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.
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 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 214nm. 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 125mM Tris, 338mM 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 0.5×1011–1.5×1011 adenovirus particles per ml (~80 to 250pmol/l). 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.
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•A CE method was development and validated for adenovirus particle determination.•No peak interference observed from cell debris, protein, surfactant or DNA.•The method is accurate and precise for downstream and upstream processing samples.•The CE method was superior to the current method, quantitative PCR, in a method comparability study.•Analytical quality by design (AQbD) method development approach.
•A validated method for quantitative protein profiling in adenovirus-based vaccines.•14 Adenovirus proteins baseline separated within 17 min by RP-UPLC.•Adenovirus-based samples directly injected, ...needing no sample pretreatment.•The method can detect and quantify protein degradants.
A method for the quantitative determination of the protein composition of adenovirus-vector based vaccines was developed. The final method used RP-UPLC with UV absorbance detection, a C4 column (300 Å, 1.7 μm, 2.1 × 150 mm), and a water- acetonitrile (ACN) gradient containing trifluoroacetic acid (TFA) as ion-pairing agent. The chromatographic resolution between the various adenovirus proteins was optimized by studying the effect of the TFA concentration and the column temperature, applying a full factorial design of experiments. A reproducible baseline separation of all relevant adenovirus proteins could be achieved within 17 min run time. Samples containing adenovirus particles could be directly injected into the UPLC system without sample pretreatment. The viruses reproducibly dissociate into proteins in the UPLC system upon contact with the mobile phase containing ACN. The new RP-UPLC method was successfully validated for protein profiling and relative quantification of proteins in vaccine products based on adenovirus vector types 26 and 35. The intermediate precision of the relative peak areas of all proteins was between 1% and 14% RSD, except for the peak assigned to protein V (26% RSD). The method proved to be stability indicating with respect to thermal and oxidation stress of the adenovirus-vector based vaccine and was successfully implemented for the characterization of adenovirus-based products.
Current methods for the identification and/or quantification of viral proteins in influenza virus and virosome samples suffer from long analysis times, limited protein coverage and/or low accuracy ...and precision. We studied and optimized capillary gel electrophoresis (CGE) in order to achieve faster and enhanced characterization and quantification of viral proteins. Sample preparation as well the composition of the gel buffer was investigated in order to achieve adequate protein separation in relatively short times. The total sample preparation (reduction and deglycosylation) could be carried out efficiently within two hours. Hydrodynamic injection, separation voltage, and capillary temperature were optimized in full factorial design. The final method was validated and showed good performance for hemagglutinin fragment 1 (HA1), hemagglutinin fragment 2 (HA2), matrix protein (M) and nucleoprotein (NP). The CGE method allowed identification of different virus strains based on their specific protein profile. B/Brisbane inactivated virus and virosome samples could be analyzed within one day. The CGE results (titers) were comparable to single radial immune-diffusion (SRID), but the method has the advantage of a much faster time to results. CGE analysis of A/Christchurch from upstream process demonstrated the applicability of the method to samples of high complexity. The CGE method could be used in the same analyte concentration range as the RP-HPLC method, but showed better precision and accuracy. Overall, the total analysis time for the CGE method was much shorter, allowing analysis of 100 samples in 4 days instead of 10 days for SRID.
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•The total sample preparation was reduced from over 16 h to less than two hours.•Both the run time and the resolution were improved by a factor of two.•4 influenza proteins were quantified in influenza virus and virosome material.•The CGE method was validated and applied for influenza viruses and virosomes.
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