Evaluation of capillary zone electrophoresis for charge heterogeneity testing of monoclonal antibodies

Moritz, Bernd; Schnaible, Volker; Kiessig, Steffen; Heyne, Andrea; Wild, Markus; Finkler, Christof; Christians, Stefan; Mueller, Kerstin; Zhang, Li; Furuya, Kenji; Hassel, Marc; Hamm, Melissa; Rustandi, Richard R.; He, Yue; Solano, Oscar Salas; Whitmore, Colin D.; Park, Sung Ae; Hansen, Dietmar; Santos, Marcia Rodrigues dos; Lies, Mark

doi:10.1016/j.jchromb.2014.12.024

Cited by 63 publications

(55 citation statements)

References 25 publications

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“…The set screws, that were evaluated in this study and elsewhere , are well suited for successful development of CZE for protein pharmaceuticals (e.g. antibodies and new formats).…”

Section: Discussionmentioning

confidence: 99%

Optimization of capillary zone electrophoresis for charge heterogeneity testing of biopharmaceuticals using enhanced method development principles

et al. 2017

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CZE is a well‐established technique for charge heterogeneity testing of biopharmaceuticals. It is based on the differences between the ratios of net charge and hydrodynamic radius. In an extensive intercompany study, it was recently shown that CZE is very robust and can be easily implemented in labs that did not perform it before. However, individual characteristics of some examined proteins resulted in suboptimal resolution. Therefore, enhanced method development principles were applied here to investigate possibilities for further method optimization. For this purpose, a high number of different method parameters was evaluated with the aim to improve CZE separation. For the relevant parameters, design of experiments (DoE) models were generated and optimized in several ways for different sets of responses like resolution, peak width and number of peaks. In spite of product specific DoE optimization it was found that the resulting combination of optimized parameters did result in significant improvement of separation for 13 out of 16 different antibodies and other molecule formats. These results clearly demonstrate generic applicability of the optimized CZE method. Adaptation to individual molecular properties may sometimes still be required in order to achieve optimal separation but the set screws discussed in this study [mainly pH, identity of the polymer additive (HPC versus HPMC) and the concentrations of additives like acetonitrile, butanolamine and TETA] are expected to significantly reduce the effort for specific optimization.

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“…The set screws, that were evaluated in this study and elsewhere , are well suited for successful development of CZE for protein pharmaceuticals (e.g. antibodies and new formats).…”

Section: Discussionmentioning

confidence: 99%

Optimization of capillary zone electrophoresis for charge heterogeneity testing of biopharmaceuticals using enhanced method development principles

et al. 2017

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“…Capillary zone electrophoresis (CZE), which separates proteins by the mass to charge ratio, has been employed to study deamidation in biotechnology products such as insulin, recombinant human growth hormone (rhGH), and monoclonal antibodies (mAbs) . A common pitfall encountered for CZE, particularly for mAbs, was reproducibility in a biopharmaceutical production setting . However, the performance of this technique can be enhanced by using sample derivatisation or laser‐induced fluorescence detection to improve sensitivity.…”

Section: Deamidation In Proteins: Mechanism and Measurementmentioning

confidence: 99%

Protein deamidation in biopharmaceutical manufacture: understanding, control and impact

Gervais¹

2015

J of Chemical Tech & Biotech

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Understanding of product-related variants, such as variants with post-translational modifications, is an important part of biopharmaceutical development. Deamidation is a common post-translational modification occurring in biopharmaceutical proteins, affecting L-asparagine (Asn) and to a lesser extent, L-glutamine (Gln) residues. The rate of deamidation reactions are influenced by factors including protein structure (primary, secondary and higher structure), temperature and pH. In the vast majority of cases, deamidation is undesirable in biopharmaceuticals, and may lead to potential changes in protein structure, function, stability and immunogenicity. Measurement and characterisation of deamidated biopharmaceutical variants may be challenging, particularly with regard to quantitation of the two L-aspartate isoforms that are created, L-aspartic acid (Asp) and isoaspartate (isoAsp). Deamidation may occur intracellularly or during biopharmaceutical manufacture and storage, and must be understood, minimised and controlled, particularly in a regulatory context. Process control strategies that have been employed to date include alterations to fermentation steps, additives to cell cultures, chromatographic separation of charge variants and protein engineering to remove deamidation-prone Asn residues. However, the impact of deamidated forms of biopharmaceuticals should also be thoroughly studied, as they may not necessarily represent deleterious changes to the function of the molecule or the quality of the final product. This mini-review provides a summary of the current understanding of the origins, control and measurement of deamidation during biopharmaceutical development.

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“…The final CZE separation is performed using PVA‐coated capillary (Agilent) with 365 μm ID, 50 μm OD, 50 cm L D , and 60.2 cm L T . The BGE contains 40 mM EACA/acetic acid with 0.2 % w/w HPMC and 0.2 mg/mL BSA . The analyte is injected from the anode with normal polarity using a 1 psi injection pressure and a 10–25 s injection time with a water plug injected before the separation is performed at 0.5 psi for 5 s. The autosampler and capillary temperatures are maintained at 20°C.…”

Section: Methodsmentioning

confidence: 99%

Development of a capillary zone electrophoresis method for dose determination in a tetravalent dengue vaccine candidate

2015

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Dengue is known to cause morbidity and mortality worldwide and currently there is neither available specific therapeutics to treat nor a vaccine to prevent this disease. Although efforts are being made, development of a vaccine against this disease remains challenging. Hawaii Biotech Inc developed a recombinant subunit envelope protein-based vaccine against all four serotypes produced in Drosophila S2 cells which were transferred over to Merck in 2010. Each subunit of the four dengue serotypes contains the N-terminal 80% of the amino acids comprising the envelope protein (DEN-80E). A Phase 1 study using only monovalent DEN1-80E was done by Hawaii Biotech Inc and most recently, a Phase 1 clinical trial of the tetravalent DEN-80E formulation (V180) was conducted. Here, we report the development of a dose assay for the tetravalent dengue vaccine-containing subunit protein of DEN1-80E, DEN2-80E, DEN3-80E, and DEN4-80E using various separation methods such as HPLC and CE. Based on the results of the comparison, the CZE separation was chosen as the most suitable method to perform the dose assay for the tetravalent dengue vaccine.

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Evaluation of capillary zone electrophoresis for charge heterogeneity testing of monoclonal antibodies

Cited by 63 publications

References 25 publications

Optimization of capillary zone electrophoresis for charge heterogeneity testing of biopharmaceuticals using enhanced method development principles

Optimization of capillary zone electrophoresis for charge heterogeneity testing of biopharmaceuticals using enhanced method development principles

Protein deamidation in biopharmaceutical manufacture: understanding, control and impact

Development of a capillary zone electrophoresis method for dose determination in a tetravalent dengue vaccine candidate

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