Bioprocess R&D, Merck Research Laboratories, PO Box 2000, 07065, Rahway, NJ, USA,
Cytotechnology. 2005 Jun;48(1-3):59-74. doi: 10.1007/s10616-005-3563-z.
There has been a recent boom of monoclonal antibodies on the market, and a significant portion of them were produced by NS0 cell lines. As regulations become more stringent in ensuring production processes are free of potential contamination by adventitious agents, it is highly desirable to further develop serum-free media into ones that do not contain any components of animal origin, or 'animal-free media'. Using a shake-flask batch culture system, recombinant proteins (human albumin and human insulin) and synthetic compounds (tropolone and ferric ammonium citrate) were identified to be capable of replacing the animal-sourced proteins commonly found in serum-free media for NS0 cell culture, namely bovine albumin, insulin and transferrin. The cholesterol requirement of NS0 cells was satisfied by the use of a commercially available non-proteinaceous, non-animal sourced cholesterol/fatty acid mix in place of bovine lipoproteins, which in effect also eliminated the need for recombinant albumin. In the animal-free medium thus formulated, NS0 cell lines, either the host or recombinant constructs, were all able to grow in batch culture to 1~ 3x10(6) viable cells/ml for multiple passages, with no requirement for gradual adaptation even when seeded from 10% serum-containing cultures. It was surprising to observe that the recombinant insulin was essentially ineffective as sodium salt compared to its zinc salt. Studies showed that the zinc deficiency in the former resulted in a rapid decline of cell viabilities. Supplementation of zinc ions greatly improved growth, and even led to the total replacement of recombinant insulin and hence the formulation of a protein-free medium. When the cell lines were adapted to cholesterol-independent growth which eliminated the need for any lipid source, a completely chemically-defined animal-free medium was formulated. In all cases, antibody production by various GS-NS0 constructs in animal-free media was stable for multiple passages and at least similar to the original serum-free medium containing the animal-sourced proteins. The medium also served well for cryopreservation of NS0 cells in the absence of serum.
最近市场上单抗药物蓬勃发展，其中很大一部分是由 NS0 细胞系生产的。随着法规越来越严格，确保生产过程不受潜在外来污染物的污染，进一步开发无动物源成分的无血清培养基，即“无动物源培养基”，变得非常重要。使用摇瓶分批培养系统，鉴定出重组蛋白（人白蛋白和人胰岛素）和合成化合物（三酮和柠檬酸铁铵）能够替代无血清培养基中常见的动物源蛋白，即牛白蛋白、胰岛素和转铁蛋白。通过使用市售的非蛋白、非动物源胆固醇/脂肪酸混合物代替牛脂蛋白，满足了 NS0 细胞的胆固醇需求，实际上也消除了对重组白蛋白的需求。在如此配制的无动物源培养基中，无论是宿主还是重组构建体的 NS0 细胞系都能够在分批培养中生长到 1~3×10(6)个活细胞/ml 进行多次传代，甚至不需要逐渐适应，即使从含 10%血清的培养物中接种也不需要。令人惊讶的是，与锌盐相比，重组胰岛素基本上作为钠盐无效。研究表明，前者中的锌缺乏导致细胞活力迅速下降。补充锌离子大大改善了生长，甚至可以完全替代重组胰岛素，从而形成无蛋白培养基。当细胞系适应不需要任何脂质来源的胆固醇非依赖性生长时，就可以配制完全化学定义的无动物源培养基。在所有情况下，各种 GS-NS0 构建体在无动物源培养基中的抗体产生在多次传代中都很稳定，至少与含有动物源蛋白的原始无血清培养基相似。该培养基也非常适合在没有血清的情况下冷冻保存 NS0 细胞。
