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CHO-K1稳定细胞株构建 CHO-K1稳定细胞株构建
细胞株

CHO-K1稳定细胞株构建

  • 高表达量
  • 经验丰富
  • CHO-K1细胞株亚授权

服务介绍

细胞株构建在抗体药物开发过程中起着至关重要的作用。细胞株的高效产能，可以为工业生产重组蛋白和抗体节省宝贵的时间并降低总体成本。

百英可以提供直接用于CMC大规模生产的稳定细胞株开发服务，我们的CHO K1细胞株来源清晰，表达量高，来源ECACC，可以全球亚授权。

CHO-K1稳定细胞株构建 Overview

服务亮点

高表达量

  • 抗体表达量>5g/L
  • 工艺完善
  • 细胞池周期2-2.5个月，单克隆4个月

经验丰富

  • 10年以上细胞株开发经验
  • 上百个细胞株项目开发
  • 根据需求，量身定做构建方案

CHO K1细胞株亚授权

  • 商业化亚授权
  • 根据项目授权
  • 一次性授权
立即咨询

CHO-K1稳定细胞株构建
服务详情

服务步骤 服务详情 周期 交付物
基因合成、亚克隆及表达纯化
  • 密码子优化和基因合成
  • 亚克隆到表达质粒
  • 质粒验证、大量抽提及表达纯化
 2-3周
  • 分子克隆及表达载体报告
  • 表达载体构建报告
  • 表达载体测序报告
细胞池构建
  • 稳定转染
  • 细胞池筛选
 6-7周
  • 转染载体Minipool报告
  • 亚克隆筛选报告
单克隆筛选
  • 亚克隆
  • 单克隆筛选
 6-7周
Primary Cell Bank收集
  • Primary Cell Bank (PCB) 准备
  • PCB稳定性鉴定
 12周
  • 候选克隆稳定性传代培养报告
  • 稳定细胞株测序报告
  • 支原体检测报告
  • 关键材料COA（细胞株构建过程中用到的试剂耗材的COA）
CHO-K1细胞亚授权
  • CHO-K1细胞株报告
  • CHO-K1细胞亚授权协议
 3-5天
  • CHO-K1细胞株文件和检测报告
  • CHO-K1细胞亚授权协议

案例分享

案例1: Tag free vaccine protein

Single cell image system is applied to confirm the monoclonality. The yield of the final obtained clone A is 2.31 g/L.

Day 0
Day 0
Day 1
Day 1
Day 2
Day 2
Day 4
Day 4
Day 7
Day 7
Day 10
Day 10
Finally

The stability of clone A is also tested by assessing the doubling time of the cells, cell density and yield.

Stability Studies
Subcultured in CD04 medium containing 25 uM MSX the doubling time:22 ± 1hours (Average: 22.6h)
Feed batch
Cells from passages 3, 8, 13, 18, and 23 were collected, and the cell density was assessed. The results indicated a consistent level of cell growth.
Doubling Time of N108-54
Cells from passages 3, 8, 13, 18, and 23 were collected, and the yield was assessed. The results indicated that over 90% productivity titer was retained for over 22 passages.

案例2: Anti-PD-1

Three single clones were selected, and their yield was assessed using three different commercial media. The results are as follows:

Subclone Medium A (g/L) Medium B (g/L) Medium C (g/L)
414203-N42-6-N1 3.19 5.5 2.63
414203-N42-7-N1 6.48 2.78 1.9
414209-N10-6-N1 7.82 9.02 7.72

Both reducing and non-reducing CE-SDS analyses were performed to assess the purity of these three clones after one step of affinity purification. The results indicated >97% purity.

R-CE-SDS
Sample Name main peak % LMW % Total %
Positive control (Pembrolizumab) 99.1 0.9 100.0
B414203-N42-6-N1 98.8 1.3 100.0
B414203-N42-7-N1 98.5 1.5 100.0
B414209-N10-6-N1 97.8 2.2 100.0
NR-CE-SDS
Sample Name main peak % LMW % HMW % Total %
Positive control (Pembrolizumab) 98.3 1.7 0.0 100.0
B414203-N42-6-N1 94.6 5.4 0.0 100.0
B414203-N42-7-N1 94.1 5.9 0.0 100.0
B414209-N10-6-N1 93.3 6.4 0.3 100.0

CEX-HPLC analyses were performed to assess the charge of these three clones. The results indicated a similar main component% compared to the positive control for the first two clones. Additionally, the first clone (B414203-N42-6-N1) shows the most similar basic component%.

