7 The Production of Biopharmaceuticals

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7 The Production of Biopharmaceuticals

B. Hughes, L.E. Hann

7.1

Introduction

The term biologics refers to a broad class of medicinal products that share a number of common features.

Unlike traditional medicines that are made by chemical synthesis, biologics are made by biosynthesis in living cells. Biologics are generally much larger than traditional synthetic medicinal products and range from highly complex inactivated vaccines and plasma- derived factors to highly purified, well characterised recombinant therapeutic proteins. As new biological therapies come to market, the term biologics may encompass a diverse portfolio and include therapeutic options such as gene and cellular therapies, therapeutic vaccines, and nucleic acid preparations. The scope of this chapter focuses primarily on therapeutic proteins produced in mammalian cell culture processes.

The use of therapeutic proteins as the treatment of choice for certain unmet medical needs was enabled by the convergence of two emerging technologies in the 1970s: genetic engineering and the science of cell culture. These technologies provided researchers with the ability to create specific recombinant DNA molecules encoding specific proteins and the methodology to introduce these recombinant DNA molecules into bacterial or animal cells that synthesised the protein. Fur- ther advances in cell culture technology permitted the development of high-viability, high-density cell cultures and the ability to scale cultures to larger volumes.

Cell cultures, maintained in large, computer-controlled, stainless steel bioreactors enabled large-scale protein production.

An interesting illustrative case history in the development of a biologic can be seen with the medicinal product alpha-interferon. In the early 1970s, interferons were heralded as promising therapeutics for a vari-

ety of disease conditions from viral infections to cancer. Initially, alpha-interferon was produced by purification of the active protein from human white blood cells. As cell culture technology advanced, a number of groups were successful in producing alpha-interferon in vitro, from cultures of transformed human lym- phoblastoid cells that spontaneously produced a range of endogenous interferons. The advent of recombinant DNA technology enabled the creation of DNA vectors containing the alpha-interferon gene and the successful expression of the gene in bacterial cells. In 1986, both nonrecombinant and recombinant alpha-interferons gained regulatory approval.

The introduction of recombinant expression systems cleared the way for several major protein products to be launched as therapeutics. Peptide hormones (erythropoietin, growth hormone, beta-interferon, reproductive hormones) (Chu and Robinson 2001;

Lubiniecki and Lupker 1994; Simson 2002; Walsh 2003a) and enzymes (tissue plasminogen activator) (Walsh 2003a, 2003b) were produced. These molecules were used as “replacement therapies” to treat patients with diseases caused by the deficiency of specific molecules; supplementation of endogenous protein levels with the recombinant product provided a therapeutic benefit. Frozen cell banks, containing recombinant cells producing these replacement proteins, provided a readily available supply of the required factor that was not dependent on rare and potentially hazardous raw materials such as human blood and tissue.

The next generation of protein therapeutics moved beyond the established strategies of managing disease states by restoring or supplementing endogenous proteins. Recombinant proteins emerged in the 1990s that included antibodies designed to bind to specific anti- gens or the cells they were attached to, permitting the removal or destruction of the antibody-bound moiety

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by the immune system or via toxic molecules attached to the antibodies. Antibodies targeting tumor markers [alemtuzumab (CamPath 2005), gemtuzumab ozogamicin (Mylotarg 2006) and trastuzumab (Herceptin 2005)] and markers of inflammatory disease [anti- tumour necrosis factor (TNF) antibodies for rheuma- toid arthritis (Humira 2005, Remicade 2005) and anti- IgE for asthma (Xolair 2005)] were successfully developed and deployed in the clinic, having a profound impact on a range of diseases. Additionally, the ability to screen patients and identify those who would respond to a particular therapy added a further refine- ment in treatment of various diseases. The trastuzumab molecule (Herceptin 2005), targeted toward the human epidermal growth factor receptor 2 (HER2) antigen present on certain tumor cells, has been described as an early example of “patient-directed- medicine”. In this model, the patient is first assessed for the presence and level of a specific cancer antigen, allowing for treatment with an antibody that binds to that specific antigen and recruits the immune system to attack the tumor cells. Additional mechanisms are sus- pected in the case of trastuzumab. Fusion proteins such as etanercept (Enbrel 2005), an anti–TNF-targeted therapy, joined the arsenal of therapeutic proteins in the late 1990s. Etanercept contains a portion of the human endogenous TNF receptor fused to the constant region (Fc) of an immunoglobulin molecule; the therapeutic effect of the molecule is to bind and sequester the proinflammatory cytokine, TNF.

