Therapeutic protein purity and fragmented species characterization by capillary electrophoresis sodium dodecyl sulfate using systematic hybrid cleavage and forced degradation

Duhamel, Lauren; Gu, Yan; Barnett, Gregory V.; Tao, Yuanqi; Voronov, Sergey; Ding, James; Mussa, Nesredin; Li, Zheng Jian

doi:10.1007/s00216-019-01942-8

Cited by 15 publications

(11 citation statements)

References 26 publications

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“…Other modes of capillary electrophoresis are also utilized for the evaluation of biological therapeutics. This includes the use of capillary gel electrophoresis size-based sieving of proteins to rapidly screen for antibody impurities − and to investigate protein fragmentation. , A more selective Western blot capillary electrophoresis approach was reported to identify antibody fragmentation . With this technique, antibody fragments, which had been separated with size exclusion chromatography prior to capillary electrophoresis, were visualized with fluorescent probes specific for the kappa light chain as well as the Fc and Fab regions of an IgG.…”

Section: Proteinsmentioning

confidence: 99%

Challenging Bioanalyses with Capillary Electrophoresis

et al. 2019

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This report covers advances in capillary electrophoresis (CE) from January 2018 through September 2019. A summary of the literature during this time period is insightful. A search performed using the SciFinder Scholar® database for journal reports (limited to English) using the term capillary electrophoresis returned approximately 1,800 publications. Further analysis of this list, depicted in Figure 1, provided a snapshot of activity in biomolecular research. Classes of biomolecules most frequently associated with CE publications were proteins, drugs, DNA and metabolites. Another measure of the impact of CE is the translation of this technology into society. A search of patent activity illustrating this process of CE technology transfer returned 346 patents published in all languages, with a substantial contribution reported only in Chinese (198 patents) or English (98 patents). The versatility of CE for biological systems is exemplified by the rise of the technique in several areas. Metabolomics research involving measurements of large sets of molecules with subtle structural differences benefits from rapid separations achieved with high peak capacity and automated instruments. Single cell and sub-cellular analyses continue to progress in CE because of the size compatibility of the technique with the sample. Other examples of areas utilizing CE that are accelerating include portable and printable instrumentation, affinity interaction, as well as proteomics. As an established analytical tool, CE instrumentation and methods have been designed to be accessible and easily adopted by researchers with expertise in areas beyond the field of separations. Generally, publications including CE measurements either outline innovations in the technique or they are compelling applications of a mature analytical approach. The goal of this review, which is limited to 180 citations, is

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Section: Proteinsmentioning

confidence: 99%

Challenging Bioanalyses with Capillary Electrophoresis

et al. 2019

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“…11,12 However, these studies largely relied on prior knowledge of possible mAb fragments generated under different conditions, estimations of peak MW from the protein standard curve, or correlations with peptide mapping results to identify the cSDS peaks. 4,[13][14][15] In general, the peak assignments have not been confidently verified and it would be difficult to identify unexpected cSDS peaks generated from novel degradation pathways of a mAb product. Furthermore, the large uncertainty in MW estimations often prevents accurate and precise peak assignments.…”

Section: Introductionmentioning

confidence: 99%

Specific and high-resolution identification of monoclonal antibody fragments detected by capillary electrophoresis–sodium dodecyl sulfate using reversed-phase HPLC with top-down mass spectrometry analysis

