Fast, selective and quantitative protein profiling of adenovirus-vector based vaccines by ultra-performance liquid chromatography

Abstract

a b s t r a c tA 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 m, 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 adenovirusvector based vaccine and was successfully implemented for the characterization of adenovirus-based products.

Characterization of Recombinant Chimpanzee Adenovirus C68 Low and High-Density Particles: Impact on Determination of Viral Particle Titer

“…The majority of the viral particle content tools presented in this manuscript harken back to the past 50 or so years of adenovirus characterization. From the early days of density gradient ultracentrifugation to separate differentially packaged capsids followed by SDS-PAGE to examine the protein content ( Prage et al, 1972 ; Burlingham et al, 1974 ; Daniell, 1976 ; Tibbetts and Giam, 1979 ; Tóth et al, 1982 ), to substitution of SDS-PAGE with RP-HPLC and mass spectrometry ( Lehmberg et al, 1999 ; Vellekamp et al, 2001 ; Chelius et al, 2002 ; Takahashi et al, 2006 ; Benevento et al, 2014 ; Perez-Berna et al, 2014 ; van Tricht et al, 2018 ) and rounding out with direct content analysis via AUC and DCS ( Bondoc and Fitzpatrick, 1998 ; Berkowitz and Philo, 2007 ; Berkowitz, 2008 ; Yang et al, 2008 ; Shih et al, 2010 ). The outlier is the introduction of direct measurement of adenoviral mass via CDMS, something that would have been thought almost impossible just a few years ago.…”

Sixteen capillary electrophoresis applications for viral vaccine analysis

“…Peaks were identified with fraction collection and peptide mapping procedure with trypsin digestion and LC-MS E analysis. See for more details van Tricht et al [53].…”

“…Double-stranded DNA is encapsidated by proteins and about 13 different types of proteins are present in the virion [91]. The individual proteins can be separated by UPLC [53]. The adenovirus vector vaccines are produced in bioreactors with cultured human cells by adenovirus vector seed inoculation (e.g., HEK293, Per.C6®) [19,[92][93][94][95][96][97][98][99][100][101][102][103].…”

“…Thermal stress (incubation at 50°C for 120 min or at 70°C for 45 min) and oxidation stress (incubation with 0.08% v:v H 2 O 2 for 24 h or 0.40% v:v H 2 O 2 for 17 h) were applied in this study example. The samples were analyzed with CZE [19], RP-HPLC [53], DLS, and ELS.…”

Identification of Recombinant Chimpanzee Adenovirus C68 Degradation Products Detected by AEX-HPLC

“…Therefore, it is desirable to have higher throughput physicochemical techniques as a part of the analytical control strategy that give an analogous assessment of product quality but can be run routinely during process development, formulation development, and on fully purified products. For adenoviruses, these options vary but include many well-known techniques such as AEX-HPLC ( Blanche et al, 2000 ; Klyushnichenko et al, 2001 ; Kuhn et al, 2007 ; Whitfield et al, 2009 ; Kramberger et al, 2015 ), RP-HPLC ( Lehmberg et al, 1999 ; Blanche et al, 2001 ; Vellekamp et al, 2001 ; Chelius et al, 2002 ; Takahashi et al, 2006 ; Benevento et al, 2014 ; Pérez-Berná et al, 2014 ; van Tricht et al, 2018 ), capillary electrophoresis (CE) ( van Tricht et al, 2017 ), analytical ultracentrifugation (AUC) ( Berkowitz and Philo, 2007 ; Berkowitz, 2008 ; Yang et al, 2008 ) and differential centrifugal sedimentation (DCS) ( Bondoc and Fitzpatrick, 1998 ; Shih et al, 2010 ). AEX-HPLC is the most widely applied of these for in-process, release and stability testing, as AEX-HPLC can simultaneously determine viral particle concentration, product purity and surface charge.…”