Characterization of a cathepsin D protease from CHO cell-free medium and mitigation of its impact on the stability of a recombinant therapeutic protein.

During purification process development of a recombinant therapeutic protein, an endoproteolytic activity endogenous to the Chinese hamster ovary (CHO) cells and leading to degradation at particular hydrophobic amino acid residues (e.g., Phe and Trp) was observed when processing at acidic pH. The presence of residual levels of protease activity in purified protein batches affected the inherent activity of the product when stored as a solution. To develop a robust purification strategy to minimize this undesirable impact, identification and characterization of this protease was essential to ultimately ensure that a solution formulation was stable for many years. A protease was isolated from CHO cell-free medium (CFM) using a combination of immobilized pepstatin-A agarose chromatography and size exclusion chromatography (SEC). The isolated protease has significant proteolytic activity at pH ∼ 3 to neutral pH and was identified as cathepsin D by mass spectrometry. Analytical SEC, chip-based capillary gel electrophoresis, imaged capillary isoelectric focusing (cIEF), and circular dichroism (CD) spectropolarimetry analyses were performed for additional characterization of the protease. The identification and characterization of this protease enabled the development of a robust purification process by implementation of a controlled temperature inactivation unit operation (heat inactivation) that enabled essentially complete inactivation of the protease, resulting in the production of a stable drug product that had not been possible using column chromatography alone. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:120-129, 2018.

Alkaline cation-exchange chromatography for the reduction of aggregate and a mis-formed disulfide variant in a bispecific antibody purification process.

During the purification development of a bispecific antibody, cation-exchange chromatography was screened for its ability to separate a prominently expressed (>12%) mis-formed disulfide bond variant, termed MAb-diabody, and aggregate from the product of interest. The influence of pH, product load (g of product per liter of resin) and linear velocity on the separations were evaluated for the strong cation-exchange resins SP Sepharose HP and POROS(®) HS50. Cation-exchange chromatography is commonly operated distant to the isoelectric point of a molecule, generally leading to acidic conditions for antibody purification. However, the results herein demonstrated improved removal of MAb-diabody with increasing pH, resulting in reduction of MAb-diabody content greater than 12-fold when operating near the alkaline pI of the product. This approach was successful over a range of linear velocities and g/L of resin loading. Aggregate removal was less affected by pH and was effectively reduced from 10.9% to less than 3% for each condition. Furthermore, this method was successfully scaled to a 60 cm diameter column using SP Sepharose HP resin.

Evaluation of microfluidics reactor technology on the kinetics of virus inactivation.

Mammalian cell lines constitute an important part in the manufacture of therapeutic proteins. However, their susceptibility to virus contamination is a potential risk to patient safety and productivity, and has led to the development of a repertoire of virus inactivation techniques. From a process development viewpoint, the challenge is to demonstrate the required log reduction in virus content without a significant loss in product titer or quality. The balance between the two is dictated by the kinetics of virus inactivation and protein degradation, both of which are critically affected by process parameters. In this study we describe a commercially available microchannel reactor (MCR) and demonstrate how it can be used to evaluate the impact of temperature on the kinetics of virus inactivation and protein product degradation. Virus spiking experiments are reported using Xenotropic Murine Leukemia Virus and REOvirus, into buffers in the absence and presence of a therapeutic protein currently under development at Lilly. The results demonstrate that the MCR is an ideal platform for evaluation of fast reactive systems and reactions that are particularly sensitive to small changes to process conditions. These conditions include heat inactivation of a virus in a mammalian cell culture process stream used in the manufacture of therapeutic proteins and antibodies.

Evaluation of viral removal by nanofiltration using real-time quantitative polymerase chain reaction.

Nanofiltration is commonly introduced into purification processes of biologics produced in mammalian cells to serve as a designated step for removal of potential exogenous viral contaminants and endogenous retrovirus-like particles. The LRV (log reduction value) achieved by nanofiltration is often determined by cell-based infectivity assay, which is time-consuming and labour-intensive. We have explored the possibility of employing QPCR (quantitative PCR) to evaluate LRV achieved by nanofiltration in scaled-down studies using two model viruses, namely xenotropic murine leukemia virus and murine minute virus. We report here the successful development of a QPCR-based method suitable for quantification of virus removal by nanofiltration. The method includes a nuclease treatment step to remove free viral nucleic acids, while viral genome associated with intact virus particles is shielded from the nuclease. In addition, HIV Armored RNA was included as an internal control to ensure the accuracy and reliability of the method. The QPCRbased method described here provides several advantages such as better sensitivity, faster turnaround time, reduced cost and higher throughput over the traditional cell-based infectivity assays.

Selective enhancement of multipotential hematopoietic progenitors in vitro and in vivo by IL-20.

In a search for novel growth factors, we discovered that human interleukin-20 (IL-20) enhanced colony formation by CD34+ multipotential progenitors. IL-20 had no effect on erythroid, granulocyte-macrophage, or megakaryocyte progenitors. IL-20 transgenic mice increased the numbers and cell cycling of multipotential but not other progenitors. IL-20 administration to normal mice significantly increased only multipotential progenitor cells, demonstrating that IL-20 significantly influences hematopoiesis, with specificity toward multipotential progenitors. This is the first cytokine with such specificity identified.