Wang

Cheung-Lau

Chen

et al. 2019

mAbs

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In recent years, capillary electrophoresis-sodium dodecyl sulfate (cSDS) has been widely used for high resolution separation and quantification of the fragments and aggregates of monoclonal antibodies (mAbs) to ensure the quality of mAb therapeutics. However, identification of the low-molecular-weight (LMW) and high-molecular-weight (HMW) species detected in cSDS electropherograms has been based primarily on the approximate MWs calculated from standard curves using known MW standards and correlations with fragments and aggregates identified by other methods. It is not easy to collect sufficient amounts of H/LMW species from cSDS for analysis by orthogonal methods and the direct coupling of cSDS with mass spectrometry (MS) is very difficult due to interference from SDS. In this study, we describe the precise identification of H/LMW species detected by cSDS using reversed-phase high performance liquid chromatography (RP-HPLC) coupled with top-down tandem MS analysis. The H/ LMW species were first identified by on-line RP-HPLC MS analysis and the RP-HPLC fractions were then analyzed by cSDS to connect the identified H/LMW species with the peaks in the cSDS electropherogram. With this method, 58 unique H/LMW species were identified from an immunoglobulin G1 (IgG1) mAb. The identified fragments ranged from 10 kDa single chain fragments to 130 kDa triple chain fragments, including some with post-translational modifications. This is the first study to clearly identify the antibody fragments, including the exact clipping sites, observed in cSDS electropherograms. The methodology and results presented here should be applicable to most other IgG1 mAbs.

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“…Peaks 2–6 labeled in Figure A were known based on prior literature . As mentioned previously, peak 1 corresponded to the ∼11 kDa clipped species resulting from truncation.…”

Section: Resultsmentioning

confidence: 95%

“…For this work, orthogonal techniques such as partial IdeS digestion and BME reduction activity were used in combination with historical knowledge of mAb CGE profiles to confidently identify peaks. 5,10,17,19,21 As shown in Figure 5 and Table 3, compared to intact NR CGE, the IdeS subunit NR CGE electropherogram contained additional peaks. Although direct characterization of profile peaks was not feasible, characterization using partial IdeS digestion and partial BME reduction was employed to identify product impurities and method-induced species, such as peaks representing the IdeS protein and any potential IdeS-induced partially digested species.…”

Section: Improved Resolution Of the Truncated Mab-1 Variant By Ides S...mentioning

confidence: 93%

Using Digestion by IdeS Protease to Improve Quantification of Degradants in Monoclonal Antibodies by Non-Reducing Capillary Gel Electrophoresis

et al. 2022

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Monoclonal antibodies (mAbs) have become predominant therapeutics by providing highly specific mechanisms of action enabling treatment of complex diseases. However, mAbs themselves are highly complex and require thorough testing and characterization to ensure efficacy and patient safety. In this regard, fragmentation is a degradation product of concern. The biotechnology industry uses capillary gel electrophoresis (CGE) to quantify fragmentation by electrophoretically resolving size variants, such as products resulting from partial reduction of interchain disulfides. However, standard CGE methods may not adequately separate less typical fragments, particularly when there is minimal size difference to the parent molecule. For mAb-1, a degradant only ∼11 kDa smaller than the intact mAb (∼149 kDa) was unable to be resolved under typical non-reducing conditions, preventing an accurate purity assessment and precluding tracking of product purity within stability studies. To address these deficiencies, a subunit-based non-reducing CGE method was developed to employ IdeS protease to produce F(ab’)2 and Fc fragments, which resulted in baseline resolution of the clipped subunit species from its parent species. This enabled more accurate trending of purity throughout stability studies. Method characterization ensured that this subunit method monitored expected impurities observed by intact non-reducing CGE and thus could suitably replace non-reducing CGE in the release and stability testing panel. It also has the potential to replace reducing CGE based on its tracking of the deglycosylated Fc species. We believe this approach of utilizing proteases to develop subunit CGE methods for release and stability can be applied to other molecules when in need of resolving analogous fragments.

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Therapeutic protein purity and fragmented species characterization by capillary electrophoresis sodium dodecyl sulfate using systematic hybrid cleavage and forced degradation

Cited by 15 publications

References 26 publications

Challenging Bioanalyses with Capillary Electrophoresis

Challenging Bioanalyses with Capillary Electrophoresis

Specific and high-resolution identification of monoclonal antibody fragments detected by capillary electrophoresis–sodium dodecyl sulfate using reversed-phase HPLC with top-down mass spectrometry analysis

Using Digestion by IdeS Protease to Improve Quantification of Degradants in Monoclonal Antibodies by Non-Reducing Capillary Gel Electrophoresis

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