The development of biologics for therapeutic pur- poses has shown a rapid series of advances over the past 25 years from the extraction of endogenous human proteins to the development and manufacture

Table 7.1. Top ten biophar- maceuticals by global sales [from IMS Health repro- duced in Lawrence S (2005) Biotech drug market steadily expands. Nat Biotechnol 23:1466]

Products (company) 2004 sales ($US millions)

Percent change

Annual

growth (%)^a 2004 market share (%) Erypo/Procrit (Johnson & Johnson) 3,989 –4.2 23.0 9.0

Epogen (Amgen) 2,897 –3.8 14.1 6.5

Enbrel (Amgen/Wyeth) 2,578 58.8 42.1 5.8

Aranesp (Amgen) 2,569 77.9 N/A 5.8

Remicade (Johnson & Johnson/

Schering-Plough)

2,506 19.8 130.8 5.6

Mabthera/Rituxan (Roche) 2,192 24.4 62.7 4.9

Neulasta (Amgen) 1,873 52.1 N/A 4.2

Avonex (Biogen Idec) 1,383 16.4 18.1 3.1

Neupogen (Amgen/Roche) 1,344 –6.8 2.5 3.0

Lantus (Aventis) 1,014 80.9 N/A 2.3

Total top10 22,346 20.7 31.3 50.4

Global biotech market 44,353 17.0 21.6 100.0

aCompound annual growth 1999 – 2003

N/A not available

of specifically designed molecules targeting specific mediators of disease processes. “Designer” antibodies, containing significant modifications and specializa- tion, add an even further level of complexity: some antibodies that target tumor cells contain a covalently bound toxin or radionuclide to replace or supplement the potency of the immune system. Additionally, advances in formulation science have allowed the development of liquid formulations that have improved patient convenience, compliance, and persistence with treatment.

7.2

The Success of Modern Biotechnology

The contribution of biotechnology to medical practice and the pharmaceutical industry can be evaluated by reviewing medical advances and product revenues, as well as looking at pipeline compositions and recent approvals. Biopharmaceuticals are a growing part of research and development pipelines across the pharmaceutical industry, with an ever increasing percent- age of discovery stage candidates being described as large molecules. Biologics that have gained regulatory approval over the past 10 years include molecules that offered new approaches to treating a range of diseases, allowing physicians to intervene close to the root cause of the disease rather than alleviating symptoms. Anti- bodies have been developed for the treatment of infec- tious disease (Synagis 2004), anemia (Epogen 2005), and allergenic asthma (Xolair 2005), and a number of anticancer antibodies have been added to the options available to oncologists (Walsh 2003a). Additionally, 60 7 The Production of Biopharmaceuticals

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specific anti-TNF therapies have made major contribu- tions to the treatment of inflammatory disease.

Data published in December 2005, based on 2004 sales of biotechnology drugs (excluding vaccines), showed that the global market for the top ten recombinant protein therapeutics was in excess of $44 billion (Table 7.1) (Lawrence 2005). In these reports, biopharmaceuticals constituted 5 of the top 20 best-selling drugs, and monoclonal antibody sales grew by 52 %.

Moreover, biotech molecules comprised 12 of 64 FDA new molecular entity approvals in 2003 and 2004 (F-D-C Reports 2005).

