg / mL. To generate the internal standard, 40 μL of EndoS2 (40 U / μL; Genovis, cat. no. A0-GL1-020) and 21.3 μL of Lactobacillus casei fucosidase (fucosidase 29A; 0.5 mg / mL; NZYtech, cat. no. CZ0566) were added to 200 μL of the antibody mixture (final concentration 2.5 mg / mL) and incubated overnight at 37°C. The sample antibody mixture was completely deglycosylated by adding the following to 200 μL of the antibody mixture: 160 μL of PNGase F (2.5 U / mL; PROzyme, cat. no. GKE-5006D) and 440 μL of MilliQ (final concentration 2 mg / mL) and incubating the sample overnight at 37° C. The antibody sample was diluted to a concentration of 120 μg / mL, after which an internal standard was added to a final concentration of 75 μg / mL. LC-MS was performed essentially as described in Example 2.

[0123] Raw data were analyzed using Genedata Expressionist Refiner MS (version 13.0) software. The retention time (RT) range was restricted to 1–16 min, and chromatograms were then aligned using a tree alignment scheme based on pairwise alignment with a gap penalty of 1 and a maximum RT shift of 50 scans. Chemical noise was subtracted using chromatographic chemical noise subtraction activity (RT window: 50; quantile: 50%, method: clipping; threshold: 0.0; RT structure removal: 6 scans). Ion maps were deconvoluted using time-resolved deconvolution of intact protein activity (minimum mass: 140 kDa; maximum mass: 150 kDa; method: harmonic suppression deconvolution; 1.0 Da steps; 7 scans, curvature-based intensity-weighted (0% threshold) peak detection with inflection points to determine boundaries; minimum peak intensity: 1%; minimum search window: 5.0 kDa; maximum search window: 500.0 kDa; relative mass window: 2%; maximum mass window: 1.0 kDa; and mass deconvolution with a 10% relative mass window and a 5.0 kDa maximum mass window). Peaks in the deconvoluted ion maps were detected using peak detection activity (summation window: 10 scans; minimum peak size: 10 scans; maximum merge distance: 25 points; merge strategy: median; smoothing: 3 points; peak detection: ascending-based; isolation filter: yes, 3 points; median calculation: local maxima; boundary determination: inflection points).Prior to detection, peaks were identified using known sequences by protein mapping activity (10 Da mass tolerance with 10 Da consolidate matches; fixed modifications of N-terminal glutamine to pyroglutamic acid and loss of C-terminal lysine; glycosylation settings: deglycosylation, Asn-binding sites only, use of consensus sequences, and filtering for core structures; fully connected disulfide bridges; multiple connectivity possibilities for each IgG; and the following mass shifts for conjugates: GlcNAc(Fuc)2:698 Δ; GlcNAc(Fuc):552 Δ; GlcNAc:406 Δ, with a maximum of 1 conjugate).

[0124] Figure 5A shows the mass spectrum of an antibody mixture composed of human IgG1-CD19-21D4-E345K, IgG1-CD22-huRFB4, IgG1-7D8, IgG1-CD37-37-3, and IgG1-CD52-Campath-E345K (labeled species A–E) and their corresponding internal standards (all species A'–E' indicated by an apostrophe). Antibody samples B (IgG1-CD22-RFB4) and D (IgG1-CD37-37-3) have nearly identical masses, and thus their mass peaks overlap. Similarly, their corresponding internal standards B' and D' have overlapping mass peaks. Mass spectra of other antibody samples and their corresponding internal standards show no overlap. LC separation resulted in four large elution peaks (Figure 5B), making it difficult to identify individual antibody samples and internal standards. However, by applying time-resolved deconvolution (TRD), in which mass (x-axis) and elution time (y-axis) are plotted, a two-dimensional view was generated, allowing for the identification of individual antibody samples and internal standards (Figure 5C). Despite the overlapping mass peaks of antibodies B and D and the accompanying internal standards B' and D', the differences in elution time between these samples allowed for peak separation after TRD. In the third dimension, peak intensities are preserved in such a way that peak volumes can be estimated for further quantitative calculations.

[0125] Example 6: Analysis of mixed samples and internal standards by time-resolved deconvolution MS allows for highly accurate IgG concentration determination The time-resolved deconvolution MS-based separation procedure described in Example 5 could be applied to quantify individual antibodies in complex antibody mixtures. Here, we describe how antibody sample concentrations can be accurately determined by measuring the MS peak volume / intensity of antibody samples containing a specified IgG-GlcNAc2 internal standard (IS).

