Mapping and partial characterization of proteases expressed by a CHO production cell line.
The proteolytic activities expressed by a Chinese hamster ovary (CHO) cell line in the cultivation supernatant during the production of recombinant factor VIII were mapped with a broad spectrum protease assay and a series of different types of protease inhibitors. The destabilizing effect on the product emanated from a metalloproteinase, which could be effectively blocked by chelating agents to lead to product stabilization. Amino acid sequences of the isolated metalloproteinase were found to have sequence homology with matrix metalloproteinases (MMPs) MMP3, MMP10, and MMP12. Several species with metalloproteinase activity were characterized and found to be related to each other. The results indicate that an MMP pro-enzyme of >/=200 kDa was released from the CHO cells during the production phase. The enzyme expressed collagenase/gelatinase activity when activated. Due to autoproteolysis, a number of smaller, less specific MMPs were formed with the smallest form, a 19.4 kDa protein, being the most active. These results may be of particular relevance for other production processes using CHO cells for the expression of recombinant proteins.
Sandberg
H
H
Department of Biopharmaceuticals, Biovitrum AB, SE-112 76 Stockholm, Sweden.
Lütkemeyer
D
D
Kuprin
S
S
Wrangel
M
M
Almstedt
A
A
Persson
P
P
Ek
V
V
Mikaelsson
M
M
D000818:Animals; D016466:CHO Cells / Q000201:enzymology; D002614:Chelating Agents / Q000494:pharmacology; D006224:Cricetinae; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D005169:Factor VIII / Q000096:biosynthesis*; D020782:Matrix Metalloproteinases / Q000302:isolation & purification / Q000378:metabolism*; D011480:Protease Inhibitors; D011994:Recombinant Proteins / Q000096:biosynthesis*
D002614:Chelating Agents; D016895:Culture Media, Serum-Free; D011480:Protease Inhibitors; D011994:Recombinant Proteins; C078147:F8 protein, human; D005169:Factor VIII; D020782:Matrix Metalloproteinases