7.3

The Science and Technology Behind Modern Biopharmaceuticals

Heterologous gene expression is possible in a variety of systems including bacteria, yeast and animal cells, as well as in transgenic animals and plants. However, to date, every commercial therapeutic protein produced utilises either a mammalian or a microbial cell culture expression platform (Walsh 2005). The first licensed human biotechnology product, recombinant human insulin (Humulin), was produced using the bacterium Escherichia coli (Johnson 1983). Today, 30 – 40 % of all approved biopharmaceuticals are made in E. coli (Walsh 2005). The small size and relatively simple structure of insulin permitted successful manufacture in E. coli. However, insolubility issues necessitating expensive refolding steps, often associated with a reduction in bioactivity, have hampered the widespread use of bacterial manufacturing platforms. Many of today’s biopharmaceuticals are more complex recombinant replacement proteins (e.g. factor VIII for haemophilia A) or monoclonal antibodies that require post-translational modifications, such as glycosylation, for biological activity and stability.

While proteins expressed in yeast and transgenic systems are glycosylated, N-glycans produced by yeast and plants are different than those present in humans, thus creating immunogenicity concerns (Wurm 2004).

Recent advances in yeast glycoengineering, utilizing humanised Pichia pastoris, have demonstrated the abil- ity to produce therapeutic glycoproteins containing nearly homogeneous human glycoforms (Gerngross 2004), paving the way for the future production of glycosylated biopharmaceuticals in nonmammalian

expression systems. As of this writing, transgenic animal systems remain commercially unproven; currently, mammalian expression systems remain the platform of choice for the manufacture of high-fidelity, soluble glycoproteins. At present, about 60 – 70 % of all licensed biopharmaceuticals are produced using mammalian cell processes. For an excellent review on protein production in mammalian systems see Wurm (2004).

The majority of mammalian cell culture processes utilise Chinese hamster ovary (CHO) cells, although alternative cell lines have been used successfully including mouse myeloma (NSO), baby hamster kidney (BHK), human embryonic kidney (HEK-293), or human retina derived (PER-C6) (Butler 2005). CHO cells are the dominant platform due to their ability to grow rapidly in single-cell suspension cultures. CHO cells produce glycan structures similar, but not identi- cal, to those found in humans due to the absence of several enzymes present in the human glycosylation path- way (Jenkins and Curling 1994). However, unlike mouse cells that generate glycan structures that are highly immunogenic in humans (Jenkins et al. 1996), nonhuman glycoforms produced by CHO cells are generally not immunogenic (Butler 2005).

Suspension cultures offer advantages in terms of scale-up. Currently, stainless steel bioreactors as large as 20,000 litres are used for the manufacture of biopharmaceuticals (Thiel 2004). In 2001, 70 % of licensed processes for the production of recombinant proteins utili- sed stirred-tank bioreactors. While animal sera were required to support the growth of high-density cell cultures in the past, approximately 50 % of current manufacturing processes employ serum-free cell culture medium. The most common production processes for the manufacture of biopharmaceuticals are perfusion and fed-batch cultures (Hu and Aunins 1997). Perfu- sion culture systems allow for the continuous or semi- continuous removal of medium containing accumulat- ed inhibitory metabolic waste products such as ammonia and lactate and add an equal volume of fresh nutrients (Griffiths 2001). Fed-batch processes utilise slow feeding of key nutrients to maintain a low concentra- tion and steady level of primary carbon sources. This results in a more efficient primary metabolism that generates lower ammonia and lactate levels (Butler 2005). Fed-batch processes are cost-effective and have greatly increased yields. With the ability to control gas and nutrient levels as well as inhibitory waste products, cell densities of greater than 10⁷cells per millilitre can

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be achieved (Butler 2005). Currently, high producing processes are in the range of 3 – 6 grams per litre (Winder 2005; Wurm 2004).

Cell culture conditions can affect product quality.

