Characterization of a novel modification of a CHO-produced mAb: Evidence for the presence of tyrosine sulfation

Abstract

Herein we describe the investigation of a Chinese hamster ovary (CHO)-expressed human mAb molecule found partially modified by a +80 Da adduct. This mass difference, suggestive of a single sulfation or phosphorylation addition, was observed by mass analysis of the intact and reduced molecule by mass spectrometry (MS). The modification was located on tyrosine 31 (Y31) of the light chain in the complementarity-determining region 1 by liquid chromatography (LC)-MS peptide mapping and electron transfer dissociation fragmentation. The complete loss of the 80 Da modification moiety during collision induced dissociation fragmentation suggested this modification could not be a tyrosine phosphorylation. Treatment of the mAb with alkaline phosphatase confirmed our hypothesis. Western blot experiment using anti-tyrosine sulfation antibody and LC retention time correlation with corresponding synthetic sulfated peptides further confirmed the identification of tyrosine sulfation on the light chain. The unique sequence motif with neighboring acidic amino acids and local secondary structure might play a role to make Y31 a substrate residue for sulfation. This type of modification, to our knowledge, has not been previously reported for CHO-produced human IgG antibodies.

Characterization and prediction of positional 4-hydroxyproline and sulfotyrosine, two post-translational modifications that can occur at substantial levels in CHO cells-expressed biotherapeutics

“…9 Also, the level of sTyr recently reported in the Lc CDR-1 of a CHO-derived antibody was determined to a relatively high (~20% at the peptide level, corresponding to~40% at intact level). 7 Together, these observations and the data presented here demonstrate that CHO cells are generally capable of introducing high levels of hydroxyproline and sulfotyrosine in therapeutic proteins, which thereby significantly increase the heterogeneity of the molecules. In a very recent study by Hou et al, 12 the impact of specific nutrients on phosphoserine (~20%) and hydroxylysine (~25%) of a CHO platform, fed batch-produced Fc-fusion protein was reported.…”

“…To our knowledge, this is the first application of MALDI-ISD FT-ICR MS for the characterization of sulfated mAbs. Antibody CDR sulfation was first reported by Zhao et al 7 using positive ion mode ETD. Often, however, sulfations have been reported to be undetectable using conventional positive ion mode fragmentation techniques due to poor ionization efficiency and sulfate loss preceding peptide backbone fragmentation.…”

“…[3][4][5] Also, complementarity-determining regions (CDRs), which have inherent variability and solvent exposure, have been identified with unusual enzyme-catalyzed PTMs like O-fucosylation and sulfotyrosine. 6,7 Further uncommon enzyme-catalyzed PTMs (based on the experience and knowledge from IgGs and monoclonal antibodies (mAbs)) have been reported in non-IgG domains and linkers of antibody-fusion proteins. [8][9][10][11][12] In therapeutic proteins, PTMs are especially critical if they negatively influence drug potency or drug safety.…”

Macro- and Micro-Heterogeneity of Natural and Recombinant IgG Antibodies

“…Cysteinylation, which frequently occurs at non-canonical cysteine residues, has also been reported at canonical cysteine residues in IgG2 [12,96] and is probably due to the relative instability of the IgG2 disulfide bond linkage around the hinge region. The presence of tyrosine sulfation resulted in the formation of a distinct acidic peak for a mAb expressed in Chinese hamster ovary (CHO) cells [204]. Modification of light chain and heavy chain N-termini by maleuric acid has been detected in a mAb expressed in transgenic goats [205].…”

Genetic engineering approaches to improve posttranslational modification of biopharmaceuticals in different production platforms

“…Only few recombinant biopharmaceuticals contain sulfation, and these are generally difficult to express due to the complexity of both the molecules and the modifications (Kumar, 2015;Suflita, Fu, He, Koffas, & Linhardt, 2015). Eukaryotic expression platforms are capable of performing this kind of PTM (Zhao et al, 2017). Heparin is the most famous example of a sulfated difficult-to-express biopharmaceutical, but clotting factors rhFVIII and rhFIX also have tyrosine sulfation sites present (Garg et al, 2003;Kumar, 2015).…”