Four‐step approach to efficiently develop capillary gel electrophoresis methods for viral vaccine protein analysis

Geurink, Lars; Tricht, Ewoud van; Dudink, Justin; Pajic, Bojana; Griend, Cari Sänger van de

doi:10.1002/elps.202000107

Cited by 19 publications

(22 citation statements)

References 34 publications

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“…16 SDS-CGE has been widely used in the biopharmaceutical industry to analyze therapeutic proteins in a simple and robust manner for drug development and commercialization; however, limited studies have been published to date on the fundamental aspects of this transitionally cross-linked chemical gel separation matrix. 17 Understanding of the migration and separation behavior of SDS−protein complexes will aid in the optimization of the analysis of specific biopharmaceuticals. This article reports on the effect of the concentration and ratio of the dextran monomer (2 MDa) and borate cross-linker on molecular sieving and separation performance in capillary SDS gel electrophoresis of proteins.…”

Section: ■ Introductionmentioning

confidence: 99%

Fundamentals of Capillary Electrophoretic Migration and Separation of SDS Proteins in Borate Cross-Linked Dextran Gels

2021

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Recent progress in the development and production of new, innovative protein therapeutics require rapid and adjustable high-resolution bioseparation techniques. Sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using a borate (B) cross-linked dextran (D) separation matrix is widely employed today for rapid consistency analysis of therapeutic proteins in manufacturing and release testing. Transient borate cross-linking of the semirigid dextran polymer chains leads to a high-resolution separation gel for SDS−protein complexes. To understand the migration and separation basis of the D/B gel, the present work explores various gel formulations of dextran monomer (2, 5, 7.5, and 10%) and borate cross-linker (2 and 4%) concentrations. Ferguson plots were analyzed for a mixture of protein standards with molecular weights ranging from 20 to 225 kDa, and the resulting nonlinear concave curves pointed to nonclassical sieving behavior. While the 2% D/4% B gel resulted in the fastest analysis time, the 10% D/2% B gel was found to produce the greatest separation window, even higher than with the 10% D/4% B gel, due to a significant increase in the electroosmotic flow of the former composition in the direction opposite to SDS−protein complex migration. The study then focused on SDS-CGE separation of a therapeutic monoclonal antibody and its subunits. A combination of molecular weight and shape selectivity as well as, to a lesser extent, surface charge density differences (due to glycosylation on the heavy chain) influenced migration. Greater molecular weight selectivity occurred for the higher monomer concentration gels, while improved glycoselectivity was obtained using a more dilute gel, even as low as 2% D/2% B. This latter gel took advantage of the dextran−borate−glycoprotein complexation. The study revealed that by modulating the dextran (monomer) and borate (cross-linker) concentration ratios of the sieving matrix, one can optimize the separation for specific biopharmaceutical modalities with excellent column-to-column, run-to-run, and gel-to-gel migration time reproducibilities (<0.96% relative standard deviation (RSD)). The widely used 10% dextran/4% borate gel represents a good screening option, which can then be followed by a modified composition, optimized for a specific separation as necessary.

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Section: ■ Introductionmentioning

confidence: 99%

Fundamentals of Capillary Electrophoretic Migration and Separation of SDS Proteins in Borate Cross-Linked Dextran Gels

2021

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“…Besides monitoring the purity and integrity of the viral particles, the method was also used to study its interaction and stoichiometry with the corresponding mAb. Other CE modes such as CGE, and SDS‐CGE were reported for identification, quantification, and purity analysis of vaccine products [130–133].…”

Section: Analysis Of New Modality Protein Therapeuticsmentioning

confidence: 99%

“…Multiple reports have been published for vaccine products characterization [130–133]. For example, one study reported CZE‐UV for quantification of a subunit vaccine [130].…”

Section: Analysis Of New Modality Protein Therapeuticsmentioning

confidence: 99%

Applications of capillary electrophoresis for biopharmaceutical product characterization

2021

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Capillary electrophoresis (CE), after being introduced several decades ago, has carved out a niche for itself in the field of analytical characterization of biopharmaceutical products. It does not only offer fast separation, high resolution in miniaturized format, but equally importantly represents an orthogonal separation mechanism to high‐performance liquid chromatography. Therefore, it is not surprising that CE‐based methods can be found in all major pharmacopoeias and are recommended for the analysis of biopharmaceutical products during process development, characterization, quality control, and release testing. Different separation formats of CE, such as capillary gel electrophoresis, capillary isoelectric focusing, and capillary zone electrophoresis are widely used for size and charge heterogeneity characterization as well as purity and stability testing of therapeutic proteins. Hyphenation of CE with MS is emerging as a promising bioanalytical tool to assess the primary structure of therapeutic proteins along with any impurities. In this review, we confer the latest developments in capillary electrophoresis, used for the characterization of critical quality attributes of biopharmaceutical products covering the past 6 years (2015–2021). Monoclonal antibodies, due to their significant share in the market, have been given prioritized coverage.

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“…The separation conditions were 20°C capillary temperature, and 80% v:v gel buffer. See Geurink et al for more details [34].…”

Section: Ce-sds For Sabin Inactivated Polio Proteinmentioning

confidence: 99%

Sixteen capillary electrophoresis applications for viral vaccine analysis

et al. 2021

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A broad range of CE applications from our organization is reviewed to give a flavor of the use of CE within the field of vaccine analyses. Applicability of CE for viral vaccine characterization, and release and stability testing of seasonal influenza virosomal vaccines, universal subunit influenza vaccines, Sabin inactivated polio vaccines (sIPV), and adenovirus vector vaccines were demonstrated. Diverse CZE, CE‐SDS, CGE, and cIEF methods were developed, validated, and applied for virus, protein, posttranslational modifications, DNA, and excipient concentration determinations, as well as for the integrity and composition verifications, and identity testing (e.g., CZE for intact virus particles, CE‐SDS application for hemagglutinin quantification and influenza strain identification, chloride or bromide determination in process samples). Results were supported by other methods such as RP‐HPLC, dynamic light scattering (DLS), and zeta potential measurements. Overall, 16 CE methods are presented that were developed and applied, comprising six adenovirus methods, five viral protein methods, and methods for antibodies determination of glycans, host cell‐DNA, excipient chloride, and process impurity bromide. These methods were applied to support in‐process control, release, stability, process‐ and product characterization and development, and critical reagent testing. Thirteen methods were validated. Intact virus particles were analyzed at concentrations as low as 0.8 pmol/L. Overall, CE took viral vaccine testing beyond what was previously possible, improved process and product understanding, and, in total, safety, efficacy, and quality.

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Four‐step approach to efficiently develop capillary gel electrophoresis methods for viral vaccine protein analysis

Cited by 19 publications

References 34 publications

Fundamentals of Capillary Electrophoretic Migration and Separation of SDS Proteins in Borate Cross-Linked Dextran Gels

Fundamentals of Capillary Electrophoretic Migration and Separation of SDS Proteins in Borate Cross-Linked Dextran Gels

Applications of capillary electrophoresis for biopharmaceutical product characterization

Sixteen capillary electrophoresis applications for viral vaccine analysis

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