RESEARCH ARTICLE – Pharmaceutical Biotechnology

Development and Application of a Robust N-Glycan Profiling Method for Heightened Characterization of Monoclonal Antibodies and Related Glycoproteins TANYA Q. SHANG,1 ANDREW SAATI,1 KELLY N. TOLER,2 JIANMING MO,3 HEYI LI,4 TONYA MATLOSZ,4 XI LIN,4 JENNIFER SCHENK,1 CHEE-KENG NG,1 TONI DUFFY,1 THOMAS J. PORTER,1 JASON C. ROUSE1 1

Analytical Research and Development, Biotherapeutics Pharmaceutical Sciences, Pfizer, Inc., Andover, Massachusetts 01810 Genzyme, A Sanofi Company, Framingham, Massachusetts 01701 3 Analytical R&D, Pfizer, Inc., St. Louis, Missouri 63017 4 Analytical R&D, Pfizer, Inc., Pearl River, New York 10965 2

Received 30 January 2014; revised 7 April 2014; accepted 17 April 2014 Published online 19 May 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24004 ABSTRACT: A highly robust hydrophilic interaction liquid chromatography (HILIC) method that involves both fluorescence and mass spectrometric detection was developed for profiling and characterizing enzymatically released and 2-aminobenzamide (2-AB)-derivatized mAb N-glycans. Online HILIC/mass spectrometry (MS) with a quadrupole time-of-flight mass spectrometer provides accurate mass identifications of the separated, 2-AB-labeled N-glycans. The method features a high-resolution, low-shedding HILIC column with acetonitrile and water-based mobile phases containing trifluoroacetic acid (TFA) as a modifier. This column and solvent system ensures the combination of robust chromatographic performance and full compatibility and sensitivity with online MS in addition to the baseline separation of all typical mAb N-glycans. The use of TFA provided distinct advantages over conventional ammonium formate as a mobile phase additive, such as, optimal elution order for sialylated N-glycans, reproducible chromatographic profiles, and matching total ion current chromatograms, as well as minimal signal splitting, analyte adduction, and fragmentation during HILIC/MS, maximizing sensitivity for trace-level species. The robustness and selectivity of HILIC for N-glycan analyses allowed for method qualification. The method is suitable for bioprocess development activities, heightened characterization, and clinical drug substance release. Application of this HILIC/MS method to the detailed C 2014 Wiley Periodicals, Inc. and the American Pharmacists characterization of a marketed therapeutic mAb, Rituxan , is described.  Association J Pharm Sci 103:1967–1978, 2014 Keywords: biopharmaceuticals characterization; HPLC; protein structure; LC–MS; liquid chromatography; mass spectrometry; monoclonal antibody; glycosylation R

INTRODUCTION The value of recombinant mAbs as pharmaceuticals has been demonstrated in a variety of therapeutic areas and the number of mAbs in development continues to grow rapidly.1–6 Structurally, IgG mAbs are glycoproteins that comprise two identical light-chain and two identical heavy-chain subunits interconnected by disulfide bonds. Each heavy-chain subunit has one N-glycosylation consensus sequence site in the CH2 domain, which is generally fully occupied by a range of complextype, variably galactosylated, core-fucosylated, biantennary structures.7,8 In addition to the oligosaccharide microheterogeneity for individual heavy-chain subunits, another level of structural diversity occurs in the mAb when each heavy chain contains different carbohydrate structures. Thus, N-glycans represent the major source of molecular heterogeneity in mAbs.

Correspondence to: Jason C. Rouse (Telephone: +978-247-2341; Fax: +978247-3456; E-mail [email protected]) Tanya Q. Shang and Andrew Saati contributed equally to this work. Xi Lin’s present address is 43 Princess Dr., North Brunswick, New Jersey 08540. This article contains supplementary material available from the authors upon request or via the Internet at http://onlinelibrary.wiley.com/. Journal of Pharmaceutical Sciences, Vol. 103, 1967–1978 (2014)  C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association

Characterization and routine analysis of N-glycosylation patterns in mAb therapeutics is important for the assessment of product quality and consistency of manufacture in the development and commercialization of protein pharmaceuticals.1,2,8–13 Commonly, the N-linked oligosaccharides are characterized and monitored through enzymatic release from the mAb with an endoglycosidase with subsequent separation of the individual native or fluorescently labeled structures by chromatographic14–19 or electrophoretic methods.11,20,21 The resulting distribution of peaks serves as an “N-glycan fingerprint” where particular carbohydrate structures are indicated by peaks at specific retention (or migration) times and their relative abundances are reflected by the peak areas. Therefore, the N-glycan profile provides a measure of production process consistency in the situation when one or more batches are analyzed side-by-side with a defined reference material. Traditionally, released, underivatized N-glycan pools from recombinant proteins22 and mAbs19 were profiled by high-pH anion-exchange chromatography with pulsed amperometric detection. In recent years, derivatization of released oligosaccharides with a fluorescent tag has become commonplace, especially for mAbs, because it facilitates sensitive, quantitative, and specific detection of carbohydrates upon separation by routine HPLC methods that have comparable or enhanced separation efficiency. Examples of approaches for profiling derivatized N-glycans from antibodies

Shang et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1967–1978, 2014

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RESEARCH ARTICLE – Pharmaceutical Biotechnology

include: normal-phase (NP) HPLC of 2-aminobenzamide (2-AB)-derivatized carbohydrates,23 anion-exchange HPLC of 2-aminobenzoic acid derivatized oligosaccharides,24 and reversed-phase HPLC25 or hydrophilic interaction liquid chromatography (HILIC)26 of 2-aminopyridine derivatized Nglycans. As a side note, a NP-HPLC method used for glycan analysis will employ water-containing mobile phases, and in this context, the term NP-HPLC is sometimes used interchangeably with HILIC. HILIC is a mode of separation based on the polar interactions between analytes in the eluent and a stationary phase that is enriched with a water layer, where retention increases proportionally with the analyte hydrophilicity and decreases with the polarity of the semi-aqueous eluents, assuming limited secondary ionic interactions.27 Any chromatography involving a polar stationary phase and a gradient of increasing aqueous content in the mobile phase will be referred to as HILIC in this work. Detailed structural characterization of N-glycans at the lowto mid-picomole level is most effective through the combined use of chromatography, mass spectrometry (MS), exo- and endoglycosidases, and bioinformatics (reviewed in Refs.28–30). Given that many chromatographic profiling methods use nonMS compatible mobile phases, mass analysis of collected fractions may be performed offline by matrix-assisted laser desorption/ionization MS,31 which is more tolerant of sample contaminants than electrospray ionization (ESI) MS. Alternatively, offline ESI–MS of the collected fractions is possible after desalting, but this is time consuming and not applicable to lowlevel species. To increase sample throughput and information content, it is desirable to couple carbohydrate profiling methods online with MS for simultaneous characterization by elution position, peak area percent, and accurate mass. Although a few online LC/MS methods have been reported,9,32–36 currently, there is no universally employed strategy for profiling and characterizing released mAb N-glycans that is highly compatible with online ESI-quadrupole time-of-flight (QTOF) MS and achieves optimal sensitivity for trace-level N-glycans. In particular, for the heightened characterization of antibody therapeutics, a primary requirement of the ideal N-glycan profiling method would be the ability to simultaneously detect and identify the major-, minor-, and trace-level (

Development and application of a robust N-glycan profiling method for heightened characterization of monoclonal antibodies and related glycoproteins.

A highly robust hydrophilic interaction liquid chromatography (HILIC) method that involves both fluorescence and mass spectrometric detection was deve...
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