ABSTRACT
When expressing complex biotherapeutic proteins, traditional expression plasmids and methods may not always yield sufficient levels of high-quality product. High-strength viral promoters commonly used for recombinant protein (rProtein) production in mammalian cells allow for maximal expression, but provide limited scope to alter their transcription dynamics. However, synthetic promoters designed to provide tunable transcriptional activity offer a plasmid engineering approach to more precisely regulate product quality, yield or to reduce product related contaminants. We substituted the viral promoter CMV with synthetic promoters that offer different transcriptional activities to express our gene of interest in Chinese hamster ovary (CHO) cells. Stable pools were established and the benefits of regulating transgene transcription on the quality of biotherapeutics were examined in stable pool fed-batch overgrow experiments. Specific control of gene expression of the heavy chain (HC):light chain (LC) of a Fab, and the ratio between the two HCs in a Duet mAb reduced levels of aberrant protein contaminants; and the controlled expression of the helper gene XBP-1s improved expression of a difficult-to-express mAb. This synthetic promoter technology benefits applications that require custom activity. Our work highlights the advantages of employing synthetic promoters for production of more complex rProteins.
ABSTRACT
Fragmentation is a major degradation pathway ubiquitous to all therapeutic monoclonal antibody (mAb) and therefore, monitored throughout the manufacturing process. Here, we describe a three-step approach to 1) detect, 2) confirm and 3) characterize partially reduced fragment species in an immunoglobulin G1 (IgG1) mAb with prolonged hold time of harvested cell culture fluid (HCCF). Microchip capillary electrophoresis (MCE) and high-performance size exclusion chromatography (HPSEC) were used as fast and efficient screening methods to detect fragmentation. HPSEC was found to be underestimating fragmentation levels. To confirm and characterize the fragments, capillary electrophoresis-sodium dodecyl sulphate (CE-SDS) was employed. Interestingly, the absence of fragments in the reduced CE-SDS analysis suggested partial reduction of disulphide bonds contributing to fragmentation in this mAb lot. This was further confirmed using reverse phase high performance liquid chromatography (RP-HPLC) coupled with mass spectrometry, which established the presence of heavy-heavy-light (HHL), heavy-heavy (HH), light-light dimer (LL), light chain (LC) and half antibody (HL) fragments with good mass accuracy. In this study, we demonstrated a readily applicable systematic strategy to support process development and investigate anomalous events in manufacturing. An additional highlight of this work is the data-driven comprehensive comparison of modern and conventional analytical techniques for fragment analysis.
