The increasing demand for antibody-based therapeutics has emphasized the need for technologies to improve recombinant antibody titers from mammalian cell lines. Moreover, as antibody therapeutics address an increasing spectrum of indications, interest has increased in antibody engineering to improve affinity and biological activity. However, the cellular mechanisms that dictate expression and the relationships between antibody sequence and expression level remain poorly understood. Fundamental understanding of how mammalian cells handle high levels of transgene expression and of the relationship between sequence and expression are vital to the development of new antibodies and for increasing recombinant antibody titers. In this work, we analyzed a pair of mutants that vary by a single amino acid at Kabat position 49 (heavy-chain framework), resulting in differential transient and stable titers with no apparent loss of antigen affinity. Through analysis of mRNA, gene copy number, intracellular antibody content, and secreted antibody, we found that while translational/post-translational mechanisms are limiting in transient systems, it appears that the amount of available transgenic mRNA becomes the limiting event on stable integration of the recombinant genes. We also show that amino acid substitution at residue 49 results in production of a non-secreted HC variant and postulate that stable antibody expression is maintained at a level which prevents toxic accumulation of this HC-related protein. This study highlights the need for proper sequence engineering strategies when developing therapeutic antibodies and alludes to the early analysis of transient expression systems to identify the potential for aberrant stable expression behavior. (C) 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012