Analysis of AdV5 samples by transmission electron microscopy (TEM) was conducted in collaboration with Vironova AB using the MiniTEM™ system. Host cell protein (HCP) was determined using an ELISA assay total protein, using a BCA assay kit host cell DNA (hcDNA), by qPCR and total DNA using Quant-iT™ PicoGreen™ dsDNA Reagent (Invitrogen).
The Biacore™ T200 system was used for determination of virus particles through binding of virus fiber or hexon protein to CAR or FX protein, respectively, immobilized on a Biacore Sensor Chip CM5. Total virus titer was determined by hexon DNA qPCR and size exclusion chromatography (SEC)-HPLC using a Superose™ 6 Increase 10/300 GL column. Infectious virus titer was determined using the 50% tissue culture infective dose (TCID 50) assay and by automated fluorescence microscopy (AFM) using the IN Cell Analyzer.
Process aliquots of the final purified bulk were sterile filtered using a syringe filter (polyethersulfone, 0.2 μm). Sample was concentrated and buffer exchanged (5 × UF/ 5 × DF) into 20 mM Tris, pH 8 + 25 mM NaCl, 2 mM MgCl 2, 2.5% glycerol. Polishing was performed using Sepharose 4 Fast Flow resin (HiScale 50 column, 382 mL ). Elution was performed in two steps using 20 mM sodium phosphate, pH 7.3 + 2 mM MgCl 2 + 2% sucrose with 500 mM + 750 mM NaCl. Polishing was conducted on Capto Core 700 resin (HiScale 16, 10 mL and 29 mL ).įor capture, Q Sepharose™ XL resin (HiScale 50 column, 249 mL ) was used. Elution was conducted with 20 mM Tris, pH 8.0 + 2 mM MgCl 2 using a linear gradient of 480–570 mM NaCl. 5–7įor capture, Capto™ Q ImpRes resin (HiScale™ 26 column, 88 mL or HiScale 50 column, 294 mL ) was used. 4Ĭolumns were operated on an ÄKTA™ pure 150 system. Concentration and buffer exchange were performed by tangential flow filtration (TFF) on a ReadyToProcess™ hollow-fiber filter with a nominal molecular weight cutoff (NMWC) of M r 300,000 (10 × ultrafiltration /5 × diafiltration into 20 mM Tris, pH 8.0 + 300 mM NaCl, 2 mM MgCl 2).
The harvest was thereafter clarified by normal flow filtration (NFF) using a combination of 2 μm and 0.6 μm ULTA GF filters. Based on analytical data, the novel downstream process was compared with a reference process regarding virus load capacity, recovery, and purity ( Figure 1).įorty-two hours post infection, the cells were lysed with 0.5% Tween 20 and treated with 20 U/mL Benzonase™ + 1 mM MgCl2 for 4 hr in the bioreactor. We used traditional analytics but also developed new sensitive and reproducible assays for virus titer. The cells were lysed using Tween™ 20 as an alternative to Triton™ X-100 that is now on the authorization list (Annex XIV) of Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH).Īnalytical methods for determination of virus titer are challenging and depend on purity and quality of the sample. Human AdV5, expressing the green fluorescent protein (GFP), was used for process development. In this study, we have combined technical evaluation of process steps and process economy calculations, from AdV production in cell culture to purified bulk product in up to 10 L scale 1-8. Process outline for the novel and reference processes for adenovirus productionĪdenovirus (AdV) vectors are commonly used in cancer gene therapy trials, evaluated in gene therapy, and used in vaccines for various diseases.
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