High-yield preparation of recombinant human α-thrombin for therapeutic use.
In this study, we established stable cell lines producing 1.5 mg/mL recombinant human prothrombin in 400-L fed-batch culture, using CHO DG44 cells as a host cell line. And we also established a recombinant human α-thrombin purification process that produces a purified product suitable for use as a biopharmaceutical, by using recombinant ecarin from CHO DG44 cells, achieving a total yield of approximately 27% of prothrombin in culture medium. The establishment of stable cell lines with high expression levels, long-term passage stability and satisfactory scale-up are essential to ensure the stable supply of biopharmaceuticals. Furthermore, biopharmaceuticals must be of high quality to assure safety and effectiveness in target applications. We had previously reported that recombinant human prethrombin-2 expression level in a stable cell line established using the mouse myeloma cells, Sp2/0-Ag14, reached 200 μg/mL using animal-free materials in 50-L fed-batch culture. However, the productivity was insufficient to completely replace α-thrombin in human plasma preparations. By employing CHO DG44 cells as a host cell line, we had established a stable cell line and achieved significant improvements in quality, productivity of recombinant human α-thrombin manufacture suitable for use as a biopharmaceutical.
R&D Division, The Chemo-Sero-Therapeutic Research Institute, KAKETSUKEN, 1314-1 Kyokushi-Kawabe, Kikuchi, Kumamoto 869-1298, Japan.
Akihiro
A
Meta
R&D Division, The Chemo-Sero-Therapeutic Research Institute, KAKETSUKEN, 1314-1 Kyokushi-Kawabe, Kikuchi, Kumamoto 869-1298, Japan.
Masaki
M
Hirashima
R&D Division, The Chemo-Sero-Therapeutic Research Institute, KAKETSUKEN, 1314-1 Kyokushi-Kawabe, Kikuchi, Kumamoto 869-1298, Japan.
Takayuki
T
Imamura
R&D Division, The Chemo-Sero-Therapeutic Research Institute, KAKETSUKEN, 1314-1 Kyokushi-Kawabe, Kikuchi, Kumamoto 869-1298, Japan.
Ryoichi
R
Kawamura
Blood Plasma Division, The Chemo-Sero-Therapeutic Research Institute, KAKETSUKEN, 1314-1 Kyokushi-Kawabe, Kikuchi, Kumamoto 869-1298, Japan.
Kentaro
K
Yano
R&D Division, The Chemo-Sero-Therapeutic Research Institute, KAKETSUKEN, 1314-1 Kyokushi-Kawabe, Kikuchi, Kumamoto 869-1298, Japan.
Kenji
K
Uehara
R&D Division, The Chemo-Sero-Therapeutic Research Institute, KAKETSUKEN, 1314-1 Kyokushi-Kawabe, Kikuchi, Kumamoto 869-1298, Japan. Electronic address: nakashima-to@kaketsuken.or.jp.
Toshihiro
T
Nakashima
D000818:Animals; D061249:Batch Cell Culture Techniques; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media / Q000737:chemistry; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011516:Prothrombin / Q000032:analysis / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000627:therapeutic use; D013917:Thrombin / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000627:therapeutic use
D003470:Culture Media; D011994:Recombinant Proteins; D011516:Prothrombin; D010450:Endopeptidases; D013917:Thrombin; C020097:ecarin; C513462:recombinant human thrombin
Biopharmaceuticals; Ecarin; High-yield preparation; Human α-thrombin; Recombinant; Therapeutic use