(81f) Minimal Purification of Recombinant Proteins for Early Developability Assessment

Characterization of protein therapeutics and subunit vaccine candidates is a critical step in the protein development phase. Selection of lead candidates can be done by performing a developability assessment, which includes applying a battery of biophysical assays and selecting for ideal properties in accordance with the target product profile. To perform a developability assessment, however, several milligrams of purified protein is required, often delaying this assessment to the later stages of the development phase or obligating the use of affinity tags. Here we describe the use minimal purification process for recombinant proteins secreted by Komagataella phaffii. A buffer exchange and filtration with a Q-membrane filter proved to be sufficient to remove key supernatant impurities like host-cell proteins and DNA. This purification process yielded acceptable purity and 1-2 milligrams to characterize an engineered SARS-CoV-2 RBD by several widely used methods like mass spectrometry and differential scanning calorimetry. Furthermore, we used this method to perform a biophysical and functional comparison of SARS-CoV-2 RBD variants. Lastly, we demonstrate broader applicability to three other protein classes: a single-domain antibody, an NRRV protein subunit, and a human growth hormone. Implementation of a minimal purification strategy could streamline feedback loops between discovery and development teams in order to design more developable protein candidates.