Therefore, identification and control of critical process parameters are essential to ensure performance consistency from batch to batch. For example, the glycosylation profile of a particular recombinant protein can vary depending upon the culture duration, nutrient level, growth state of the cells, pH, temperature, and dissolved gas. Thus, process control and accurate ana- lytical methods to monitor process controls can greatly affect manufacturing process robustness and product quality.

Future technological advances will significantly influence the biopharmaceutical industry’s approach to manufacturing. Presently, mammalian cell culture processes remain competitive relative to other seem- ingly less expensive platforms due to the proven ability of mammalian processes to deliver high-fidelity, soluble, efficacious proteins. It is expected that further improvements, such as increases in overall productivity through increased volumetric productivity and increased yields via streamlined chromatographic purification trains, will enable mammalian expression systems to remain economically competitive with other expression platforms. However, as alternative manufacturing platforms demonstrate production and eco- nomic advantages through higher yields and shorter production times, while maintaining product safety and efficacy, these new production platforms may offer a competitive advantage. The development and application of new technologies to current manufacturing paradigms will be key in determining operational flexi- bility of manufacturing facilities, influencing future capital investments, overall manufacturing costs, and ultimately patient access to important new therapeutic proteins.

7.4

Process Development

The delivery of a new biopharmaceutical to the marketplace requires an extensive and extended period of process development involving the application of advanced techniques in molecular biology, cell culture, separation technology, and formulation science, taking several years to complete. Prior to regulatory approval,

development of the manufacturing process consumes considerable resources in terms of equipment and material as well as the people that are needed to prepare and characterise the clinical trial material. Once the manufacturing process is finalised, the process details are transferred to the manufacturing facility where the material is to be made. Details of the manufacturing process and the characterisation of the material produced are provided to the regulatory agencies to pro- vide evidence of a stable and reproducible process, molecule, and product. Process changes after regulatory approval require additional process and molecular analyses as well as additional filings in many cases.

The creation of the Master Cell Bank (MCB) is a critical milestone in biopharmaceutical process development. The MCB is a cryopreserved, long-term store of recombinant cells, either bacterial or mammalian, containing the gene that encodes the desired protein. Fol- lowing transfection of host cells with a DNA plasmid containing the desired gene, the cells are subjected to a cloning procedure to ensure genetic uniformity and then are screened to identify clones that have a stable, high-level expression of the desired protein. Once iden- tified, the desired clone is expanded and cryopreserved in vials, creating the MCB. The MCB is the source of all cells used to manufacture the medicinal product, either directly by thawing MCB vials or via an intermediate working cell bank (WCB) derived from the MCB. Cell banks are usually laid down as hundreds of vials and stored at multiple redundant locations to ensure secu- rity of supply. These cell banks are extensively tested and characterised to ensure that they are fit for pur- pose, stably express the desired protein over the manufacturing period, and do not contain microbiological contaminants.

Another major component of process development is defining the cell culture process that expands the small number of cells in the MCB or WCB vials into the large volumes of cells required to produce economically viable amounts of proteins in a production facility.

For example, the process may require expanding 3 million cells in a 1-millilitre vial to a 20,000-litre volume in a stainless steel bioreactor to achieve 10 million cells per millilitre. The cell culture development process establishes the appropriate nutrient media and specific physiological conditions for cell growth including O2

and CO2levels and the pH of the medium, as well as any specific manipulations required to achieve high levels of protein production. Processes may be separated into 62 7 The Production of Biopharmaceuticals

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Fig. 7.1. Overview of the production process for a biopharmaceutical product. [Reprinted with permission from Walsh, G (ed) (2003) Biophar- maceuticals: Biochemistry and biotechnology, 2nd edn. John Wiley & Sons, Chichester]

two phases including an early rapid cell growth phase to maximise the number of cells available to make protein and a second production phase to maximise protein output. The phase transition can be triggered by changes in bioreactor conditions or by the addition of certain induction molecules to the cell culture medium.

