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. Biotechnol. Bioeng. 2010; 107: 163-171. (C) 2010 Wiley Periodicals, Inc.