Recombinant Protein Therapeutics from CHO Cells - 20 Years ...

1 CHO Consortium SBE Special Section Recombinant Protein Therapeutics from CHO Cells 20 Years and Counting The CHO cell is at its height of technological Karthik P. Jayapal prominence thanks to its adaptability to various Katie F. Wlaschin culture conditions and plasticity in the context Wei-Shou Hu University of Minnesota of genetic alterations. With further research, the application of cell culture strategies based on Miranda G. S. Yap Bioprocessing Technology Institute, scientific reasoning, rather than heuristics, is in Biomedical Sciences Institutes the not-so-distant future. R. ecombinant Protein Therapeutics have changed the array of highly successful CHO-based Therapeutics face of modern medicine in the past decade, and (Table), which continue to revolutionize the field of med- they continue to provide innovative and effective icine to this day. The knowledge and expertise amassed therapies for numerous previously refractory illnesses.

2 Over the past two decades will most certainly ensure that Today, they are used in the treatment of a variety of CHO Cells continue to remain the industry's premier human diseases, ranging from cancers to infertility. These workhorse for therapeutic Protein production, at least in proteins are generally synthesized by large-scale cultiva- the near future. tion of genetically engineered host Cells , which harbor In this article, we revisit the evolution of CHO Cells , artificially transfected genes encoding for the proteins of tracing from their origins to the development of current interest. For Protein Therapeutics to be effective, they must production cell lines, and discuss some important aspects be synthesized in biologically active forms, requiring that make them robust and versatile Protein expression proper folding and post-translational modifications. In hosts. Unlike some of its rodent relatives (mouse and rat), many cases, this includes glycosylation, a type of modifi- the generation of genomic resources for Chinese hamster cation where certain carbohydrate moieties are added to has been quite limited.

3 Genomic and proteomic tools specific amino acid residues of the Protein . using these resources can potentially aid in understand- Glycoproteins, as these are usually called, are general- ing and improving Recombinant Protein production ly synthesized in mammalian Cells , because common processes leading to a significant enhancement in the microbial hosts like Escherichia coli lack the requisite speed with which these Therapeutics transition from lab- machinery to synthesize appropriate glycoforms. Several oratory molecules to life-saving medicines. We, therefore, rodent- or human-derived Cells like 3T3, CHO, BHK, devote special attention to the value and need for such HeLa and HepG2 are frequently used in biomedical information in meeting the challenges of future research research for heterologous Protein expression. Despite the and manufacturing processes. availability of a plenitude of cell lines, nearly 70% of all Recombinant Protein Therapeutics produced today are From hamster to tissue cultures made in Chinese Hamster Ovary (CHO) Cells .

4 The cur- Chinese hamsters were first used as laboratory rent annual sales for biologics produced using CHO Cells specimen in 1919 in place of mice for typing pneumo- alone exceed US$30 billion worldwide. cocci (Box). Subsequent efforts at domestication by Dr. The first Recombinant therapeutic Protein produced in George Yerganian and others in the mid-20th century mammalian Cells , tissue plasminogen activator (r-tPA, led to the development of spontaneous hereditary dis- Activase) synthesized using CHO Cells , was approved for eases due to inbreeding, spurring research interest in clinical use in 1987. This marked the beginning of an hamster genetics (1, 2). It was noted during that time 40 CHO Consortium SBE Special Section Table. Selected list of approved biologics produced in Chinese Hamster Ovary cell lines. Product Type Therapeutic use Manufacturer Year of approval (FDA). Vectibix Anti-EGFR mAb Metastatic colorectal cancer Amgen 2006.

5 Myozyme -glucosidase Pompe disease Genzyme 2006. Aldurazyme Laronidase Mucopolysaccharidosis I Genzyme 2006. Orencia Ig-CTLA4 fusion Rheumatoid arthritis Bristol-Myers Squibb 2005. Naglazyme N-acetylgalactosamine-4-sulfatase Mucopolysaccharidosis VI BioMarin Pharmaceutical 2005. Luveris Luteinizing hormone Infertility Serono 2004. Avastin Anti-VEGF mAb Metastatic colorectal cancer & lung cancer Genentech 2004. Advate Factor VIII (engineered) Hemophilia A Baxter 2003. Xolair Anti-IgE mAb Moderate/severe asthma Genentech 2003. Raptiva Anti-CD11a mAb Chronic psoriasis Genentech 2003. Fabrazyme -galactosidase Fabry disease Genzyme 2003. Rebif Interferon- Relapsing multiple sclerosis Serono 2002. Humira Anti-TNF mAb Rheumatoid arthritis Abbott 2002. Aranesp Erythropoietin (engineered) Anemia Amgen 2001. Campath Anti-CD52 mAb Chronic lymphocytic leukemia Genzyme, Bayer 2001. ReFacto Factor VIII Hemophilia A Wyeth 2000.

