Department of Early Stage Cell Culture, Genentech, Inc., South San Francisco, California 94080, USA.
Biotechnol Bioeng. 2010 Sep 1;107(1):163-71. doi: 10.1002/bit.22780.
An amino acid sequence variant is defined as an unintended amino acid sequence change and contributes to product heterogeneity. Recombinant monoclonal antibodies (MAbs) are primarily expressed from Chinese Hamster Ovary (CHO) cells using stably transfected production cell lines. Selections and amplifications with reagents such as methotrexate (MTX) are often required to achieve high producing stable cell lines. Since MTX is often used to generate high producing cell lines, we investigated the genomic mutation rates of the hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT) gene using a 6-thioguanine (6-TG) assay under various concentrations of MTX selection in CHO cells. Our results show that the 6-TG resistance increased as the MTX concentration increased during stable cell line development. We also investigated low levels of sequence variants observed in two stable cell lines expressing different MAbs. Our data show that the replacement of serine at position 167 by arginine (S167R) in the light chain of antibody A (MAb-A) was due to a genomic nucleotide sequence change whereas the replacement of serine at position 63 by asparagine (S63N) in the heavy chain of antibody B (MAb-B) was likely due to translational misincorporation. This mistranslation is codon specific since S63N mistranslation is not detectable when the S63 AGC codon is changed to a TCC or TCT codon. Our results demonstrate that both a genomic nucleotide change and translational misincorporation can lead to low levels of sequence variants and mistranslation of serine to asparagine can be eliminated by substituting the TCC or TCT codon for the S63 AGC codon without impacting antibody productivity.
氨基酸序列变异体定义为非预期的氨基酸序列变化，并导致产品异质性。重组单克隆抗体（Mab）主要通过使用稳定转染的生产细胞系从中国仓鼠卵巢（CHO）细胞中表达。为了获得高产的稳定细胞系，通常需要使用甲氨蝶呤（MTX）等试剂进行选择和扩增。由于 MTX 常用于生成高产细胞系，我们使用 6-硫代鸟嘌呤（6-TG）测定法在 CHO 细胞中不同 MTX 选择浓度下研究了次黄嘌呤鸟嘌呤磷酸核糖转移酶（HGPRT 或 HPRT）基因的基因组突变率。我们的结果表明，在稳定细胞系开发过程中，随着 MTX 浓度的增加，6-TG 抗性增加。我们还研究了在表达不同 Mab 的两个稳定细胞系中观察到的低水平序列变异。我们的数据表明，抗体 A（Mab-A）轻链中丝氨酸 167 被精氨酸（S167R）取代是由于基因组核苷酸序列变化所致，而抗体 B（Mab-B）重链中丝氨酸 63 被天冬酰胺（S63N）取代则可能是由于翻译时错读所致。这种错译是密码子特异性的，因为当 S63 AGC 密码子变为 TCC 或 TCT 密码子时，不会检测到 S63N 错译。我们的结果表明，基因组核苷酸变化和翻译时错读都可能导致低水平的序列变异，并且可以通过用 TCC 或 TCT 密码子替代 S63 AGC 密码子来消除丝氨酸到天冬酰胺的错译，而不会影响抗体的生产力。
