Comparative evaluation of integrated purification pathways for bacterial modular polyomavirus major capsid protein VP1 to produce virus-like particles using high throughput process technologies

An integrated and continuous downstream process for microbial virus‐like particle vaccine biomanufacture

“…For the PCC step in bind‐elute mode, out of 31.0 ± 0.62 mg VP1 applied on Capto™ MMC, 27.6 ± 1.38 mg (89.0%) was recovered in the elution pool and approximately 0.73 ± 0.12 mg (2.4%) remained in the flow through. The product loss in the flow‐through probably corresponds to aggregated VP1‐J8 capsomeres, as we have recently shown that VP1‐J8 aggregates do not bind to Capto™ MMC and remain in the flow‐through (Gerstweiler et al, 2021c). The remaining 8.4% is either strongly bound to the matrix or noneluted with selected conditions or stripped in the washing step with 1 M NaOH.…”

“…Murine polyomavirus major capsid protein VP1 with inserted Group A Streptococcus antigen J8 was constructed and expressed as described in our earlier paper (Gerstweiler et al, 2021c). In brief, GCN4‐J8 was inserted with flanking G4S linkers into VP1 and cloned into pETDuet‐1 and transformed by heat shock transformation into Rosetta™ 2(DE3) Singles™ competent cells (Merck KGaA) and stored as 25% glycerol stocks.…”

“…RP‐HPLC was used to determine VP1‐J8 concentration as previously described (Gerstweiler et al, 2021c). In brief samples were combined 1:4 with a denaturing buffer (8 M guanidine [GE1914, ChemSupply], 50 mM DTT, 20 mM Tris pH 8) and heated for 10 min at 75°C.…”

“…A gradient elution with water, containing 0.5% TFA (Buffer A) (TS181, ChemSupply), and acetonitrile (LC1005, ChemSupply), containing 0.4% TFA (Buffer B), was used to separate the sample (3 µl) on a Vydac Protein C4 column 2.1 × 100 mm, 5 µm (214TP521), at a flow of 1 ml min −1 and 60°C column temperature. The elution program was as following: 6 min gradient from 35% B to 60% B, 30 s gradient from 60% B to 100% B, 1 min 100% B, 30 s from 100% B to 35% B, and 4 min of 35% B (Gerstweiler et al, 2021c). A Shimadzu UFLC‐XR system (pump: LC‐20AD‐XR, autosampler: SIL‐20AXR, diode array detector: SPD‐M20A, column oven: CTO‐20) with detection at 280 nm was used for HPLC experiments.…”

“…To further extend the field of continuous bio‐processing we developed and here report an automated continuous and integrated purification process for microbially‐expressed VLP vaccines based on an integrated production pathway developed by our group (Gerstweiler et al, 2021c). The process couples a flow through Capto™ Q chromatography step followed by a bind‐elute multimodal (Capto™ MMC) PCC process with subsequent in‐line assembly of VLPs.…”