The ReFOLD assay for protein formulation studies and prediction of protein aggregation during long-term storage

Abstract

The formulation of novel therapeutic proteins is a challenging task which aims at finding formulation conditions that will minimize protein degradation during long-term storage. One particularly important and difficult-to-predict protein degradation pathway is the so-called non-native aggregation. The qualitative and quantitative prediction of the latter has been a subject of extensive research over the past two decades. An increasing body of evidence shows that the widely-used short-term biophysical techniques cannot accurately rank formulation conditions in order of their effect on the aggregation during long-term storage of some therapeutic proteins, e.g. monoclonal antibodies. Here we suggest a novel approach for the selection of formulation conditions that will suppress the formation of protein aggregates during long-term storage. We postulate that conditions (i.e. pH, buffer type, ionic strength) that reduce the isothermal aggregation of various denaturant-induced partially folded protein species will be conditions that impede protein aggregation during long-term storage. To test our hypothesis, we developed an isothermal microdialysis-based unfolding/refolding assay, named ReFOLD, which we use to induce moderate aggregation of partially folded proteins. Next, we assessed the relative monomer yield after isothermal unfolding/refolding of two monoclonal antibodies, each formulated in 12 different conditions. Using the proposed approach, we were able to accurately rank the formulations in order of their effect on the amount of protein aggregates detected after storage for 12 months at 4 ⁰C and 25 ⁰C, while widely-used stability-indicating parameters like protein melting and aggregation onset temperatures failed to provide accurate predictive formulation rankings.

Orthogonal Techniques to Study the Effect of pH, Sucrose, and Arginine Salts on Monoclonal Antibody Physical Stability and Aggregation During Long-Term Storage

“…The bulk buffer was exchanged to 10 mM histidine/histidine hydrochloride with pH 5.0, 5.75, and 6.5 at 25 C using extensive dialysis as described earlier. 20 The absorption of PPI13 at 280 nm was measured with a Nanodrop 2000 UV spectrophotometer (Thermo Fisher Scientific, Wilmington, DE) and the protein concentration was calculated using the protein extinction coefficient. Stock solutions of the additivesdsucrose, ArgHCl, ArgGlu, guanidinium hydrochloride (GuHCl), and NaCldwere prepared in the respective histidine buffer and spiked to the dialyzed protein solution.…”

“…The assay was performed as described earlier. 20 Briefly, 50 mL of 5 g/L PPI13 solution in the respective buffer (or buffer plus additive) were filled in Pierce™ microdialysis devices (3.5 kDa MWCO). The samples were dialyzed in a deep multiwell plate against 1.5 mL of 9 M urea dissolved in the respective formulation buffer (or buffer plus additive).…”

“…The relative monomer yield (RMY) of the protein after isothermal unfolding/refolding in urea was calculated after dividing the area of the monomer peak of the refolded sample by the area of the monomer peak of the sample before unfolding/refolding. 20 The soluble protein yield after refolding was calculated by dividing the area of all protein peaks detected with SEC after refolding by the area of all protein peaks before refolding. The value is then multiplied by 100 to obtain the soluble protein yield as a percentage.…”

“…The relative area of aggregates and the monomer recovery of PPI13 during long-term storage were calculated as earlier described. 20 Briefly, the area of the monomer peak after storage was divided by the area of the monomer peak measured at the beginning of the stability study and multiplied by 100. Thus, the monomer recovery will account for monomer loss during storage both due to the formation of small soluble aggregate and subvisible particles.…”

“…We recently presented an unfolding/refolding assay, named ReFOLD, that can be used to assess the aggregation of urea-induced partially unfolded protein species. 20 We applied this assay to study whether the additives tested here suppress the aggregation of the partially unfolded protein at pH 5.0 and 6.5. The 9 M concentration of urea was selected because it causes significant perturbations in the protein structure as shown by the change in the circular dichroism protein spectra (Fig.…”