Antibodies now make up the fastest growing category of therapeutic drugs. Even with today's highly-defined recombinant humanized antibodies, the control and analysis of micro-heterogeneities relating to functional impact have become critical to the quality by design paradigm.1 Alain Beck and his colleagues at the Centre d'Immunologe at Pierre Fabre are leading the efforts to identify and characterize the criteria for therapeutic antibody analysis and quality control.2,3
Among the areas of antibody peptide and glycan analysis currently used or under investigation:
Several modalities of Mass Spectroscopy (MS) analysis have emerged as core technologies for antibody peptide and glycan characterization. The following are some examples:
Dr. Beck and others are investigating not only new methodologies for improving the quality control of existing therapeutic antibodies, but for the development of biosimilars, biobetters, and next generation antibodies.2 In his recent review article, Dr. Beck highlights two new enzymes from Genovis Enzyme Technologies for antibody-specific glycan and peptide analysis:3
Peptide hydrolysis of antibodies has historically utilized proteases such as papain, pepsin, and endoproteinase Lys-C, which can suffer from limited specificity. Recently, the immunoglobulin-degrading IdeS cysteine protease (FaBRICATOR) from Streptococcus pyogenes has provided an improvement in specificity and digestion time. Since FabRICATOR cleaves under the hinge domain, antibodies with glycosylated Fab regions will yield glycans associated with two separate peptide fragments. This enables more effective glycan analysis, particularly relating to the effector functions of Fc fucosylation and galactosylation.4
Because of its exceptionally high specificity for IgG protein sequences, Goetze, A.M. et al. were able to utilize the FaBRICATOR enzyme to cleave IgG in serum to produce Fc fragments for analysis by LC-MS to identify individual haplotypes.5
Figure 1.FabRICATOR cleaves IgG isotypes just below the hinge region, generating an intact F(ab')2 fragment and a Fc fragment.
Figure 2.Cleavage of a therapeutic antibody with FabRICATOR just below the hinge region, generating an intact F(ab')2 fragment and a Fc fragment. This provides simplified MS analysis of antibodies that have two different glycosylation sites on the heavy chain, by yielding two separate peptide fragments, each with one glycosylation site.
Enzymatic glycan hydrolysis of antibodies has historically utilized glycanases such as PNGase F, and other endo and exoglycosidases. IgGZero (EndoS), also from Streptococcus pyogenes, has demonstrated exceptional hydrolytic activity specific for IgG bound glycans.6,7 The enzyme cleaves the chitobiose core of the glycan on IgG from various sources, such as human, rabbit, mouse, Rhesus monkey, goat, sheep, rat, horse, dog, porcine, and more.
Figure 3.IgGZERO shows specific endoglycosidase activity on native IgG because it hydrolyzes the conserved glycans attached to Asp297 on IgG heavy chains.
Figure 4.Cleavage sites of IgGZERO and PNGase F
Therapeutic antibody analysis is evolving, particularly as liquid chromatography, electrophoresis, and MS workflows are being refined. FabRICATOR and IgGZero enzymes are adding another dimension of accuracy and ease-of use to these methods.
Find out more about products for antibody characterization including the Genovis enzymes, and Maxi and Minispin Columns on our Antibody Characterization Website.
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