Fig 1.
Analysis of proline substitutions observed in recombinant antibodies produced in CHO cell lines.
(a) Sum of sequence variant percentages across all proline residues of the recombinant antibody sequence produced from four cell lines A, B, C and D. (b) Hydroxyproline substitution levels were directly proportional to the number of proline residues on peptides analyzed. (c) Alanine substitution levels were directly proportional to the number of proline residues on peptides analyzed. (d) Extracted ion chromatogram (XIC) chromatogram of a peptide showing proline sequence variants with hydroxyproline and alanine replacing proline.
Fig 2.
Analysis of cell culture performance and the relationship between proline amino acid levels and substitutions observed for cell line D.
Cell culture performance for a single production bioreactor is shown (a) Viable cell density, (b) Viability, (c) Titer. (d) Proline concentration and proline substitutions in the harvest cell culture fluid (HCCF) from the same bioreactor, measured at different time points across culture duration. (e) Relationship between proline substitutions and minimum proline concentration. Multiple bioreactor runs of cell line D were analyzed to derive this relationship.
Fig 3.
Cell culture performance of bioreactors treated with labeled hydroxyproline compared with other control conditions.
(a) Viable cell density (b) Viability (c) Titer (d) Integrated capillary electrophoresis and mass spectrometry analysis of cell culture samples from experimental bioreactors showing unlabeled and labeled hydroxyproline present in the media.
Fig 4.
Detection of labeled hydroxyproline in recombinant antibody sequence.
Hydroxyproline misincorporation was observed in the antibody as shown by the XIC ratios for a representative peptide (inset). Both unlabeled and labeled hydroxyproline were observed. MS analysis showed the presence of D5 and D4 hydroxyproline.
Table 1.
Percentage of labeled hydroxyproline misincorporations observed in a set of random peptides chosen from the recombinant antibody sequence.