Mass spectrometry (MS) has gained importance in the anlytics of biopharmaceuticals, taking different complementary approaches ranging from peptide-based sequencing to direct analysis fo intact proteins and protein assmeblies. (Rosati, Nature Protocols, 9(4), 20140)p

A variety of “hyhenated chromatography-mass spectrometry” methods have demonstrated the capability of detecting low-abundance impurities in mAb products and providing highly detailed analyses that cannot be acheived by either SDS-PAGE or CE-SDS methods.  (Wang, US 16/223,463, published as US 2019/0194298).

Hydrophilic interaction chromatography (HILIC) + MS:

for characterization of glycosylation:

Glycosylation is one of the most common postranslational modifications of proteins. Protein heterogeneity introduced by glycosylation includes differences in the nature and structure of the attached oligosacharides, and the number and position of the glycans. Common methods to study glycosylation involve glycan release or protein digestion, followed by analysis of the reuslting glycans or clycopeptides. These methods offer detailed information about the sites of clycosylation and/or the carbohydrate composition. Electrospray ionization mass spectrometry (ESI-MS) enalbes the precise mass determination and characterization of intact (glyco) proteins. However, seperation prior to MS detection often is essential to acheive reliable assignment of intact glycoproteins and detection of low abundant glycoforms. HILIC-MS has shown highly useful for the analysis of a wide range of compounds including metabolites and pepties. (Somsen, “high-resolution glycofrom profiling of intact therapeutic proteins by hydrophilic interaction chromatography-mass spectrometry” Talanta 184 (2018) 375-381). 

Glycosylation is a post-translational modificaiton of proteins that occurs in all eukaryotic cells. The sugar chains on glucoproteins can mediate biologial activity, play a role in receptror-mediated recognition, increase solubility, regulate half-life and exert a stabilizing influence upon conformation. (Gillece-castro, “N-linked glycans of glycoproteins: a new column for improved resolution” Water Corporation (2009) .ueous mobile phase used in HILIC, which overcomes the drawbacks of the poor solubility often encounted in NP-LC. Expensive ion pair reagents are not required in HILIC, and it can be conveniently coupled to mass spectromtry (MS). (Busewski, “Hydrophilic interaction liquid chromatogrpahy (HILIC) – a powerful spearation technique, Anal Bioanal Chem (2012) 402: 231-247). 

HILIC has many specific advantages over conventional NP-LC and RP-LC. For example, it is suitable for anlyzing compounds in complex systems that always elute near the void in reserved-phase chromatography. Polar samples always show good solubility in the aq

D’Atri (Hydrophilic Interaction Chromatography hyphenated with mass spectrometry: a powerful analytical tool for the comparison of originator and biosimilar therapeutic monocloanl antibodies at the middle-up level of analysis” Anal. Chem 2017, 89, 2086-2092) discloses that using a sationary phase in combination with a highly organic mobile phase, HILIC is fully compatible with electrospray ionization mass spectrometry (ESL-MS) and offers a complementary retntion mechaism compared to RPLC, since analytes are eluted based on their hydrophilicity. 

Lauber (US 2015/0316515) discloses characterization of large biological molecules modified with polar groups such as antibody glycorms by hydrophilic interaction chromatography (HILIC) which further includes the step of identifying the glycopeptie with mass spectrometry. In one embodiment the sample is first prepared by reductyion, enzymatic digeston, denaturation, fragmentation, ehmical cleavage or a combination thereof. In another emobdiment the method for analyzing a glycosylated protenaceous sample, the chromatographic material is a porous material which includes at least one hydrophilic monomer and a poly-amide bonded pahse. In certain embodimetns, the method includes the step of denaturing the glycosylated proteinaceous sample at a temperature 80C or greater and deglycosylating the denatured sample. In certain embodiments, the method includes a mobile phase eluent such as acetonitrile, isopropanol or ethanol. 

Wang (J Pharmaceutical and Biomedical Analysis, 154: 468-476 (2018) disclsoes that hydrophilic interaction chromatography (HILIC) coupled with mass spectrometry analysis can characterize LMW impurites present within a purified mAb drug product sample. After mreoval of N-linked glycans, the HILIC method separates mAb related LMW impurites with asize based elution order. Free ligh chains, half-antibody, H2L species (antibody possessing a single light chain) and protein backbone truncated species can all be confidently identifed and elucidatesd in great detal, including the truncation sites and associated post translational modificaitons. 

Wang, (US 16/223,463, published as US 2019/0194298) discloses methods for charaterizing low molecular weight (LMW) protein drug product impurities which uses hydrophilic interaction chromatography (HILIC) coupled to mass spectrometry analysis. After removal of the N-linked glycans from teh protein drug product, for example an antibody drug product, the elution of LMW impurities from the HILIC column is determend by the size of the molecular weight species. 

IEX + Mass Spectrometry: 

Cation exchange (CEX) + MS:

Wang (Anal Cehm 87: 914-921 (2015) disclsoes drug product related impurities of an anti-Clostridium difficile IgG1 mAb were profiled by CEX liquid chromatogrpahy followed by teh CEX peaks being fraction collected for MS characterization. A reversed-phase liquid chromatography/mass spectrometry (LC/MS) methology was developed on a Thermo Q-Exactive orbitrap mass spectrometer for mass measurements of the mAb, its CEX fractioanted impurties and their respective H and L chains. 

Wang (US 16/259095, published as US 2019/0234959) discloses using IEX, preferably strong CEX  with an aqueous mobile phase coupled with native mass spectrometry analysis to detect and characterize size variant protein drug product imprities. In one embodiment the protein is first deglycosylated such as by treating the sample with IdeS.

SEC + Mass Spectrometry:

Kukrer (Pharm Res, 27: 2197-2204 (2016) discloses mass spectrometric analysis (MS of mAg aggregates fractionated by size-exclusion chromatography (SEC). IgG aggregates (dimers, trimers, tetramers and HMW oligomers) were created by subjecting an IgG formulation to several pH jumps. Protein oligomer fractions were isolated by high performance size exclusion chromatogrpahy (HP-SEC) dialyzed agaisnt ammonium acetate pH 6.0 and analyzed by native electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). 

Wang (US 16/259095, published as US 2019/0234959) discloses using SEC with an aqueous mobile phase coupled with native mass spectrometry analysis to detect and characterize size variant protein durg product imprities. In one embodiment the protein is first deglycosylated such as by treating the sample with IdeS. 

Wollacot (mAbs, 5(6): 925-935 (2013) discloses using seize exclusion high-performance liquid chromatogrpahy (SE-HPLC) analysis of a hmAb to identify a mAb species that eluted between the monomeric and dimeric species.