Contents:

Chapter 1: Overview of Large-Scale Cell Culture Two Decades of Experience Chapter 2: Mammalian Cells Cell Line Generation Cell Line Selection Post Translational Modifications (PTMs) Cell Line Types and Characteristics - Chinese Hamster Ovary - PER.C6 - NS0 - BHK - Hybridomas - Insect Cell Culture Choice of Cell Line - Blood Factors - Antibodies - Enzymes - Hormones Chapter 3: Cell Line Engineering Vectors and Host Cells Cell Culture Health - Bacterial and Fungal - Mycoplasmas - Viral Chapter 4: Cell Culture Methods and Design Small Scale Large Scale Culture Modeling Media Selection Bioreactor Design - Physical Configurations - Batch and Fed-batch - Perfusion - Centrifugal Bioreactor Process Monitoring and Control Chapter 5: Product Characterization Process Impact on Product Species Variation Regulatory Environment Chapter 6: References Table of Exhibits Exhibit 1.1 Technologies Used to Prepare Commercial Products Exhibit 1.2 Major Licensed Recombinant Therapeutics Expressed by Mammalian Cell Lines Exhibit 2.1 Typical Steps in the Creation of a Mammalian Cell Line and Process Design Exhibit 2.2 Example Antibody Products (Adapted from BioPharm International, 2004) Exhibit 3.1 Contribution of Various Stages to the Time and Titer During Cell Line Development Exhibit 3.2 Examples of Viral Contamination of Commercial Cell Cultures Exhibit 3.3 Viral Tests Exhibit 4.1 Comparison of in vivo and in vitro Systems for the Production of Monoclonal Antibodies Exhibit 4.2 Comparison of Recombinant Factor VIII Products: Presence of Humanand/ or Albumin-Derived Plasma/albumin Exhibit 4.3 Comparison of Recombinant Factor VIII Products

Summary:

This Guide presents an overview of current techniques used for the production of protein therapeutics. The focus is on the use of large scale mammalian cell culture systems. The industry, which began in the late 1980s from the utilization of recombinant DNA technology and cell hybridization, is now a significant production process of the biopharmaceutical industry. As of 2003 the biopharmaceutical market represented over $40 billion in sales annually contributed by over 90 products approved for 130 disease indications. With over 350 additional products in clinical development and approximately 10 new products approved per year, growth for this market is expected to continue at >20% annually. Today, a quarter of all new drug approvals are for biopharmaceuticals. The approved drugs include hormones, blood factors, thrombolytics, vaccines, interferons, monoclonal antibodies (MAb), and therapeutic enzymes. The primary method of producing many of these biopharmaceuticals is mammalian cell culture. When Amgen launched Epogen in 1989 it set a precedent that many of its competitors were to follow. Erythropoietin (EPO) is a hormone made in the kidneys that stimulates production of red blood cells. Amgen had found a way of making EPO by recombinant DNA technology, which involved inserting the human gene for EPO into a non-human host, cell and harvesting the EPO protein from this gene. Today the worldwide sales for this single product have been reported to have exceeded $1 billion. Their choice of manufacturing technique—roller bottles—may seem archaic by today's standards but it allowed Amgen to produce clinical-grade material in sufficient quantity in little time. A typical antibody manufacturing facility requires a production capacity of 50-250 kg protein per year with periodic investments of $300-500 million every 5-10 years to turn a "greenfield" site into a fully geared manufacturing facility. In addition to the capital investment, 400-500 employees must be hired and trained. Start-up costs in the range of $100 million will be incurred for commissioning a plant with 6 x 15,000 L bioreactors. Validation costs alone can be as high as 15% of the final cost of the plant. Although new technologies such as transgenics are achieving regulatory acceptance mammalian cell culture is a faster production process to bring "on-line". It takes about 5 months from genetic engineering the cell line to producing enough material for comprehensive testing. Transgenic systems can take 1 to 3 years depending on whether plants or animals are considered. However, there are still many hurdles and challenges in creating a successful cell culture manufacturing system despite the relative ease in generating cell mass and product titers. This Guide provides an introduction and overview of current manufacturing practices associated with large scale mammalian cell culture. Topics covered include cell line selection, cell engineering, cell culture health, culture methods, potential contamination sources, and product characterization.