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ABSTRACT In‐depth analytical characterization of biotherapeutics originating from different production batches is mandatory to ensure product safety and consistent molecule efficacy. Previously, we have shown unintended incorporation of tyrosine (Tyr) and leucine/isoleucine (Leu/Ile) at phenylalanine (Phe) positions in a recombinant produced monoclonal antibody (mAb) using an orthogonal MASCOT/SIEVE based approach for mass spectrometry data analysis. The misincorporation could be avoided by sufficient supply of phenylalanine throughout the process. Several non‐annotated signals in the primarily chromatographic peptide separation step for apparently single Phe→Tyr sequence variants (SVs) suggest a role for isobar tyrosine isoforms. Meta‐ and ortho‐ Tyr are spontaneously generated during aerobic fed‐batch production processes using Chinese hamster ovary (CHO) cell lines. Process induced meta ‐ and ortho ‐Tyr but not proteinogenic para ‐Tyr are incorporated at Phe locations in Phe‐starved CHO cultures expressing a recombinant mAb. Furthermore, meta ‐ and ortho ‐Tyr are preferably misincorporated over Leu. Structural modeling of the l‐phenylalanyl‐tRNA‐synthetase (PheRS) substrate activation site indicates a possible fit of non‐cognate ortho ‐Tyr and meta ‐Tyr substrates. Dose‐dependent misincorporations of Tyr isoforms support the hypothesis that meta ‐ and ortho ‐Tyr are competing, alternative substrates for PheRS in CHO processes. Finally, easily accessible at‐line surrogate markers for Phe→Tyr SV formation in biotherapeutic production were defined by the calculation of critical ratios for meta ‐Tyr/Phe and ortho ‐Tyr/Phe to support early prediction of SV probability, and finally, to allow for immediate process controlled Phe→Tyr SV prevention. Biotechnol. Bioeng. 2015;112: 1187–1199. © 2014 Wiley Periodicals, Inc. Non‐proteinogenic meta ‐ and ortho ‐tyrosine are spontaneously generated in production processes using Chinese hamster ovary cells. Both tyrosine isomers are misincorporated at phenylalanine positions in a recombinantly expressed antibody due to promiscuity of l‐phenylalanyl‐tRNA‐synthetase. The findings described offer mechanistic insights for Phe→Tyr sequence variant formation in biotherapeutic production processes and suggest an alternative approach to Phe→ meta ‐Tyr and Phe→ ortho ‐Tyr sequence variant prevention.
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