Comparison of two types of cell cultures for preparation of sTNFRII-gAD fusion protein.
- Author:
Shigao HUANG
1
;
Yuting YIN
;
Chunhui XIONG
;
Caihong WANG
;
Jianxin LÜ
;
Jimin GAO
Author Information
1. Zhejiang Provincial Key Laboratory for Technology & Application of Model Organisms, Wenzhou Medical College, Wenzhou 325035, Zhejiang, China.
- Publication Type:Journal Article
- MeSH:
Adiponectin;
biosynthesis;
genetics;
Animals;
Bioreactors;
CHO Cells;
Cell Culture Techniques;
methods;
Cricetinae;
Cricetulus;
Receptors, Tumor Necrosis Factor, Type II;
biosynthesis;
genetics;
Recombinant Fusion Proteins;
biosynthesis;
genetics
- From:
Chinese Journal of Biotechnology
2013;29(1):115-118
- CountryChina
- Language:Chinese
-
Abstract:
In this study we used two types of cell cultures, i.e., anchorage-dependent basket and full suspension batch cultures of sTNFRII-gAD-expressing CHO cells in the CelliGen 310 bioreactor (7.5 L) to compare their yields in order to optimize the culturing conditions for efficient expression of sTNFRII-gAD fusion protein consisting of soluble tumor necrosis factor receptor II and globular domain of adiponectin. The anchorage-dependent basket culture was performed in 4L 10% serum-containing medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 3 days, and then switched to 4 L serum-free LK021 medium to continue the culture for 4 days. The full suspension batch culture was carried out in the 4 L serum-free LK021 medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 7 days. The culturing conditions were monitored in real-time to maintain pH and dissolved oxygen stability through the whole process. The supernatants were collected by centrifuge, and the protein was concentrated through Pellicon flow ultrafiltration system and then purified by DEAE anion exchange. The results showed that the yields of sTNFRII-gAD fusion protein were 8.0 mg/L with 95% purity and 7.5 mg/L with 98% purity in the anchorage-dependent basket and the full suspension batch cultures, respectively. The study provided the framework for the pilot production of sTNFRII-gAD fusion protein.