[0126] An antibody mixture was generated as an external calibration standard by mixing IgG1-CD19-21D4-E345K, IgG1-CD22-huRFB4, IgG1-7D8, IgG1-CD37-37-3, and IgG1-CD52-Campath-E345K at defined antibody concentrations as described in Example 5. A portion of the mixture was treated with EndoS2 and Lactobacillus casei fucosidase (29A) as described in Example 5 to generate the IS, and another portion was completely deglycosylated with PNGase F as described in Example 2 to generate the antibody external standard. A dilution series of the external standard was then generated and added to each standard at one fixed IS concentration (i.e., XXX μg / mL for each IgG-GlcNAc2). The antibody external calibration standard varied from 15 to 80 μg / mL depending on the individual antibody. 50 μL of each calibration standard was pipetted into a Q-sert vial (Sigma). Data were acquired and then processed by TRD MS as described in Examples 2 and 5.

[0127] The MS peak volumes / intensities of deglycosylated IgG1-CD19-21D4-E345K, IgG1-CD22-huRFB4, IgG1-7D8, IgG1-CD37-37-3, and IgG1-CD52-Campath-E345K, as well as calibration standards containing the reference IS IgG-GlcNAc2 variant, were measured by LC-MS, and the data were processed by time-resolved deconvolution.

[0128] First, a calibration curve was generated by calculating the ratio of the MS peak intensity of the deglycosylated antibody to the MS peak volume / intensity of the IS (Ext st. / IS). Figures 6A-E show excellent linearity when the resulting ratio (Y axis) is plotted against the individual standard antibody concentrations. A coefficient of determination (R) of 0.99 was obtained for the standard curve of each generated antibody. 2 ) were found, demonstrating the suitability of the IgG-GlcNAc2IS for obtaining calibration curves. Figures 6F-J show the calculated antibody sample concentrations for each antibody, as deduced from the calibration curves, relative to the expected antibody concentrations, demonstrating the high accuracy of the measurements.

[0129] Example 7: In-process IgG concentration determination by time-resolved deconvolution MS IgG quantification can be accelerated by integrating the method described in Example 5 into an in-process workflow. To this end, test samples containing cell culture supernatants are deglycosylated with PNGase F for 2-24 hours (using a method similar to that described in Example 2). The deglycosylated cell supernatant samples are then spiked with the previously prepared IS antibody (Example 5), preferably in a range close to the expected sample IgG concentration. The prepared sample will then be injected into an LC-MS system. The LC-MS system includes an affinity purification trap column. In this example, the affinity trap column contains immobilized Protein A, which binds to the Fc portion of the antibody. Injection of the prepared cell culture sample captures the antibody onto the Protein A column. Subsequently, all host cell proteins are washed off the column using a wash buffer. In the next step, the trapped antibody is eluted from the column in one step using a low-pH elution buffer and then separated on a reversed-phase (RP) analytical column, which is inline with the mass spectrometer. The acquired MS data will be processed to detect antibody masses and concentration values ​​will be calculated from signal intensities according to Example 6.

Claims

1. 1. A method for the quantification of one or more glycoproteins in a sample, comprising: each glycoprotein to be quantified contains one or more Asn-linked glycans; The method comprises the steps of: a. providing a sample containing the one or more glycoproteins to be quantified; b. adding an internal standard to the sample, the internal standard comprising a variant form of each of the one or more glycoproteins to be quantified, the variant forms containing only core Asn-linked GlcNAc moieties, each core Asn-linked GlcNAc moiety being an N-acetylglycosamine moiety attached directly to an Asn acceptor residue in the polypeptide chain of the variant, and no fucose moiety being linked to the core GlcNAc moiety; and c. quantitating said one or more glycoproteins by comparison to said internal standard. wherein the method comprises treating a sample with an enzyme, wherein the enzyme is capable of completely removing Asn-linked glycans from the one or more glycoproteins, but is incapable of removing core Asn-linked GlcNAc moieties from the variant forms.

2. 2. The method of claim 1, wherein the enzyme treatment is carried out prior to step b.

3. 3. The method of claim 2, wherein the enzyme treatment is performed after the addition of an internal standard in step b.

4. The method of any one of claims 1 to 3, wherein the enzyme is PNGase F (EC 3.5.1.52).

5. 2. The method of claim 1, further comprising, prior to step b., treating the sample with an enzyme capable of completely removing Asn-linked glycans from said one or more glycoproteins, and optionally, subsequently removing or inactivating said enzyme.

6. The method of any one of claims 1 to 5, wherein the quantification in step c. is performed using mass spectrometry.

7. 7. The method of any one of claims 1 to 6, wherein the quantification in step c. is performed using a combination of mass spectrometry and non-mass-based separation techniques.

