Expression and purification optimization of an N-terminal Pfs230 transmission-blocking vaccine candidate.

In an effort to control and eventually eliminate malaria, the development of transmission-blocking vaccines has long been sought. However, few antigens have been evaluated in clinical trials, often due to limitations in the expression and purification of the antigen in sufficient yield and quality. Pfs230, a surface antigen of gametocytes, has recently advanced to clinical evaluation as a conjugate vaccine using the Pseudomonas aeruginosa exoprotein A carrier protein. Here we continue to build upon prior work of developing a Pfs230 candidate in the baculovirus system, Pfs230C1 (aa 443-731), through systematic process development efforts to improve yield and purity. Various insect cells including High Five, Sf9 and Super Sf9 were first evaluated for quality and quantity of antigen, along with three insect cell media. In the selection of Sf9 cells, an intact Pfs230C1 was expressed and harvested at 48 h for downstream development. A downstream process, utilizing immobilized metal affinity column (IMAC), followed by ion exchange (IEX) membranes (Mustang S) and finally IEX chromatography (DEAE) yielded a pure Pfs230C1 protein. The complete process was repeated three times at the 20 L scale. To support the eventual chemistry manufacturing and controls (CMC) of Pfs230C1, analytical tools, including monoclonal antibodies, were developed to characterize the identity, integrity, and purity of Pfs230C1. These analytical tools, taken in combination with the optimized process, were implemented with Current Good Manufacturing Practices (cGMP) in mind with the ultimate objective of Phase I clinical trials.

AbSep--an amino acid based pseudobioaffinity adsorbent for the purification of immunoglobulin G.

The present work deals with the development and characterization of a tryptophan based pseudobioaffinity adsorbent for the purification of monoclonal and polyclonal antibodies. Tryptophan as a ligand was selected based on molecular docking and experimental screening studies of the amino acids involved in IgG-Protein A interaction. The ligand was coupled to a polymethacrylate based rigid, porous SEPABEADS beaded matrix to obtain the desired affinity adsorbent, which was named AbSep. Characterization studies with regards to the effect of matrix properties (pore size, particle size, nature of matrix, spacer arm) and the medium properties (pH, conductivity, additives) were performed to elucidate the nature of IgG-AbSep interactions and to determine the optimal conditions for obtaining high binding and purity of IgG. The equilibrium binding capacity of AbSep and dissociation constant was found to be 78 mg/ml and 5.31×10(-6)M respectively. AbSep was able to successfully purify polyclonal human IgG from plasma and monoclonal antibody (chimeric IgG1) from CHO cell culture supernatant. Both binding and elution steps were performed at near neutral pH resulting in a purity and recovery of more than 90% and 85% respectively. Additionally, AbSep was shown to be stable to 0.5M NaOH solutions, the preferred agent for cleaning and sanitization of chromatographic media.