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Vaccines that are safe, efficacious, and can be rapidly developed are needed to prevent and to react to emerging global infectious disease threats such as influenza, Polio, and Coronavirus diseases. Fast and reliable analytical methods are required without delay to support vaccine process and product development, characterization, and quality control testing. The traditional analytical methods for vaccines are laborious and often lack analytical power, causing slow, expensive, or sometimes failing vaccine development. Capillary electrophoresis (CE) is a technique that has great potential for biopharmaceutical analysis, although there has been limited application in vaccine development.

Several novel CE methods were explored, developed, and applied for viral vaccine analysis, making use of the analytical quality by design (AQbD) process and tools. AQbD is a framework of science- and risk-based decision making to achieve in-depth method understanding and to set up fit-for-purpose and in-control analytical methods. 

Commercial kits for capillary gel electrophoresis (CGE) and imaging capillary isoelectric focusing (icIEF) for antibodies analysis were applied and improved for vaccine analysis. Analytical mechanisms were studied, such as the effect of gel buffer composition on separation, and an AQbD CGE method development strategy was established. The strategy was successfully applied to develop CGE methods for the analysis of seasonal and universal influenza, and sabin inactivated polio vaccine proteins. An icIEF method was also developed, validated, and applied for the universal influenza vaccine protein. 

A capillary zone electrophoresis (CZE) method development for intact adenovirus concentration determination started with background electrolyte (BGE) and capillary design and screening. An BGE with tris and tricine and a neutral capillary resulted in optimal and robust separation and limited adsorption. The CZE method was validated for seed release, in-process control, product release, and stability testing. The precise, accurate, fast, and robust CZE method was applied for all process intermediates and used at different locations. Process impurities and product degradation could also be characterized.

Additionally, CZE methods for chloride and bromide analysis in complex matrices, and a CGE method for host cell DNA characterization were developed for characterization as well as to support process development.

Development of CE methods using AQbD reduced lead times and costs. The developed CE methods were easier to use, were more accurate and precise, and were more selective for product and process impurities compared to the previously used analytical methods for vaccines. The use of CE and AQbD helped improve on vaccine safety, efficacy, and quality.

Vaccines against infectious diseases are urgently needed. Therefore, modern analytical method development should be as efficient as possible to speed up vaccine development. The objectives of the study were to identify critical method parameters (CMPs) and to establish a set of steps to efficiently develop and validate a CE-SDS method for vaccine protein analysis based on a commercially available gel buffer. The CMPs were obtained from reviewing the literature and testing the effects of gel buffer dilution. A four-step approach, including two multivariate DoE (design of experiments) steps, was proposed, based on CMPs and was verified by CE-SDS method development for: (i) the determination of influenza group 1 mini-hemagglutinin glycoprotein; and (ii) the determination of polio virus particle proteins from an inactivated polio vaccine (IPV). The CMPs for sample preparation were incubation temperature(s) and time(s), pH, and reagent(s) concentration(s), and the detection wavelength. The effects of gel buffer dilution revealed the CMPs for CE-SDS separation to be the effective length, the gel buffer concentration, and the capillary temperature. The four-step approach based on the CMPs was efficient for the development of the two CE methods. A four-step approach to efficiently develop capillary gel electrophoresis methods for viral vaccine protein analysis was successfully established.

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.

A CZE method was validated and implemented for fast and accurate in-process determination of adenovirus concentrations of downstream process samples obtained during manufacturing of adenovirus vector-based vaccines. An analytical-quality-by-design approach was embraced for method development, method implementation, and method maintenance. CZE provided separation of adenovirus particles from sample matrix components, such as cell debris, residual DNA and proteins. The intermediate precision of the virus particle concentration was 6.9% RSD and the relative bias was 2.3%. In comparison, the CZE method is intended to replace a quantitative polymerase chain reaction method which requires three replicates in three analytical runs to achieve an intermediate precision of 8.1% RSD. Given that, in addition, the time from sampling till reporting results of the CZE method was less than 2 h, whereas quantitative polymerase chain reaction requires 3 days, it follows that the CZE method enables faster processing times in downstream processing.

During development of adenovirus-based vaccines, samples have to be analyzed in order to either monitor the production process or control the quality and safety of the product. An important quality attribute is the total concentration of intact adenoviruses, which currently is determined by quantitative polymerase chain reaction (qPCR) or anion exchange-HPLC. Capillary Electrophoresis (CE) was evaluated as alternative to the current methods with the aim to have one single method that allows reliable and fast quantification of adenovirus particles throughout the full process. Intact adenoviruses samples from downstream processing and upstream processing were analyzed directly by CE with UV-detection at 214 nm. Only the samples with high amounts of DNA required a simple sample pretreatment by benzonase. Adenovirus particles were separated from matrix components such as cell debris, residual cell DNA, and/or proteins on a PVA-coated capillary using a BGE consisting of 125 mM Tris, 338 mM tricine and 0.2% v/v polysorbate-20 at pH 7.7. Full factorial design of experiments was used for method optimization as part of the analytical quality by design (AQbD) method development approach. The method was validated for the quantification of adenoviruses on five representative samples from the manufacturing process in the range of 0.5 x10(11)-1.5 x10(11) adenovirus particles per ml (similar to 80 to 250 pmo1/1). The CE method showed intermediate precision of 7.8% RSD on concentration and an accuracy (spiked recovery) of 95-110%. CE proved highly useful for process development support and is being implemented for in-process control testing for adenovirus vaccine manufacturing.