Bioreactor scale up and protein product quality characterization of piggyBac transposon derived CHO pools

Abstract

Chinese hamster ovary (CHO) cells remain the most popular host for the production of biopharmaceutical drugs, particularly monoclonal antibodies (mAbs), bispecific antibodies, and Fc-fusion proteins. Creating and characterizing the stable CHO clonally-derived cell lines (CDCLs) needed to manufacture these therapeutic proteins is a lengthy and laborious process. Therefore, CHO pools have increasingly been used to rapidly produce protein to support and enable preclinical drug development. We recently described the generation of CHO pools yielding mAb titers as high as 7.6 g/L in a 16 day bioprocess using piggyBac transposon-mediated gene integration. In this study, we wanted to understand why the piggyBac pool titers were significantly higher (2-10 fold) than the control CHO pools. Higher titers were the result of a combination of increased average gene copy number, significantly higher messenger RNA levels and the homogeneity (i.e. less diverse population distribution) of the piggyBac pools, relative to the control pools. In order to validate the use of piggyBac pools to support preclinical drug development, we then performed an in-depth product quality analysis of purified protein. The product quality of protein obtained from the piggyBac pools was very similar to the product quality profile of protein obtained from the control pools. Finally, we demonstrated the scalability of these pools from shake flasks to 36L bioreactors. Overall, these results suggest that gram quantities of therapeutic protein can be rapidly obtained from piggyBac CHO pools without significantly changing product quality attributes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:534-540, 2017.

Evaluation of piggyBac‐mediated CHO pools to enable material generation to support GLP toxicology studies

“…At Gen 0, all the PB CHO pools showed a single peak of expressing population which corresponds to previously reported data . Each PB CHO pool, expressing mAb‐A, mAb‐B, mAb‐C, or mAb‐D, consistently showed the same profile over generations with no appearance of a low or non‐expressing population.…”

“…We have recently reported very high mAb titers using PB CHO pools . In this specific study (Part I) we wanted to test the expression stability of PB CHO pools over time.…”

“…We have recently reported that PB‐mediated gene integration to highly productive CHO pools with appropriate PQAs . Here we performed a thorough evaluation of the PB CHO pools expressing three hIgG4 mAbs and one hIgG1 mAb and compared to pooled Top 4 CDCLs.…”

“…In a subsequent manuscript we showed that the PQAs from mAbs produced from PB‐mediated CHO pools (PB CHO pool) were very similar to PQAs from CHO pools based on a random gene integration cell line generation approach (control conditions). We also demonstrated successful scale‐up of these PB pools in 36 L bioreactors …”

Generation of High Expressing Chinese Hamster Ovary Cell Pools Using the Leap‐In Transposon System

“…CHO‐GS knock out cells were cultured and maintained as previously described . Briefly, the CHO cells were maintained in a proprietary DMEM‐based medium with 8 mM l ‐glutamine (LM‐Growth) (Cat.59202C‐100, SAFC, St. Louis, MO) in shake flasks at 37 °C and 8% CO2 and passaged every 3–4 days by dilution.…”