Automated method for quantification of 20 amino acids in cell culture media during biopharmaceutical development.

Herein, a step-by-step protocol for simultaneous detection of 20 amino acids commonly present in cell culture media is described. The protocol facilitates detection of both primary and secondary amino acids through a two-step precolumn derivatization strategy using ortho-phthalaldehyde and 9-fluorenylmethyl chloroformate as derivatizing agents. The separation of derivatized amino acids with varying hydrophobicity is achieved through reverse-phase chromatography. The amino acids are simultaneously detected in a single workflow through the use of Variable Wavelength Detector at 338 and 262 nm. The protocol is applicable for both mammalian and bacterial cell culture matrices with an option for automation of precolumn derivatization.

Ion exchange chromatography hyphenated with fluorescence detector as a sensitive alternative to UV detector: Applications in biopharmaceutical analysis.

Cation-exchange chromatography (CEX) is widely used for analysis of charge heterogeneity in monoclonal antibodies (mAbs) in the biopharmaceutical industry. Charge variant separation is typically achieved by either alteration of ionic strength or pH, and detection of the various species is performed using diode array detectors (DAD) at 280 nm. In this study, we investigate the suitability of fluorescence detector (FLD) as a sensitive alternative to traditional UV280 detection-based charge variant analysis. The objective of this work is to compare the performance of charge variants detection through cation exchange chromatography between FLD and DAD detectors. This was demonstrated for a mass spectroscopy compatible, pH based CEX method utilizing volatile ammonium acetate salt. The stability of FLD detection was tested as per method validation criteria set in the ICH Q2-(R1). LODFLD and LOQFLD were 59.07 and 59.64 times lower in comparison to LODUV and LOQUV, respectively (LODFLD: 0.13 µg, LOQFLD: 0.39 µg). The LinearityFLD was obtained in the range of 1-200 µg. Accuracy calculated as recovery value was between 93.38% and 103.35% and highest RSD value obtained for robustness was 2.54% (area under the curve). The application of FLD detection based CEX as a Process Analytical Technology (PAT) enabler in mAb biopharmaceutical analysis was demonstrated through direct charge variant profiling of CHO cell harvest supernatant (without pre purification through Pro-A). The method was also applied to and found suitable for biosimilarity assessment of drug products. While the present study is focused on analysis of trastuzumab biosimilars, the proposed method is expected to be applicable to any mAb product after suitable gradient modification.

Impact of various factors on the kinetics of non-enzymatic fragmentation of a monoclonal antibody.

Non-enzymatic hinge fragmentation of monoclonal antibodies (mAb) is considered a critical quality attribute since it changes the primary sequence of the proteins, thereby leading to structural changes which can affect stability, function, and efficacy. While peptide bonds are exceptionally stable under physiological conditions, reactive side chains of a few residues, the flexibility of the backbone, and physicochemical parameters such as pH, temperature, and the reaction of radicals and metal ions can promote the cleavage of peptide bonds. In this study, the relative extent and rate of fragmentation are compared with respect to the presence of several different factors (including hydrogen peroxide, metal ion, and temperature) as measured by size exclusion chromatography. A kinetic model of monomer degradation as a function of time (mAb only) is created. In the presence of either H2O2 or Cu2+, or both, the reaction kinetics follow different orders depending on the reaction conditions. The half-life for peptide bond cleavage of the mAb hinge region was 385 days at 40 °C and decreases to 250, 48, and 45 days in the presence of H2O2, Cu2+, and a combination of H2O2 and Cu2+, respectively. A temperature dependence of peptide bond cleavage at 35 °C, 40 °C, 45 °C, and 50 °C showed Arrhenius behavior with an apparent activation energy of 76.9 ± 16.4 kJ/mol. The reaction rates obtained from the Arrhenius equation were then extrapolated to predict fragmentation rates under real storage conditions (e.g., at 2-8 °C). We demonstrate that trace levels of impurities including peroxide left after surface sterilization or degradation of non-ionic surfactants or metal ions from the buffer components can significantly affect the stability of a mAb.