Freeze thaw and lyophilization induced alteration in mAb therapeutics: Trastuzumab as a case study.

Long-term stability of therapeutic monoclonal antibody (mAb) products is necessary for their successful commercialization. Freeze-thaw (F/T) operations are often performed for a mAb product during processing, storage and distribution. Lyophilization (Lyo) is another unit operation that is commonly used for drug product manufacturing of mAbs. This paper aims to explore the impact of these operations on structure and function of a mAb therapeutic, as well as of biosimilars. Trastuzumab innovator and its five biosimilars were analysed for aggregation, charge heterogeneity, secondary structure, binding kinetics, and potency after each freeze-thaw and lyophilization cycle. It is observed that both F/T and Lyo induce protein aggregation, which in turn causes perturbations in the biological potency of the mAb therapeutic. The average value of the percentage of aggregation increased from 0.6 % (week 1) to 5.3 % (week 10) in F/T study and from 0.8 % (week 1) to 10.1 % (week 10) in Lyo study. The acidic pool increased from 26.5 % (week 1) to 44.4 % (week 10) and the basic variants from 13.9 % (week 1) to 24.0 % (week 10) in F/T study. Similarly, acidic pool increased from 27.1 % (week 1) to 42.0 % (week 10) and basic variants from 14.8 % (week 1) to 24.4 % (week 10) in Lyo study. The average percentage of beta-sheet increased from 58.4 % (week 1) to 60.9 % (week 10) in F/T study and from 59.7 % (week 1) to 72.6 % (week 10) in Lyo study. Lower binding affinity was found in week 7 as compared to week 1 in Lyo study whereas no change in binding affinity was observed in the F/T study. The average potency value gradually decreased from 0.97IU/ ml (week 1) to 0.75IU/ ml (week 10) in F/T study and from 1.0IU/ ml (week 1) to 0.66IU/ ml (week 10) in Lyo study. Results indicate that lyophilization has a bigger impact on binding affinity than freeze thaw and as expected, the impact was comparable across the innovator and biosimilar products.