34 BioProcess International JUNE 2008 SUPPLEMENT C H A P T E R FIVE Expression of a Fab Fragment in CHO and Pichia pastoris A Comparative Case Study by Renate Kunert, Johannes Gach, Hermann Katinger M ammalian cell expression systems are currently essential for production of glycosylated biopharmaceuticals such as monoclonal antibodies or molecules requiring even more complex glycan structures. Various host cell and vector systems aimed at improving expression levels and quality have been established ( 1, 2). Development of biopharmaceutical product candidates from genes to clinical trials should be based on technology platforms that will require no major changes in the entire development chain, including manufacturing once a product candidate has successfully progressed through phase 1–2 clinical testing. The intrinsic cost structure thus is widely determined by the category of technology platform chosen very early in development. Development time is currently considered by modern management to be of utmost importance. Antibody fragments (Fabs) represent an interesting category of potential biopharmaceuticals (3, 4). About 20% of all Fabs have glycosylation sites that putatively might contribute to their biological in vivo performance (5). We investigated two different state-of-the- art technology platforms — the CHO mammalian system and the Pichia pastoris yeast system — to produce Fab fragments with identical primary sequence and compared the results. We analyzed the time needed for development of different technology platforms and intrinsic parameters such as specific and volumetric productivity as well as quality criteria based on preclinical and in vitro properties. MATERIALS AND METHODS 3H6 Fab Expression Vectors: A human/ mouse Fab fragment, 3H6/Fab (6), was expressed in CHO cells by using a two plasmid strategy (7). We cloned the light-chain vL-cκ into the pIRESdhfr vector derived from pIRESneo from Clontech Laboratories, Inc. (www. clontech.com) by exchanging the neom ycinphosphotransferase with the dihydrofolate reductase (dhfr) at 3 of the internal ribosomal entry site (IRES), resulting in the cotranscription of 3H6 light chain and dhfr under control of the human CMV promoter (8). The heavy chain was separately cloned into a eukaryotic expression vector under control of the SV40 early promoter. Expression of 3H6/Fab in P. pastoris was accomplished by the pPICZalphaA vector. Both chains were cloned at 3 of the alpha mating factor leader sequence, and expression was controlled by the methanol- inducible AOX promoter (9). Clone Selection, Screening, and Recombinant Production of Fab Fragments: We transfected protein-free– cultivated CHO cells (DUKX-B11, ATCC CRL-9096) with a nucleofector from Amaxa AG (www.amaxa.com) and started selection (hypoxanthine/ thymidine-deficient medium) 24 hours later in combination with limiting dilution to select predominantly single clones in 96-well plates ( 10). Transfection was done in four independent experiments, each with 4,000,000 cells plated at 1,000,000 cells per plate. After three weeks, 75% of seeded wells showed a minimum of semiconfluent growth, and we screened the supernatants of such selected clones using ELISA for human Fab productivity. Those identified as best producers (less than 10% of screened wells) were further propagated in 50 nM methotrexate (MTX) and in the next passage adapted to 100 nM MTX for gene amplification. Following stabilization of clones, we subcloned the best clone by limiting dilution and implemented a second subcloning step with 400 nM MTX before adapting the best clones to 1.6 µM MTX. The best clone was then evaluated according to specific growth rate and productivity. For expression in P. pastoris, we used the wild-type strain X33 as our host strain and transformed it with the linearized pPICZalphaA3H6Fab introduced into the cells by electroporation ( 11). We then used POLYMUN SCIENTIFIC GMBH (WWW.POLYMUN.COM)