Animal Cell Biotechnology

Hansjörg Hauser, Roland Wagner, Hansjörg Hauser, Roland Wagner

• Publisher: De Gruyter
• Year: 2014
• ISBN:
  9783110381429
• Edition: 1
• Topic: Scienze biologiche
• Subtopic: Biologia molecolare

This book introduces fundamental principles and practical application of techniques used in the scalable production of biopharmaceuticals with animal cell cultures. A broad spectrum of subjects relevant to biologics production and manufacturing are reviewed, including the generation of robust cell lines, a survey of functional genomics for a better understanding of cell lines and processes, as well as advances in regulatory compliant upstream and downstream development. The book is an essential reference for all those interested in translational animal cell-based pharmaceutical biotechnology.

1 Introduction

1.1 Industrial Use and Perspectives of Animal Cell Culture

Roland Wagner and Hansjörg Hauser

1.1.1 Introduction

In 2013, it was estimated that there were more than 900 products in clinical phases up to submission and several thousand products in preclinical development ^[1]. The global pharmaceutical sales increased dramatically during the last 2 decades, e.g., it more than doubled from 2000 to 2009. The USA, with around 37 %, is still the world’s biggest single market. Growth in this market, which was above average until the first years of the new millennium, has tended to approximate the moderate dynamics of European markets in the past 3 years. Latin American and Asian markets have grown most strongly. Europe’s share of the world market declined in 2009 to 31 % (from 32 % in the previous year), due mainly to the Euro’s weaker exchange rate against the U.S. dollar. Germany’s global market share also fell very slightly in 2009 from 4.5 to 4.3 %. In real terms, assuming a constant exchange rate, Germany’s share of the world market decreased from 5.0 to 3.5 % over the past decade. Additionally, the product diversity is broadened by the placement of gene and viral therapeutics as well as cell therapeutics and antisense products to the portfolio. From the overall 907 products in development, 338 count for the largest group of monoclonal antibodies and 250 for vaccines.

In the mid-1980s the average accumulated protein product concentration ranged at ca. 200 mg/L. For producing today’s necessary amount of the anti-cancer drug Avastin^® of nearly 3 t per year several million liters bioreactor capacity would have been needed based on such a process. Subsequent developments particularly concerned cell culture nutrient media selection of suitable cell lines, cell line engineering, and adapted modular feeding strategies. A combination of achievements led to substantial extension of the cultivation time and hence the available time period for the enrichment of a product such that the amount of producing cells over the prolonged cultivation interval, the cell integral, based on a distinctly higher amount of viable cells, and cell specific productivity is increased. With these methods antibody product titer of more than 5 g/L and above can be achieved within 2 weeks of culture.

1.1.2 Cells as Bioreactors for the Production of Biologics

With the approval of the tissue plasminogen activator (tPA, Activase^®) in 1986 mammalian cell culture and particularly the Chinese hamster ovary (CHO) cell line became the most popular production system for the manufacturing of protein therapeutic products. Even 20 years after tPA approval, CHO cells remained as the preferred mammalian cell line for the production of recombinant protein therapeutic for several reasons. CHO cells are easy to handle and can grow in suspension culture, a prerequisite for a homogenous large-scale culture in the industry. Moreover, and very important, CHO cells pose less risk as few human viruses are able to propagate in them ^[2]. They can grow in serum-free and chemically defined media, which ensures reproducibility between different batches of cell culture and minimizes risk of contamination and impurities. Last, but not least, CHO cells are capable to perform post-translational modifications to recombinant proteins, which are compatible and bioactive in humans ^[3]. Specifically, glycosylation of glycoproteins produced by CHO cells are more human-like, with the absence of immunogenic α-galactose epitope ^[4]. Several gene amplification systems are well established to make use of the genome instability of CHO cells to allow for gene amplification, which ultimately result in higher yield of recombinant protein. Currently, recombinant protein titers from CHO cell culture have reached the gram per liter range, which is a 100-fold improvement over similar processes in the 1980s. The significant improvement of titer can be attributed to progress in establishment of stable and high producing clones as well as optimization of culture process. Due to these reasons, CHO cells are established host cell lines for regulatory approvals of therapeutic glycoprotein products ^[2], ^[3], ^[5]. Beside CHO, there are a handful of other cell lines accepted by the regulatory authorities derived from other the Syrian baby hamster, the Muscovy duck, insects and human tissue.

