Evaluation of a glycoengineered monoclonal antibody via LC-MS analysis in combination with multiple enzymatic digestion

ABSTRACT Glycosylation affects the efficacy, safety and pharmacokinetics/pharmacodynamics properties of therapeutic monoclonal antibodies (mAbs), and glycoengineering is now being used to produce mAbs with improved efficacy. In this work, a glycoengineered version of rituximab was produced by chemoenzymatic modification to generate human-like N-glycosylation with α 2,6 linked sialic acid. This modified rituximab was comprehensively characterized by liquid chromatography-mass spectrometry and compared to commercially available rituximab. As anticipated, the majority of N-glycans were converted to α 2,6 linked sialic acid, in contrast to CHO-produced rituximab, which only contains α 2,3 linked sialic acid. Typical posttranslational modifications, such as pyro-glutamic acid formation at the N-terminus, oxidation at methionine, deamidation at asparagine, and disulfide linkages were also characterized in both the commercial and glycoengineered mAbs using multiple enzymatic digestion and mass spectrometric analysis. The comparative study reveals that the glycoengineering approach does not cause any additional posttranslational modifications in the antibody except the specific transformation of the glycoforms, demonstrating the mildness and efficiency of the chemoenzymatic approach for glycoengineering of therapeutic antibodies.

[1]  Fredrik Olsson,et al.  EndoS and EndoS2 hydrolyze Fc-glycans on therapeutic antibodies with different glycoform selectivity and can be used for rapid quantification of high-mannose glycans , 2015, Glycobiology.

[2]  M. Butler,et al.  Production of α2,6-sialylated IgG1 in CHO cells , 2015, mAbs.

[3]  A. Manning,et al.  Controlled tetra-Fc sialylation of IVIg results in a drug candidate with consistent enhanced anti-inflammatory activity , 2015, Proceedings of the National Academy of Sciences.

[4]  E. Park,et al.  Terminal sialic acid linkages determine different cell infectivities of human parainfluenza virus type 1 and type 3. , 2014, Virology.

[5]  M. Ratner.  Genentech's glyco-engineered antibody to succeed Rituxan. , 2014, Nature biotechnology.

[6]  B. Karger,et al.  Comparability analysis of anti-CD20 commercial (rituximab) and RNAi-mediated fucosylated antibodies by two LC-MS approaches , 2013, mAbs.

[7]  W. Somers,et al.  Pyroglutamate and O-Linked Glycan Determine Functional Production of Anti-IL17A and Anti-IL22 Peptide-Antibody Bispecific Genetic Fusions , 2012, The Journal of Biological Chemistry.

[8]  H. Gadgil,et al.  Evaluation of effects of Fc domain high-mannose glycan on antibody stability. , 2012, Journal of pharmaceutical sciences.

[9]  Wei Huang,et al.  Chemoenzymatic glycoengineering of intact IgG antibodies for gain of functions. , 2012, Journal of the American Chemical Society.

[10]  F. Wurm,et al.  Glycan variability on a recombinant IgG antibody transiently produced in HEK-293E cells. , 2012, New biotechnology.

[11]  F. Rüker,et al.  Significant Impact of Single N-Glycan Residues on the Biological Activity of Fc-based Antibody-like Fragments* , 2012, The Journal of Biological Chemistry.

[12]  Alain Van Dorsselaer,et al.  Biosimilar, biobetter, and next generation antibody characterization by mass spectrometry. , 2012, Analytical chemistry.

[13]  Lai-Xi Wang,et al.  Emerging technologies for making glycan-defined glycoproteins. , 2012, ACS chemical biology.

[14]  B. Karger,et al.  Complete Mapping of a Cystine Knot and Nested Disulfides of Recombinant Human Arylsulfatase A by Multi-Enzyme Digestion and LC-MS Analysis Using CID and ETD , 2012, Journal of The American Society for Mass Spectrometry.

