Redissolution of recombinant antibodies precipitated by ZnCl2

Abstract

Precipitation of antibodies by means of suitable precipitation agents is considered as an alternative to state-of-the-art affinity chromatography, although redissolution of the precipitated product with minimal formation of aggregates still poses challenges. One of these is to find an appropriate buffer composition, promoting the rapid redissolution of antibodies at high yields, with minimal impact on product quality. Here we report the development of a robust method for efficient redissolution of two industrially relevant antibodies, namely adalimumab and trastuzumab, which were precipitated by ZnCl2. Using this method, yields over 90% were achieved. Furthermore, the effect of various parameters such as pH and ionic strength on the redissolution were investigated. A mechanism regarding zinc-precipitated proteins, in which the deprotonated buffer species are the main actors responsible for redissolution due to their high affinity for zinc is hypothesized. We further evaluated the impact of ZnCl2 precipitation and redissolution at low pH on high molecular weight impurities and additionally assessed protein stability in these conditions. Glycoanalysis and nano-differential scanning calorimetry confirmed that glycostructure and tertiary and quaternary structure are not affected by the redissolution method.