Biochemical analysis of retroviral structural proteins to identify and quantify retrovirus expressed by an NS0 murine myeloma cell line.
A subclone of the NS0 murine myeloma cell line, frequently used to produce recombinant monoclonal antibodies, was found by a transmission electron microscopy method to express a surprisingly high titer of 10(11) retroviral particles per ml of culture supernatant. Infectivity assays showed a very low infectious titer with the restricted host range expected for a murine amphotropic retrovirus. A Western blot assay for the viral capsid protein was developed to confirm the high titer values and provide a means for monitoring batch consistency and virus removal during the purification process. Mass spectrometry of several of the viral Gag proteins demonstrated that the cell line appeared to produce at least two closely related retroviruses. N-terminal sequencing of three of the Gag proteins demonstrated that these retroviruses were members of the murine leukemia retroviral family. Western blot detection with an antibody for the capsid protein gave a linear standard curve over the range of 0.1-3 ng per lane. This allows the detection of viral titers as low as 6x10(7) virions per ml without the need to concentrate the sample. The Western blot method has higher throughput and less variability than transmission electron microscopy methods and has potential for monitoring viral titer and clearance during development of manufacturing processes.
Taylor
F R
FR
Biogen Inc., 14 Cambridge Center, Cambridge, MA 02142, USA. fred_taylor@biogen.com
Ferrant
J L
JL
Foley
S F
SF
Zeng
C
C
Sernatinger
J
J
Juffras
R
R
Pepinsky
R B
RB
D000595:Amino Acid Sequence; D000818:Animals; D019149:Bioreactors; D015153:Blotting, Western; D002213:Capsid / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D015683:Gene Products, gag / Q000032:analysis*; D009052:Leukemia Virus, Murine / Q000737:chemistry* / Q000302:isolation & purification*; D007942:Leukemia, Experimental; D013058:Mass Spectrometry; D051379:Mice; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009101:Multiple Myeloma; D012192:Retroviridae Infections; D012191:Retroviridae Proteins / Q000032:analysis*; D014407:Tumor Cells, Cultured / Q000648:ultrastructure / Q000821:virology; D014412:Tumor Virus Infections; D019562:Viral Load
D015683:Gene Products, gag; D012191:Retroviridae Proteins