Simultaneous Assessment of Asp Isomerization and Asn Deamidation in Recombinant Antibodies by LC-MS following Incubation at Elevated Temperatures

Abstract

The degradation of proteins by asparagine deamidation and aspartate isomerization is one of several chemical degradation pathways for recombinant antibodies. In this study, we have identified two solvent accessible degradation sites (light chain aspartate-56 and heavy chain aspartate-99/101) in the complementary-determining regions of a recombinant IgG1 antibody susceptible to isomerization under elevated temperature conditions. For both hot-spots, the degree of isomerization was found to be significantly higher than the deamidation of asparagine-(387, 392, 393) in the conserved CH3 region, which has been identified as being solvent accessible and sensitive to chemical degradation in previous studies. In order to reduce the time for simultaneous identification and functional evaluation of potential asparagine deamidation and aspartate isomerization sites, a test system employing accelerated temperature conditions and proteolytic peptide mapping combined with quantitative UPLC-MS was developed. This method occupies the formulation buffer system histidine/HCl (20 mM; pH 6.0) for denaturation/reduction/digestion and eliminates the alkylation step. The achieved degree of asparagine deamidation and aspartate isomerization was adequate to identify the functional consequence by binding studies. In summary, the here presented approach greatly facilitates the evaluation of fermentation, purification, formulation, and storage conditions on antibody asparagine deamidation and aspartate isomerization by monitoring susceptible marker peptides located in the complementary-determining regions of recombinant antibodies.

Engineering deamidation‐susceptible asparagines leads to improved stability to thermal cycling in a lipase

“…These studies have reported loss of stability, activity of proteins, and also increased aggregation properties as shown in crystallins, 7,8,11,12,23,24,[51][52][53] superoxide dismutase, 49 and immunoglobins. 14,34,54 Reported properties of mutC suggest that this protein has retained the secondary and tertiary structure with a partial loss in activity but with a significant improvement in the ability to withstand multiple heat cycles.…”

“…Modified work flows are being developed to avoid process-induced deamidation. [34][35][36] In the case of 6B, incubation at 37 C during proteolysis did not result in deamidation, and reduction step is not required as 6B lipase does not have a cysteine. Most of the proteomic methods use electrospray ionization in combination with electron capture dissociation 37 or electron transfer dissociation 38 for qualitative determination of the product type, that is, aspartate or isoaspartate.…”

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

“…A common mAb PTM is Nglycosylation at Asn 297 of the CH2 region, resulting in considerable proteoform heterogeneity from the combination of glycans at the two glycosylation sites [14]. Other PTMs, further increasing proteoform heterogeneity, include Met 1 -loss, formation of pyroglutamic acid, Cterminal lysine clipping, carboxymethylation of lysine, oxidation, deamidation, disulfide bond formation, glycation, single site amino acid replacement, and sequence truncation, amongst others [13,[15][16][17][18] . .…”

In vivo characterization of therapeutic monoclonal antibodies

“…After administration, the accumulated succinimide hydrolyzes to Asp and/or isoAsp under the physiologic pH. In mAbs, deamidation has been reported in the Fc regions [4,5] and the CDRs [6][7][8][9][10][11][12]. Deamidation in the CDR regions could affect drug efficacy [10,12].…”