8. The method of any one of claims 1 to 7, wherein the quantification in step c. is carried out using liquid chromatography-mass spectrometry (LC-MS).

9. The method described in claim 8, wherein the quantification in step c. is performed using reverse phase based LC-MS.

10. 10. The method of any one of claims 1 to 9, which does not include a step of proteolytically digesting or otherwise fragmenting the amino acid chains of the glycoprotein to be quantified.

11. 11. The method according to any one of claims 1 to 10, wherein both the glycoprotein(s) to be quantified and their variant form(s) used for internal standard are used in their full length, i.e. in a form having the full length amino acid chain and comprising 50% or more of their full length amino acid sequence.

12. The method described in claim 11, wherein both the glycoprotein(s) to be quantified and their variant form(s) used as internal standards comprise 75% or more of their full-length amino acid sequences.

13. The method described in claim 11, wherein both the glycoprotein(s) to be quantified and their variant form(s) used as internal standards comprise 90% or more of their full-length amino acid sequences.

14. The method described in claim 11, wherein both the glycoprotein(s) to be quantified and their variant form(s) used as internal standards comprise 95% or more of their full-length amino acid sequences.

15. The method of any one of claims 1 to 14, wherein the glycoprotein or glycoproteins to be quantified are antibodies.

16. 16. The method of any one of claims 1 to 15, comprising the quantification of two or more different antibodies.

17. A method according to any one of claims 1 to 16, comprising quantifying three or more different antibodies.

18. A method according to any one of claims 1 to 16, comprising quantifying four or more different antibodies.

19. A method according to any one of claims 1 to 16, comprising quantifying five or more different antibodies.

20. 17. The method of claim 16, wherein the two or more antibodies are IgG antibodies.

21. 21. The method of any one of claims 1 to 20, wherein at least one of the one or more glycoproteins to be quantified contains fucose in a core Asn-linked GlcNAc moiety.

22. 22. The method of any one of claims 1 to 21, wherein the sample is a cell culture sample.

23. 23. The method of any one of claims 1 to 22, wherein the sample comprises purified, recombinantly produced glycoprotein.

24. 24. The method of claim 22 or 23, wherein steps b. and c. of the method are performed in an automated manner.

25. 22. The method of any one of claims 1 to 21, wherein the sample is a blood sample, a plasma sample, or a serum sample.

26. 1. A process for monitoring the production of a glycoprotein in a cell culture, comprising: Culturing a host cell that produces the glycoprotein; Carrying out the method of claim 22 or 24 The process comprising:

27. 1. A process for quality control of purified recombinantly produced glycoproteins, comprising: Carrying out the method of claim 23 or 24 The process comprising:

28. 1. A method for the quantification of one or more glycoproteins in a sample, comprising: each glycoprotein to be quantified contains one or more Asn-linked glycans; The method comprises the steps of: a. preparing an internal standard by treating the one or more glycoproteins with one or more enzymes to obtain variant forms of each of the one or more glycoproteins, wherein the variant forms contain only core Asn-linked GlcNAc moieties, each core Asn-linked GlcNAc moiety being an N-acetylglycosamine moiety attached directly to an Asn acceptor residue in the polypeptide chain of the variant, and no fucose moiety being linked to the core GlcNAc moiety; b. providing a sample containing the one or more glycoproteins to be quantified; c. adding the internal standard to the sample; and d. quantitating said one or more glycoproteins by comparison to said internal standard; wherein said treatment with one or more enzymes in step a. comprises treatment with an endo-β-N-acetylglucosaminidase and a fucosidase capable of hydrolyzing alpha-1,6 linkages from Asn-linked fucose-alpha-1,6-GlcNAc moieties.

29. 29. The method of claim 28, wherein said treatment with one or more enzymes in step a. comprises treatment with endo-β-N-acetylglucosaminidase.

30. The method described in claim 29, wherein the endo-β-N-acetylglucosaminidase is EndoS2 or EndoS.

31. A method described in any one of claims 28 to 30, comprising treating a sample with an enzyme, wherein the enzyme is capable of completely removing Asn-linked glycans from the one or more glycoproteins but is incapable of removing the core Asn-linked GlcNAc moiety from the variant forms.

32. A method described in any one of claims 28 to 31, wherein the enzyme is PNGase F (EC 3.5.1.52).

33. A method according to any one of claims 28 to 32, wherein the one or more glycoproteins to be quantified are antibodies.

34. A method described in any one of claims 28 to 32, comprising quantifying two or more different antibodies.

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