Method for Large Scale Production and Purification of Parvovirus

Content Provider	The Lens
Abstract	Described is a reproducible, effective and scalable process for parvovirus production including characterization strategies, preferably production of H-1PV.
Related Links	https://www.lens.org/lens/patent/011-870-236-492-696/frontpage
Language	English
Publisher Date	2019-04-23
Access Restriction	Open
Content Type	Text
Resource Type	Patent
Jurisdiction	United States of America
Date Applied	2016-06-22
Agent	Leason Ellis Llp
Applicant	Deutsches Krebsforsch
Application No.	201615739684
Claim	A method for producing empty inactive or full active parvovirus particles, wherein the parvovirus is H-1PV, said method comprising: (a) providing the producer cell line NB-324K; (b) growing the cell line under suitable conditions and infecting the cells at a cell density from 2.0 to 5.0×10 4 cells/cm 2 with the parvovirus at a MOI of 0.5 to 2×10 2 PFU/cells; (c) harvesting the cells 2 to 6 days post-infection and obtaining a cell pellet by centrifugation; (d) subjecting the resuspended cell pellet to a mechanical, physical or chemical cell lysis method for obtaining a parvovirus containing cell lysate; (e) sonicating the cell lysate and subjecting it to DNAse treatment; (f) clarifying the DNAse-treated parvovirus harvest by filtration; and (g1) purifying the parvovirus by two successive density gradient ultracentrifugations, wherein the first gradient is a Iodixanol/PBS step gradient and the second gradient is a Iodixanol/Ringer step gradient or a Iodixanol/Ringer continuous gradient for obtaining full active parvovirus particles in one fraction and empty parvovirus particles in another fraction. The method of claim 1 , wherein the cell density of step (b) is from 3.0 to 4.0×10 4 cells/cm 2 . The method of claim 1 , wherein for step (f) a 0.2-μm filter with prefilter is used. The method of claim 1 , wherein the producer cell line NB-324K is characterized by (a) a viability of at least 95%; (b) a passage number below 20; and/or (c) lack of mycoplasma contamination. The method of claim 1 , wherein virus production is performed in a collection system. The method of claim 5 , wherein the collection system is a 10-layer cell culture chamber. The method of claim 1 further comprising determining the ratio of native parvovirus capsids to non-assembled capsid proteins or denatured capsids. The method of claim 7 , wherein the ratio is determined by using a monoclonal antibody. The method of claim 8 , wherein the monoclonal antibody is the antibody BL-H1 (DSM ACC 3030).
CPC Classification	MICROORGANISMS OR ENZYMES;COMPOSITIONS THEREOF;PROPAGATING; PRESERVING; OR MAINTAINING MICROORGANISMS;MUTATION OR GENETIC ENGINEERING;CULTURE MEDIA PEPTIDES Investigating Or Analysing Materials By Determining Their Chemical Or Physical Properties
Examiner	Michelle S Horning
Extended Family	090-612-564-627-936 146-044-548-345-098 036-857-322-464-561 117-247-071-353-952 004-459-184-545-499 186-333-644-187-351 148-883-976-237-907 144-874-934-365-767 029-857-128-340-529 096-510-366-158-930 085-998-497-978-712 170-775-552-937-271 011-870-236-492-696
Patent ID	10266810
Inventor/Author	Leuchs Barbara Roscher Mandy Müller Marcus Rommelaere Jean
IPC	C12N7/00 C07K16/08 C12N7/02 G01N33/569
Status	Active
Simple Family	090-612-564-627-936 146-044-548-345-098 036-857-322-464-561 117-247-071-353-952 004-459-184-545-499 186-333-644-187-351 148-883-976-237-907 144-874-934-365-767 029-857-128-340-529 096-510-366-158-930 085-998-497-978-712 170-775-552-937-271 011-870-236-492-696
CPC (with Group)	C12N7/00 C12N2750/14323 C12N2750/14351 C12N2750/14361 C12N7/02 C07K16/081 G01N33/56983
Issuing Authority	United States Patent and Trademark Office (USPTO)
Kind	Patent/New European patent specification (amended specification after opposition procedure)