The cell culture process (”upstream process”) pre- sents the expressed protein to the “downstream process” for further processing to a pure active ingredient (Fig. 7.1). This downstream processing is usually com- posed of a harvest step that separates the cells from the protein product as well as a number of further separation steps that purify the protein product from the remaining cell culture-derived impurities. The process development team evaluates a wide range of separation technologies and experimental conditions to determine the optimal conditions for separating the protein product from the process impurities to ensure that all products made using the process will meet predeter- mined quality specifications.

An important activity in most process development projects is the definition of the formulation in which the protein is delivered to the clinic or marketplace. Chemi- cal solutions are assessed for their ability to maintain stable, intact, and biologically active protein for the desired shelf life of the biopharmaceutical. Biopharma- ceuticals generally are relatively fragile at room temperature and require cold chain transport and storage.

Long-term stability studies (several years in duration) are carried out on the protein using a range of tempera- tures followed by detailed characterisation to detect any changes in chemical structure or biological activity.

7.5

Biopharmaceutical Manufacturing

Over the past 2 decades, the manufacture of biopharmaceuticals has progressed dramatically to the highly complex, state-of-the-art operations that epitomise the industry today. Modern biopharmaceutical production facilities comprise multiple departments that function together to produce, test, and assure the quality of the biological drug (usually called the drug substance) before release to a fill/finish facility for drug product manufacture. Drug product manufacturing involves the preparation of the final, sterile presentation of the biological drug substance and can include lyophilised or liquid products in vials or syringes. The drug product is packaged before ultimate delivery to the patient.

Biopharmaceutical manufacturing is carried out under the philosophy and approach of good manufacturing practice (GMP). GMP is an aspect of quality assurance that ensures that the biological drug is con- sistently produced and controlled to a standard appropriate for its intended use. The tight controls present in modern biopharmaceutical manufacturing plants ensure consistency in the manufacture of the biological drug. Raw materials such as media, water, and gases are tested against multiple specifications before being released for use in the process. Cleaning procedures are validated to ensure that process residues or by-products are removed from equipment between successive batches. Sterilisation procedures are verified for all equipment, such as bioreactors and automated process equipment, governed by a central or distributed con-

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trol system, which helps to remove human error in the process step execution. Air quality is regulated by envi- ronmental control systems to maintain pressure differ- entials and high-quality, low-particulate air in processing zones that require clean operations such as late- stage purification operations. Taken together, these steps assure that all aspects of manufacture are tightly controlled to deliver a biological drug that meets a pre- defined series of pharmaceutical biochemical and functional specifications.

No aspect of the manufacturing process may be changed without proper technical assessment of its impact by authorised groups within the manufacturing organisation, and where relevant, regulatory authori- ties. This process, known as change control, is overseen by the quality organisation. During its manufacture, the biological drug is transferred between stages of the manufacturing process (e.g., drug substance to drug product manufacturing) only after all quality control test criteria are met and a quality assurance-led batch release process is executed.

7.6

Quality Assurance and Quality Control

Quality systems, including Quality Control (QC) and Quality Assurance (QA), ensure that patients receive a safe, pure, potent, and stable product. While there are considerable similarities between biopharmaceuticals and chemically synthesised drug products, the QA/QC issues and challenges are very different in some respects. Raw materials of biological origin are careful- ly selected and extensively tested to minimise the risk of microbial contamination of the cell culture systems.

Although biopharmaceuticals can be characterised chemically to varying degrees, it is generally accepted that the structure-function relationships and key deter- minants of activity for biologics are not fully deter- mined. For these reasons, most biopharmaceuticals require some element of biological testing in addition to chemical testing, as part of routine quality control processes. Immunological, biochemical, or bioassay techniques are often used to determine the concentra- tion and activity of the active ingredient. There is heavy reliance on in-process sampling and testing as well as testing of the final drug substance and drug product due to the complexity of the molecules. As most biopharmaceuticals are provided as injectable solutions

and are administered parenterally, careful attention is paid to the maintenance of sterility of the final product.