[15]  Lai-Xi Wang,et al.  Chemoenzymatic synthesis and Fcγ receptor binding of homogeneous glycoforms of antibody Fc domain. Presence of a bisecting sugar moiety enhances the affinity of Fc to FcγIIIa receptor. , 2011, Journal of the American Chemical Society.

[16]  W. Hancock,et al.  Characterization and comparison of disulfide linkages and scrambling patterns in therapeutic monoclonal antibodies: using LC-MS with electron transfer dissociation. , 2011, Analytical chemistry.

[17]  Jian Zhang,et al.  The evaluation of a novel approach for the profiling and identification of N-linked glycan with a procainamide tag by HPLC with fluorescent and mass spectrometric detection. , 2010, Journal of pharmaceutical and biomedical analysis.

[18]  I. Ishiwata,et al.  Expression of α2,6-sialic acid-containing and Lewis-active glycolipids in several types of human ovarian carcinomas. , 2010, Oncology letters.

[19]  Hongwei Xie,et al.  Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies , 2010, mAbs.

[20]  Alain Beck,et al.  Identification and characterization of asparagine deamidation in the light chain CDR1 of a humanized IgG1 antibody. , 2009, Analytical biochemistry.

[21]  Liang-Yu Shih,et al.  An improved trypsin digestion method minimizes digestion-induced modifications on proteins. , 2009, Analytical biochemistry.

[22]  A. Varki,et al.  Human-specific evolution of sialic acid targets: Explaining the malignant malaria mystery? , 2009, Proceedings of the National Academy of Sciences.

[23]  S. Sanglier-Cianférani,et al.  Extending mass spectrometry contribution to therapeutic monoclonal antibody lead optimization: characterization of immune complexes using noncovalent ESI-MS. , 2009, Analytical chemistry.

[24]  S. Kaveri,et al.  DC-SIGN and α2,6-sialylated IgG Fc interaction is dispensable for the anti-inflammatory activity of IVIg on human dendritic cells , 2009, Proceedings of the National Academy of Sciences.

[25]  Gregory C Flynn,et al.  The effect of Fc glycan forms on human IgG2 antibody clearance in humans. , 2008, Glycobiology.

[26]  Lai-Xi Wang,et al.  Glycoengineering of human IgG1-Fc through combined yeast expression and in vitro chemoenzymatic glycosylation. , 2008, Biochemistry.

[27]  Robert M. Anthony,et al.  Recapitulation of IVIG Anti-Inflammatory Activity with a Recombinant IgG Fc , 2008, Science.

[28]  K. Vorauer-Uhl,et al.  Influence of a Reduced CO2 Environment on the Secretion Yield, Potency and N-Glycan Structures of Recombinant Thyrotropin from CHO Cells , 2008, Molecular biotechnology.

[29]  David Passmore,et al.  Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor , 2006, Nature Biotechnology.

[30]  M. Kiso,et al.  Enhanced Expression of an α2,6-Linked Sialic Acid on MDCK Cells Improves Isolation of Human Influenza Viruses and Evaluation of Their Sensitivity to a Neuraminidase Inhibitor , 2005, Journal of Clinical Microbiology.

[31]  F. Dorner,et al.  Comparison of N-glycan pattern of recombinant human coagulation factors II and IX expressed in Chinese hamster ovary (CHO) and African green monkey (Vero) cells , 2004, Journal of Thrombosis and Thrombolysis.

[32]  R B Freedman,et al.  Metabolic control of recombinant protein N-glycan processing in NS0 and CHO cells. , 2001, Biotechnology and bioengineering.

[33]  D. Harvey.  N-(2-diethylamino)ethyl-4-aminobenzamide derivative for high sensitivity mass spectrometric detection and structure determination of N-linked carbohydrates. , 2000, Rapid Communications in Mass Spectrometry.

[34]  R. Chammas,et al.  Linkage-specific Action of Endogenous Sialic Acid O-Acetyltransferase in Chinese Hamster Ovary Cells* , 1996, The Journal of Biological Chemistry.

[35]  M. Pang,et al.  Characterization of Terminal Sialic Acid Linkages on Human Thymocytes , 1996, The Journal of Biological Chemistry.