Investigation of the effect of salt additives in Protein L affinity chromatography for the purification of tandem single-chain variable fragment bispecific antibodies

Tandem single-chain variable fragment (scFv) bispecific antibodies (bsAb) are one of the most promising bsAb formats reported thus far. Yet, because of their increased aggregation propensity, high impurity content due to low expression level, smaller size and lack of the Fc region, it is challenging to isolate these products with high yield and purity within a limited number of purification steps in a scalable fashion. A robust purification process that is able to circumvent these issues is therefore of critical importance to allow effective isolation of this group of antibodies. We investigated the addition of sodium chloride (NaCl), calcium chloride (CaCl2), and L-arginine monohydrochloride (Arg center dot HCl) to the elution buffer of Protein L affinity chromatography, and propose here a novel mechanism for the modification of Protein L binding avidity that can lead to enhanced high molecular weight (HMW)-monomer separation, a preferential strengthening effect of the HMW-Protein L interaction compared to the monomer-Protein L interaction. In particular, we found Arg center dot HCl to be the most effective salt additive in terms of purity and recovery. The mechanism we propose is different from the widely reported chaotropic effect exerted by salt additives observed in Protein A chromatography. We also demonstrate here that a final eluate containing <1% HMW species and <100 ppm host cell proteins can be obtained within a two-step process with an overall yield of 65%, highlighting the promising suitability of Protein L affinity chromatography for the purification of kappa light chain-containing tandem scFv bsAb.