cGMP Recombinant human FGF-basic (155 amino acid)
(Email John Keach for ordering information)
Synonyms: FGF-basic, bFGF, FGFb, hFGF-2
100 mg/vial, 0.1 mg/mL in aqueous buffer
The Waisman Clinical Biomanufacturing Facility (WCBF) is now offering human FGF2 produced under cGMP conditions for use in culturing stem cells for human clinical trials or in research applications that are anticipated to lead to clinical research.
Background
Basic fibroblast growth factor (FGF2) is a member of the fibroblast growth factor family. Proteins in this family bind heparin and show broad mitogenic and cell survival activities, and are involved in a variety of biological processes, including embryonic development, cell growth, angiogenesis and wound healing (Beenken and Mohammadi, 2009). In addition, FGF2 has proven useful in human ES cell culture applications where high levels of FGF2 (100 ng/mL) are commonly used in feeder-independent culture systems (Levenstein et al., 2006; Ludwig et al., 2006). FGF2 has also demonstrated utility in culturing other human stem cell types including neural, mesenchymal, and hematopoietic stem cells (Caldwell and Svendsen, 1998; Kashiwakura and Takahashi, 2004; Lee et al., 2009).
The Need for cGMP Reagents
Cell therapeutics that are manufactured for human clinical trials in the U.S. are regulated by current Good Tissue Practice (cGTP) guidelines as detailed in the code of federal regulations (21 CFR 1271). These guidelines include requirements for all materials that are used during the manufacturing process. In addition, the U.S. Pharmacopeia (USP <1043>) provides guidelines and production and testing standard for ancillary reagents such as media and growth factors. The availability of research-grade growth factors with identical cGMP-grade products will enable a smooth transition from research into human clinical trials.
Manufacturing Process
This protein has been produced under cGMP conditions and is appropriate for use in culturing stem cells for use in human clinical trials. The protein is expressed in E. coli using animal-free media components and process conditions that are suitable for a clinical-grade product. The purification process consists of two chromatography steps and diafiltration into phosphate buffered saline resulting in levels of host impurities that are suitable for producing human cell therapeutics for clinical applications. In addition, this protein has been used in producing the first publicly available cGMP embryonic stem cell bank.
Quality Control Testing
The final rhFGF2 undergoes extensive testing to demonstrate that it meets strict requirements for identity, purity, and potency. In addition to standard assays to monitor the overall purity of the product, two cell culture potency assays are performed on each lot: MTT and Human ES Cell Proliferation Assay. The Human ES Cell Proliferation Assay consists of culturing human ES cells (WA09) for five passages using feeder-independent culture conditions (TeSR media, 100 ng/mL rhFGF2). At the end of five passages, the human ES cells are assessed for proliferation, colony morphology, and differentiation based on flow cytometry analysis of human ES cell marker expression (> 85% Oct4/SSEA4 double positive).
Release testing specifications for the rhFGF2 are summarized in the table below.
| Test | Specification |
| Purity (by HPLC) | Total Purity ≥ 90% Monomer Purity ≥ 75% |
| Protein Concentration by UV | 0.10 – 0.13 mg/mL |
| Fill Volume | 1.1 +/- 0.1 mL |
| SDS-PAGE | Compares favorably to standard |
| Sterility | No Growth |
| Endotoxin | < 0.1EU/µg |
| Activity (MTT assay) | EC50 < 2.0 ng/mL |
| Human ES Cell Proliferation Assay | Supports growth of undifferentiated human ES cells |
Reference List
- Beenken,A. and Mohammadi,M. (2009). The FGF family: biology, pathophysiology and therapy. Nature Reviews Drug Discovery 8, 235-253.
- Caldwell,M.A. and Svendsen,C.N. (1998). Heparin, but not other proteoglycans potentiates the mitogenic effects of FGF-2 on mesencephalic precursor cells. Experimental Neurology 152, 1-10.
- Kashiwakura,I. and Takahashi,T.A. (2004). Fibroblast growth factor and ex vivo expansion of hematopoietic progenitor cells. Leukemia & Lymphoma 46, 329-333.
- Lee,S.Y., Lim,J., Khang,G., Son,Y., Choung,P.H., Kang,S.S., Chun,S.Y., Shin,H.I., Kim,S.Y., and Park,E.K. (2009). Enhanced Ex Vivo Expansion of Human Adipose Tissue-Derived Mesenchymal Stromal Cells by Fibroblast Growth Factor-2 and Dexamethasone. Tissue Engineering Part A 15, 2491-2499.
- Levenstein,M.E., Ludwig,T.E., Xu,R.H., Llanas,R.A., VanDenHeuvel-Kramer,K., Manning,D., and Thomson,J.A. (2006). Basic fibroblast growth factor support of human embryonic stem cell self-renewal. Stem Cells 24, 568-574.
- Ludwig,T.E., Levenstein,M.E., Jones,J.M., Berggren,W.T., Mitchen,E.R., Frane,J.L., Crandall,L.J., Daigh,C.A., Conard,K.R., Piekarczyk,M.S., Llanas,R.A., and Thomson,J.A. (2006). Derivation of human embryonic stem cells in defined conditions. Nature Biotechnology 24, 185-187.