Impact of Glycosylation on the Local Backbone Flexibility of Well-Defined IgG1-Fc Glycoforms Using Hydrogen Exchange-Mass Spectrometry

Abstract

We have used hydrogen exchange-mass spectrometry to characterize local backbone flexibility of 4 well-defined IgG1-Fc glycoforms expressed and purified from Pichia pastoris, 2 of which were prepared using subsequent in vitro enzymatic treatments. Progressively decreasing the size of the N-linked N297 oligosaccharide from high mannose (Man8-Man12), to Man5, to GlcNAc, to nonglycosylated N297Q resulted in progressive increases in backbone flexibility. Comparison of these results with recently published physicochemical stability and Fcγ receptor binding data with the same set of glycoproteins provide improved insights into correlations between glycan structure and these pharmaceutical properties. Flexibility significantly increased upon glycan truncation in 2 potential aggregation-prone regions. In addition, a correlation was established between increased local backbone flexibility and increased deamidation at asparagine 315. Interestingly, the opposite trend was observed for oxidation of tryptophan 277 where faster oxidation correlated with decreased local backbone flexibility. Finally, a trend of increasing C'E glycopeptide loop flexibility with decreasing glycan size was observed that correlates with their FcγRIIIa receptor binding properties. These well-defined IgG1-Fc glycoforms serve as a useful model system to identify physicochemical stability and local backbone flexibility data sets potentially discriminating between various IgG glycoforms for potential applicability to future comparability or biosimilarity assessments.

Recombinant Subunit Rotavirus Trivalent Vaccine Candidate: Physicochemical Comparisons and Stability Evaluations of Three Protein Antigens

“…Although the potential impact of physicochemical structural alterations on immunogenicity is unknown at this time, developing such stability-indicating analytical tools and applying the structural knowledge gained in this work will be useful to (1) set critical manufacturing process parameters to ensure consistency, (2) monitor key structural attributes during comparability assessments, and (3) develop stable formulations for the bulk drug substance and adjuvanted final drug product. The pharmaceutical stability profiles encountered during manufacturing and storage are not only dependent on such intrinsic properties (e.g., primary sequence/post-translational modifications, 33 conformational stability, 34 solubility 35 ), but also extrinsic stress factors (e.g., storage temperature, freeze-thaw, agitation). 36,37 Primary Structure, Post-Translational Modifications, and Chemical Stability Profile An additional Met residue identified at N-terminus of each antigen was not unexpected because these antigens were expressed in E. coli and the efficiency of methionine aminopeptidase, enzyme responsible for excision of N-terminal methionine during translation, is about 30%-60% depending on the host.…”

Effects of Glycan Structure on the Stability and Receptor Binding of an IgG4-Fc

“…33 Products 1-5, resulting from photoinduced Tyr side-chain fragmentation reveal relatively higher yields in IgG4-Fc with smaller sizes of Asn 297 glycans (e.g., in N297Q and GlcNAc 1 ). The smallersize glycans may permit a more dynamic protein conformation 18 resulting in a closer approach between Tyr and Trp þ . Interestingly, the photochemical yields of products 1-5 approximately correlate inversely with the minor tryptophan fluorescence red shift, indicating that radical cations generated from Trp residues more exposed to water may also enter competitive pathways, possibly the reaction with H 2 O/HO À to yield hydroxytryptophan.…”

“…15 Glycosylation can be critical for effector function 16,17 and glycan heterogeneity should be tightly controlled during manufacturing. 18 Several analytical approaches have been developed to qualitatively and quantitatively monitor oligosaccharide profiles of mAbs, including fluorescence detection of 2-aminobenzamide labeled glycans and HPLC-MS analysis of released glycans, glycopeptides, and glycoproteins. 19 Analytical results suggest that variations of glycan structures often exist among biosimilars and reference products.…”

The Impact of Immunoglobulin G1 Fc Sialylation on Backbone Amide H/D Exchange

“…Further, the glyco-peptide uptake differences were not determined between the different variants due to the diversity of glycan structures. The varying number of H/D exchanging acetamido groups on the individual glycans makes it impossible to quantitatively compare peptides within the C'E-loop area, i.e., the glycosylation site, as has been described in detail by Guttman et al and More et al [46,55]. Interestingly, the α-2,3-linked G2S2F variant (ST3) revealed an opposite trend for H/DX and FcγR receptor binding activity compared to the ST6 variant.…”

“…Many examples of H/DX-MS applied to structural changes in biopharmaceutical proteins [37,38], including mAbs [17,[39][40][41][42] exposed to extreme stress conditions, have been published. The impact of chemical modifications and PTMs on the backbone amide hydrogen exchange behavior have been studied intensively [17,21,40,43]; e.g., for methionine oxidation [17,40,43], de-glycosylation [44,45], de-/hyper-galactosylation [17], high mannose variants [21,46], and afucosylation [17]. Likewise, the applications of H/DX-MS to characterizing mAb disulfide isoforms [47] and aggregates [48] have also been reported.…”