1.1.3 Protein Expression Technologies

The product is expressed in cells by applying appropriate genetic engineering techniques and transfection of the cells with the expression vector bearing the desired genes of interest. The cellular productivity can be modulated by selecting special DNA regulatory elements carried on the vector and by targeting its integration site on the host cell genome. Productivity can also be increased through improving cell culture characteristics via cell line engineering ^[6].

Antibodies Make up the Biggest Group

Monoclonal antibodies are the fastest growing category starting from 1 % in 1995 to 14 % in 2001 and more than 70 % in 2013. Based on their molecular structure and the resulting binding properties, they have the skills to specifically recognize antigens and cellular markers. An incredible amount of variants and their distinct action is the basis for a nearly inexhaustible therapeutic potential. For the year 2015 the market volume of monoclonal antibodies is estimated to 64 billion U.S. dollars covering at least 38 % of the total biotechnology-based pharmaceutical market which will be shared with other proteins and vaccines then encompassing around 170 billion U.S. dollars. From the 38 billion U.S. dollars in 2009 an approximate double in growth for the antibody market is prognosticated ^[7].

Personalized Medicine Drives Industry to the Development of new Drugs and More Efficient Manufacturing Processes

Personalized medicine will lead to the renunciation from standard therapy approaches looming today, that has been offered for all patients. At the same time this will require a drastic increase in the diversity of therapeutics. Further, this trend will reduce the market share of so-called blockbuster drugs as the therapeutic antibodies Avastin^®, Herceptin^®, Rituxan^®, or Enbrel^®. Industry reacts to this situation by efficiency increase in the development of new drugs and by a substantial reduction of the development period and/or by the application of innovative economic manufacturing processes.

The Biological Potential and the Technological Effort Assign the Process Limit

The rapid and straight increase in the productivity of biologics-producing processes during the last 5 years up to now easily tempts to assume that this progress could keep on with practically endless constancy. While the theoretical end of cellular productivity is not yet reached, from the industrial point of view a final titer will be sufficient when a stable platform process at maximum economy is reached. The maximum cellular productivity might be estimated by the production capacity of a natural high producer cell that has developed throughout biologic evolution. An antibody-producing B lymphocyte is able to produce up to 20,000 antibody molecules per second ^[8]. Related to a contemporary cell culture in a bioreactor this is equivalent to a production of 6 grams per liter and day at a representative cell concentration of 10 million cells per milliliter culture broth. Translating this capacity to a typical fed-batch process over a cultivation period of 14 days, 80 grams antibody product per liter culture would be produced. This considerable amount is still more than 10-fold higher than a state of the art process today (see above).

Increase in Productivity and Process Realization Have to Form a Unit

In general, product maximization leads to a higher yield. However, this advantage is often accompanied by an increase in rather undesired side products and reduction of product quality that take out the shine from the laudable but unreflected titer numbers. A higher protein concentration imposes additional molecule interactions leading to a higher formation of aggregates and substantially impeding following process steps, which in extreme result in an uneconomic process by a costly and inefficient product purification. Therefore, the economic reason will aim to a balanced situation, which will be adjusted by the competition between the biochemical and cell physiological potency as well as the height of the production capacity and the resulting possibilities. In reality, production bioreactors for cell cultures of today have a working volume of about 10,000 L. Such a volume is integrated in the flow of the total process in a way that the upstream and downstream process form a procedural unit. A significantly higher product concentration will induce new challenges for the purification concept. Chromatographic systems must take enormous dimensions and the purification efficiency has to be substantially increased, such that the higher amount of incompletely or even incorrectly processed, possibly denaturated or aggregated protein will be robustly separated from the desired product in order to guarantee a constant high quality. Primarily, such an additional investment for the manufacturing of new antibody therapeutics would be hardly justifiable. Therefore, the development of cell-based expression systems will preferably focus on the optimization and intensification of the product quality and titers ranging in the one-digit gram range will probably dominate future processes in view of economics. Nevertheless, the request of achieving high product titers will mostly drive the process expectations, especially when competitive biosimilars are developed which shall displace existing products from the market after patent closure.

Biosimilars Require Processes of Highest Robustness

Generics must contain the same active ingredients as the original formulation as they are considered identical in dose, strength, route of administration, safety, efficacy, and intended use (U.S. Food and Drug Administration on generic drugs). This term is only used for small molecule-based drugs produced by simple processes. In contrast, biologics generally exhibit high molecular complexity and are quite sensitive to changes in manufacturing processes since they are made by or derived from a living organism. Differences in impurities and/or breakdown products can have serious health implications. This has created a concern that copies of biologics might perform differently than the original branded version of the product. Consequently, only a few versions of follow-up biologics have been licensed in t...