Method for further upscaling the large-scale production of the oncolytic h-1 protoparvovirus (h-1pv) using a carrier-based production process combined with an optimized cell culture medium

A method for producing parvovirus H-1 (H-1PV), said method comprising:

• (a) providing the producer cell line NB-324K;
• (b) producing the seed train by growing the NB-324K cells under suitable conditions;
• (c) providing a culture vessel containing animal component-free cell culture medium supplemented with 2% animal serum and microcarrier or macrocarrier at the seeding time point;
• (d) seeding the NB-324K cells of step (b) in the culture vessel of step (c);
• (e) performing cell expansion of the NB-324K cells;
• (f) performing 100% medium exchange at the infection time point with animal component-free cell culture medium including microcarrier or macrocarrier, wherein the animal component-free cell culture medium is supplemented with 1% animal serum or without animal serum;
• (g) harvesting the NB-324K cells 3 to 8 days post-infection with lysis buffer containing 1-100 mM Tris, 1-10 mM MgCl2, 2.5-10% TrypLE™, pH 9-10 with or without 0.1-1% Tween® 80, and agitation of the lysis buffer, followed by wash with wash buffer containing 1-100 mM Tris, 1-10 mM MgCl2, pH 9-10, wherein the lysis and wash buffer are pooled together;
• (h) clarifying the parvovirus harvest by filtration;
• (i) eliminating non-encapsidated viral DNA and contaminating host cell DNA by DNAse treatment;
• (j) performing tangential flow filtration for buffer exchange and concentration;
• (k) performing anion exchange chromatography to eliminate empty particles and most impurities;
• (l) performing tangential flow filtration for buffer exchange to 0-3% Visipaque and 97-100% Ringer solution and concentration;
• (m) obtaining a final formulation in 48% Visipaque/Ringer solution.