Identification and measurement of isoaspartic acid formation in the complementarity determining region of a fully human monoclonal antibody

Dick, Lawrence W.; Qiu, Difei; Cheng, Kuang-Chuan

doi:10.1016/j.jchromb.2009.09.031

Cited by 22 publications

(18 citation statements)

References 25 publications

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“…Canonical motifs are specified using the standard n, n + 1 notation, 32 and include motifs that have been previously implicated in CDR deamidation and isomerization events, and/or have otherwise demonstrated modification rates in time-scales aligned with our experiments. Thus, the canonical motifs for isomerization are DG, 16,20,22,23,25,29,33,34 DS, 10,23,35,36 DD, 23,36 DT, 23 and DH. 37 For deamidation, the canonical motifs are NG, 20,22,23,32,34,38 NS, 22,23,32,39 NN, 23,32 NT, 22,23,32,34,40 and NH.…”

Section: Resultsmentioning

confidence: 99%

“…For example, deamidation rates at pH 5.5 are approximately 40fold slower than at pH 8.0, 9,11,15,21 while the rate of Asu intermediate formation increases 6-fold over the same pH range. 15 Although several studies have been conducted where both products are induced at a single pH, 12,[22][23][24] others have preferentially induced isomerization at low pH 16,25,26 or deamidation at high pH. [26][27][28] The reaction conditions used in this study employ the latter approach to amplify the observable modified populations with concurrent simplification of isomerization detection.…”

Section: Introductionmentioning

confidence: 99%

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Deamidation and isomerization liability analysis of 131 clinical-stage antibodies

Nobrega

Lynaugh

et al. 2018

mAbs

115

112

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Contemporary in vivo and in vitro discovery platform technologies greatly increase the odds of identifying high-affinity monoclonal antibodies (mAbs) towards essentially any desired biologically relevant epitope. Lagging discovery throughput is the ability to select for highly developable mAbs with drug-like properties early in the process. Upstream consideration of developability metrics should reduce the frequency of failures in later development stages. As the field moves towards incorporating biophysical screening assays in parallel to discovery processes, similar approaches should also be used to ensure robust chemical stability. Optimization of chemical stability in the early stages of discovery has the potential to reduce complications in formulation development and improve the potential for successful liquid formulations. However, at present, our knowledge of the chemical stability characteristics of clinical-stage therapeutic mAbs is fragmented and lacks comprehensive comparative assessment. To address this knowledge gap, we produced 131 mAbs with amino acid sequences corresponding to the variable regions of clinical-stage mAbs, subjected these to low and high pH stresses and identified the resulting modifications at amino acid-level resolution via tryptic peptide mapping. Among this large set of mAbs, relatively high frequencies of asparagine deamidation events were observed in CDRs H2 and L1, while CDRs H3, H2 and L1 contained relatively high frequencies of instances of aspartate isomerization.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Deamidation and isomerization liability analysis of 131 clinical-stage antibodies

Nobrega

Lynaugh

et al. 2018

mAbs

115

112

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“…[63][64][65][66] Other techniques for the separation of charged protein variants are isoelectric focusing (IEF), hydrophobic interaction chromatography (HIC), reversedphase HPLC, and free flow electrophoresis (FFE). [65][66][67][68][69][70][71][72] Catumaxomab charged variants were separated and isolated using FFE fractionation and analyzed using LC/MS methods. We identified a deamidation site in catumaxomab that is almost exclusively responsible for the appearance of different charged variants.…”

Section: Glycanmentioning

confidence: 99%

Structural and functional characterization of the trifunctional antibody catumaxomab

Chelius¹,

Rosenberger²,

Gruber³

et al. 2010

mAbs

125

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“…These heterogeneities include aggregation, fragmentation, deamidation, oxidation, N-and C-terminal differentiations. [2][3][4][5][6][7][8][9][10][11] Several analytical methods have been developed for these purposes, for example, SDS polyacrylamide gel electrophoresis for the determination of the purity of proteins, size exclusion chromatography for aggregation, ion exchange chromatography or hydrophobic interaction chromatography for the detection of deamidation and oxidation variants, and Ellman's assay for quantification of free thiol groups. 12,13 Capillary-based SDS electrophoresis (CE-SDS) is one version of the capillary gel electrophoresis (CGE) using soluble linear polymers.…”

Section: Introductionmentioning

confidence: 99%

Application of Microchip Electrophoresis Sodium Dodecyl Sulfate for the Evaluation of Change of Degradation Species of Therapeutic Antibodies in Stability Testing

et al. 2014

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We evaluated the performance of a commercial microchip electrophoresis instrument (LabChip ® GXII) for the evaluation of change of degradation species of therapeutic antibodies in stability testing. This system requires a sample volume of only 5 μg, and indicates fine resolution of size variant species such as light chain, heavy chain, non-glycosylated heavy chain and various degradation species. Precision and accuracy were high; the intermediate precision of 18 determinations was only 2.1% or less as RSD and recoveries ranged from 97.8 to 103.0% for major species as heavy chain, light chain and intact molecule of a therapeutic antibody. The applicability of this method was demonstrated by applying the method for the analysis of heat-degraded products of three pharmaceutical antibodies. Though some fragment peaks commonly appeared and increased according to temperature regardless of the source of preparations, one of them indicated specific peaks implying the cleavage of the peptide chain of the heavy chain. We also compared the performance of the method with those using conventional capillary-based SDS electrophoresis. Although the absolute purity values expressed as peak area % were different for the two methods, probably due to the difference in the detection methods, similar quality profiles were obtained within 40 s by microchip-based SDS electrophoresis. In addition, the degradation manner of three marketed antibodies depending on temperature was almost the same for the two methods. At the first stage in the development of manufacturing antibody pharmaceuticals, various factors including cell selection, cell cultivation, and formulation development should be evaluated using limited sample amounts. The stability testing using microchip-based electrophoresis seems suitable for these purposes.

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Identification and measurement of isoaspartic acid formation in the complementarity determining region of a fully human monoclonal antibody

Cited by 22 publications

References 25 publications

Deamidation and isomerization liability analysis of 131 clinical-stage antibodies

Deamidation and isomerization liability analysis of 131 clinical-stage antibodies

Structural and functional characterization of the trifunctional antibody catumaxomab

Application of Microchip Electrophoresis Sodium Dodecyl Sulfate for the Evaluation of Change of Degradation Species of Therapeutic Antibodies in Stability Testing

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