6 Tenecteplase tissue plasminogen activator (engineered) Myocardial infraction Genentech 2000. Herceptin Anti-HER2 mAb Metastatic breast cancer Genentech 1998. Enbrel TNF receptor fusion Rheumatoid arthritis Amgen, Wyeth 1998. Benefix Factor IX Hemophilia B Wyeth 1997. Follistim/Gonal-F Follicle stimulating hormone Infertility Serono/NV Organon 1997. Rituxan Anti-CD20 mAb Non-Hodgkin's lymphoma Genentech, Biogen Idec 1997. Avonex Interferon- Relapsing multiple sclerosis Biogen Idec 1996. Cerezyme -glucocerebrosidase Gaucher's disease Genzyme 1994. Pulmozyme Deoxyribonuclease I Cystic fibrosis Genentech 1993. Epogen/Procrit Erythropoietin Anemia Amgen/Ortho Biotech 1989. Activase tissue plasminogen activator Acute myocardial infraction Genentech 1987. that the low chromosome number of Chinese hamsters A Brief History of the Chinese Hamster (2n = 22) made them particularly useful models in radi- ation cytogenetics and tissue culture studies.

7 In 1957, Chinese hamsters (scientific while investigating the usefulness of various Cells in name, Cricetulus griseus) belong to somatic cell genetics, Dr. Theodore T. Puck of the Dept. a family of rodents that are native to the deserts of northern China of Medicine at the University of Colorado isolated an and Mongolia. They have been ovary from a female Chinese hamster and established used in life-saving biomedical the Cells in culture plates (3). It soon became obvious research ever since they were first that these Cells were quite resilient and lent themselves introduced into the laboratory in 1919 for typing pneumococci. In readily to in vitro cultivation with relatively fast gener- the early 1920's, they gained repu- ation times. Karyotype heterogeneity among these cell tation as valuable tools in epidemiological research, because they were populations evoked particular interest in the context of known as carriers of the deadly parasite Leishmania causing kala-azar studying chromosomal abnormalities.

8 (also known as black fever or leishmaniasis). In 1948, they were literal- ly smuggled into the by Dr. C. H. Hu and Dr. Robert Watson, who were later accused for war crimes' by the Germ Warfare Commission Contribution to basic biomedical research of China, leading to imprisonment of the former (1). Amid the turmoil Until the later part of 20th century, isolation and char- between China and the in the early cold war days, it was thought acterization of mammalian cell mutants for cytogenetic that the would use them as agents of biological warfare by infect- studies was a challenging exercise, fraught with failures ing them with deadly diseases like cholera or plague and parachuting them over Manchuria. Instead, Chinese hamsters are now credited with because, unlike microbes, mammalian Cells are generally saving thousands of lives from illnesses like cancer every year. diploid. The establishment of CHO Cells in tissue cultures enabled researchers to overcome this difficulty because cell cycle to toxicology studies, so much so, that they have these Cells were functionally hemizygous for many genes, been termed as the mammalian equivalent of the model primarily due to gene inactivation (4, 5).

9 CHO Cells have, bacterium, E. coli (6). thereafter, been used in numerous biomedical studies Among the historically important medical and cell biol- ranging from analysis of intermediary metabolisms and ogy studies conducted in CHO, it was the early work CHO Consortium SBE Special Section 41. CHO Consortium SBE Special Section involving mutagenesis of these Cells and isolation of certain netic and pharmacodynamic properties of the products, auxotrophs (7) that facilitated their migration from labora- and hence their solubility, stability, biological activity and tory benches to industrial reactors. These mutants exhibited residence time in humans. Product safety is another key particular nutritional requirements for maintaining growth aspect that must be considered in choosing host Cells . The and viability over long culture periods. Mutants with vary- production host must not allow the propagation of any ing degrees of deficiencies in metabolic enzymes like ade- adventitious pathogenic agents that may eventually find nine phosphoribosyl transferase (APRT) and dihydrofolate their way into humans.

10 From an industrial perspective, the reductase (DHFR) were isolated in this manner (8, 9). In ability to adapt and grow Cells in suspension instead of addition, mutants defective in transcription, translation and adherent cultures is highly desirable as it allows volumetric machineries for certain amino acid and polyamine biosyn- scalability and use of large stirred-tank bioreactors. Finally, thesis were also isolated (10 14). the host Cells must be amenable to genetic modifications Whilst the primary motive behind the isolation of these allowing easy introduction of foreign DNA and expression mutants was fundamental research, it was, perhaps, fortu- of large amounts of desired Protein . itous that the nutritional requirements of auxotrophs could Twenty Years of experience with CHO Cells in the bio- be put to use for selection of Cells expressing exogenous pharmaceutical industry has demonstrated that, to a large proteins.