CEX-HPLC
Sample Name Acidic component % Main component % Basic component % Total %
Positive control (Pembrolizumab) 19.5 64.1 16.4 100.0
B414203-N42-6-N1 16.0 69.7 14.3 100.0
B414203-N42-7-N1 17.4 69.2 13.4 100.0
B414209-N10-6-N1 18.1 44.6 37.3 100.0

Currently, we have sublicensed the CHOK1BN cell line to dozens of customers, and the status of some customers' projects is as follows:

Customer Type CLD PD Pilot Nonclinical IND Phase I Phase II
1 Customer A ADC
2 Customer G R-vaccines
3 Customer H Mab
4 Customer B Mab
5 Customer C Fab
6 Customer J R-vaccines
7 Customer F R-glycoprotein
“百英公司CHOK1BN细胞株是从ECACC获得贴壁培养的CHO-K1细胞，并取得商业化亚授权权益。按照项目授权，一次授权，一次性授权。”
Jing Cheng
成静
细胞株构建团队

FAQs

  • 什么是CHO-K1细胞？

    中国仓鼠卵巢细胞（Chinese hamster ovary，CHO）。自1958年Puke实验室将此连续传代细胞进行重新克隆后，建立了我们现在所用的CHO细胞最原始细胞系。随着时间的沉淀和历史的选择，以及对原始CHO细胞系的不同的培养、改造后，现已出现多种生长、表达、代谢以及基因组等具有差异的细胞系。 1

  • 为什么选择CHO细胞？

    之所以CHO细胞被选择，除了具有和人类似地翻译后的修饰，还能够在无血清及化学限定培养基悬浮培养中以高密度生长；具有产物胞外分泌功能，并且很少分泌自身的内源蛋白，便于下游产物分离纯化，并且在延长的发酵周期中维持高水平的蛋白质表达。 2

  • 为什么CHO细胞常用于治疗抗体药物生产？

    现阶段用于生产治疗性抗体（Abs）的宿主细胞往往是哺乳动物细胞，主要包括：Sp2/0 骨髓瘤细胞、NS0 小鼠骨髓瘤细胞、HEK293人胚胎肾细胞和中国仓鼠卵巢细胞（Chinese hamster ovary，CHO），其中CHO细胞已然成为需要复杂翻译后修饰的治疗性抗体首选制造细胞，占比超过70%。例如，曲妥珠单抗是一种由CHO细胞表达的治疗性抗体，对人表皮生长因子受体2 (HER2)具有特异性。

资料下载

参考文献

  • Fischer, S., Handrick, R., & Otte, K. (2015). The art of CHO cell engineering: A comprehensive retrospect and future perspectives. Biotechnology Advances, 33(8), 1878-1896. https://doi.org/10.1016/j.biotechadv.2015.10.015
  • Xu, X., Nagarajan, H., Lewis, N. E., Pan, S., Cai, Z., Liu, X., Chen, W., Xie, M., Wang, W., Hammond, S., Andersen, M. R., Neff, N., Passarelli, B., Koh, W., Fan, H. C., Wang, J., Gui, Y., Lee, K. H., Betenbaugh, M. J., . . . Wang, J. (2011). The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line. Nature Biotechnology, 29(8), 735-741. https://doi.org/10.1038/nbt.1932
  • Li, F., Vijayasankaran, N., Kiss, R., & Amanullah, A. (2010). Cell culture processes for monoclonal antibody production. MAbs, 2(5), 466-477. https://doi.org/10.4161/mabs.2.5.12720
  • Zhang, J., Shan, L., Liang, F., Du, C., & Li, J. (2022). Strategies and Considerations for Improving Recombinant Antibody Production and Quality in Chinese Hamster Ovary Cells. Frontiers in Bioengineering and Biotechnology, 10, 856049. https://doi.org/10.3389/fbioe.2022.856049

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