Generation and Comparative Characterization of Glycosylated and Aglycosylated Human IgG1 Antibodies

Abstract

Monoclonal antibodies (mAbs) are the fastest growing class of biopharmaceuticals reflecting their diverse applications in research and the clinic. The correct glycosylation of mAbs is required to elicit effector functions such as complement-dependent and antibody-dependent cell-mediated cytotoxicity, although these may be undesirable for the treatment of certain chronic diseases. To gain insight into the properties of glycan-deficient mAbs, we generated and characterized six different aglycosylated human IgG1 mAbs (carrying the N297A mutation) and compared them to their glycosylated counterparts. We found no differences in solubility or heterogeneity, and all mAbs the remained stable in stress tests at 4 and 37 °C. Surface plasmon resonance spectroscopy showed no differences in binding affinity, and the in vivo terminal serum half-life and plasma clearance were similar in rats. However, differential scanning calorimetry revealed that the aglycosylated mAbs contained a less stable C(H)2 domain and they were also significantly more susceptible to pH-induced aggregation. We conclude that aglycosylated mAbs are functionally equivalent to their glycosylated counterparts and could be particularly suitable for certain therapeutic applications, such as the treatment of chronic diseases.

A novel in vitro assay to predict neonatal Fc receptor-mediated human IgG half-life

“…It has been reported that the absence of an Fc glycan in the germline C H 2 domain does not alter the half-life of antibodies, [66][67][68][69] and thus we were interested in confirming this conclusion with our predictive model. Following PNGase treatment, 4 antibodies were confirmed to be aglycosylated by size shift and western blot analysis (Fig.…”

“…Early reports were conflicted regarding effects of glycosylation on antibody half-life, 18,[72][73][74][75] but our results support the most recent reports suggesting glycosylation does not affect FcRn-mediated PK. [66][67][68][69] In our study, this was confirmed by removing all N-glycosylation (only the natural germline Fc glycosylation site exists in the antibodies tested) and the in vitro FcRn binding measurement was used to compare the aglycosylated mAb to the naturally occurring glycosylated form produced in CHO cells. This finding further suggests the in vitro model described here is capable of recapitulating data produced from more complicated, expensive and time-consuming in vivo studies that were previously required for reliable PK data.…”

Preclinical Antitumor Efficacy of BAY 1129980—a Novel Auristatin-Based Anti-C4.4A (LYPD3) Antibody–Drug Conjugate for the Treatment of Non–Small Cell Lung Cancer

“…The C4.4A-Ab (BAY 1112623) was generated by phage display panning using the n-CoDeR library of BioInvent International AB (24) as described in the Supplementary Methods and previously (24)(25)(26)(27). All positions in the complementaritydetermining regions (CDR) of the C4.4A-Ab not required for antigen binding were reverted to human germline sequences as previously described (27).…”

Rational design of therapeutic mAbs against aggregation through protein engineering and incorporation of glycosylation motifs applied to bevacizumab

“…It has been demonstrated on several occasions that this N-glycan participates in the stabilization of mAbs against aggregation caused by various stresses. 8,24,26,27,56,57 Natural IgGs present in human serum are all glycosylated in the Fc domain whereas only less than a third are glycosylated in the Fab domain. 55 N-glycans are found attached to the variable region of the LC, the HC or both.…”