|
|
| ■ Hematology & Endocrinology |
|
Recombinant Factor IX
Hemophilia is a bleeding disorder caused by a deficiency in one of the blood clotting
factors. Hemophilia A is a congenital deficiency in clotting factor VIII and accounts for about 80% of all
hemophilia cases. Hemophilia B is also an inherited bleeding disorder caused by a deficiency in factor IX. The
bleeding typically occurs in several sites, such as muscle, joint, brain and intestine. Infusing the missing
clotting factors has been known to be one of the best ways to treat hemophilia, at present there are only two
available sources for factor IX: highly purified plasma-derived factor IX (pdFIX) and recombinant factor
IX (rFIX), produced by CHO cells.
 Factor IX is a
complex protein with several posttranslational modifications required for biological activity and
pharmacokinetics, which include propeptide removal, vitamin K-dependent-carboxylation, Ser-phosphorylation,
Tyr-sulfation and glycosylation. Because of the complicated modifications required, however, only a small
portion of rFIX produced in CHO cells is in fact biologically active. Hence, mass production of fully active
rFIX is desirable. In this regard, we have been investigating the production of high quality rFIX from CHO cell
using our proprietary expression system. At present, we are developing the production process of rFIX in CHO
cells using gene amplification, which includes the development of a production cell line, animal source-free
media for CHO cells, a serum-free suspension cell culture process bioreactor, the purification process of rFIX
and formulation.
 To express rFIX,
our proprietary CHO cell expression vector, pMSG, containing MAR elements and new termination codon, was used as
an expression vector backbone. FIX cDNA with or without a partially truncated intron 1 of FIX was then inserted
into pMSG. Intron 1 of FIX was found to improve the rFIX secretion, during stable expression using CHO cells
under G418 selection, by about 10 fold.
Recombinant CHO-rFIX cell lines were developed using a gene amplification system. Currently, studies on process
development are under investigation for the production of rFIX from suspension cultures using serum free- and
animal protein-free media at the bioreactor level, in addition to preclinical studies.
Publications
Kim JM, Kim JS, Park DH, Kang HS, Yoon J, Baek K, Yoon Y. (2004) Improved recombinant gene expression in CHO
cells using matrix attachment regions. Journal of Biotechnology. 107:95-105
[PubMed Citation]
Kim D, Kim JD, Baek K, Yoon Y, Yoon J. (2003) Improved mammalian expression systems by manipulating
transcriptional termination regions. Biotechnol Prog. 19:1620-2
[PubMed Citation]
|
|
|
