Large-Scale Propagation of Recombinant Adherent Cells That Secrete a Stable Form of Human Glandular Kallikrein, hK2

Abstract

Human glandular kallikrein 2 (hK2) is a trypsin-like serine protease that is expressed predominantly in the prostate epithelium and has 78% aa identity with prostate-specific antigen (PSA). hK2 has been recognized as a potential prostate cancer marker and has been demonstrated to be highly expressed in prostate cancer compared to benign prostatic tissue. Purification and characterization of hK2 have been impeded due to its lower expression in bodily fluids and tissues compared to PSA and its ability to autodegrade. Therefore, to study biochemical and biological characteristics of hK2, a stable and enzymatically inactive mutant form of hK2, hK2A217V, was expressed in a hamster cell line, AV12-664 (AV12-hK2A217V). AV12-hK2A217Vcells secreted prohK2A217V(phK2A217V) in the spent medium at ∼2.5 μg/ml. Since AV12-hK2A217Vare adherent cells, it was necessary to develop an efficient system to propagate large numbers of cells to obtain significant quantities of phK2A217V. In this paper, we compared ceramic core bioreactor and microcarrier beads as alternatives to static culture to propagate adherent cells. Considering production levels, ease of operation, cost effectiveness, and labor, microcarrier beads were found to be a better alternative. Our findings led to the development of a general protocol for large-scale propagation of adherent cells on microcarrier beads eliminating the need for propagating AV12-hK2A217Vin culture flasks or bioreactors. Microcarrier beads coated with AV12-hK2A217Vcells could be propagated in 1- or 3-liter spinner flasks and were passed from one spinner to the next in a manner analogous to static culture or could be frozen and later used as inoculum for subsequent spinners. Using this protocol, >40 liters of spent medium was harvested within 30 days, which in turn was used to purify phK2A217V. phK2A217Vpurified from spent medium of cells grown either on microcarrier beads or in culture flasks were biochemically similar as indicated by HIC–HPLC profile followed by sequencing of relevant peaks.