Purification and characterization of a new recombinant factor VIII (N8).
A new recombinant factor VIII (FVIII), N8, has been produced in Chinese hamster ovary (CHO) cells. The molecule consists of a heavy chain of 88 kDa including a 21 amino acid residue truncated B-domain and a light chain of 79 kDa. The two chains are held together by non-covalent interactions. The four-step purification includes capture, affinity purification using a monoclonal recombinant antibody, anion exchange chromatography and gel filtration. The specific clotting activity of N8 was 8800-9800 IU mg(-1). Sequence and mass spectrometry analysis revealed two variants of the light chain, corresponding to two alternative N-terminal sequences also known from plasma FVIII. Two variants of the heavy chain are present in the purified product, namely with and without the B-domain linker attached. This linker is removed upon thrombin activation of N8 rendering an activated FVIII (FVIIIa) molecule similar to plasma FVIIIa. All six known tyrosine sulphations of FVIII were confirmed in N8. Two N-linked glycosylations are present in the A3 and C1 domain of the light chain and two in the A1 domain of the heavy chain. The majority of the N-linked glycans are sialylated bi-antennary structures. An O-glycosylation site is present in the B-domain linker region. This site was glycosylated with a doubly sialylated GalNAc-Gal structure in approximately 65% of the product. In conclusion, the present data show that N8 is a pure and well-characterized FVIII product with biochemical properties that equal other FVIII products.
Thim
L
L
Biopharmaceutical Research Unit, Novo Nordisk A/S, Maaloev, Denmark. lth@novonordisk.com
Vandahl
B
B
Karlsson
J
J
Klausen
N K
NK
Pedersen
J
J
Krogh
T N
TN
Kjalke
M
M
Petersen
J M
JM
Johnsen
L B
LB
Bolt
G
G
Nørby
P L
PL
Steenstrup
T D
TD
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D005169:Factor VIII / Q000737:chemistry* / Q000302:isolation & purification; D006023:Glycoproteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
D006023:Glycoproteins; D011994:Recombinant Proteins; D005169:Factor VIII; D014357:Trypsin