7.7

Facility Considerations

The continued development of the biopharmaceutical industry has resulted in a significant increase in demand for manufacturing capacity. The construction of a biopharmaceutical manufacturing facility is a major undertaking, even for large, established pharmaceutical companies. Wyeth’s Grange Castle facility in Dublin, Ireland, is an example of a modern integrated biopharmaceutical campus. For smaller start-up companies, the risk level and levels of human and financial capital investment required to build a manufacturing facility can be prohibitive. A large biopharmaceutical plant with the ability to generate bulk drug substance and manufacture final drug product can take up to 5 years to build and can cost up to $1 billion. Due to timeline considerations, the construction of these expensive facilities is often underway before the efficacy of the medicine or the marketplace demand is known. Because of the inherent risk and high cost associated with building a manufacturing facility, many companies decide to partner with another company with existing capacity or use a contract manufacturer.

There is a complex interplay between facility design, process development, and market assessment. It is not unusual for market volume predictions to vary by 50 – 100 % or more during the development of an innovative new biopharmaceutical. For new facilities, where emerging market data exceed the initial design capacity, a difficult series of decisions must be made. A change to the facility design during the detailed plan- ning phase will usually incur cost and time penalties. A similar change once construction has commenced can involve major increases in project cost and timelines. In this environment, the value of an effective process development unit and a sophisticated approach to project management are invaluable. An innovative process development group can help mitigate extra expense by increasing process output or recovery, in the bioreactor or downstream process respectively, increasing plant output without additional capital spending.

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7.8

Biosimilar Products (or Follow-on Biologics)

The era of follow-on biopharmaceuticals has arrived. A number of early biopharmaceuticals that lost patent pro- tection in 2006 are already in production by noninnova- tor companies (Walsh 2003a). Some of the follow-on biologics currently on the market include human growth hormone and alpha-interferon in eastern Europe, and colony stimulating factor in China (Walsh 2003a).

The terminology used for generic biopharmaceuticals is complex, with different terms having different regulatory implications. The terms “generic biopharmaceuticals”, “biogenerics”, or “generic biologics” are very similar to the small-molecule generic paradigm and suggest no requirement for clinical trials. Regula- tory agencies in the United States (Food & Drug Administration, FDA) and Europe (European Medi- cines Agency, EMEA) have adopted the terms “follow- on biologic” and “biosimilar” respectively. Implied in this terminology is the understanding that clinical trials would be required to assess safety and efficacy prior to approval. The EMEA has already issued guidelines for regulatory approval of biosimilars where limited clinical trials would be required for simple, less complex products, and more extensive clinical trials required for more complex products. While numerous stakeholders have asked that the FDA develop guidance paving the way for approval of certain “generic” biologics, as of this writing, the agency has yet to offer guidelines for generic biopharmaceutical production.

Many believe that the paradigm for chemical identi- ty between biopharmaceuticals and their generic counterparts is different from small molecule drugs. For small-molecule generics, pharmaceutical and bio- equivalence predict an equal therapeutic equivalence.

This certainly may not be the case for many generically manufactured biopharmaceuticals. Hurdles will be high for a generic biopharmaceuical to be approved as interchangeable with an innovator’s product, especially in terms of safety and efficacy profiles: the use of a different cell line for the manufacture of a generic biopharmaceutical may result in a different product profile with respect to heterogeneity, impurities, and glycosylation, raising potential immunogenicity and other concerns. The increased immunogenicity observed with reformulated erythropoietin (Eprex) is a caution- ary example illustrating how changes in stabilisers, storage, and route of administration can influence

human clinical immunogenetic responses, experi- ences, and safety (Casadevall and Rossert 2005).

The probability that follow-on biologics or biosimilars will require limited clinical trials to prove “compa- rability” will make the cost of these molecules higher than their small-molecule generic counterparts. While the targets for biosimilars are known and proven, the development of generic versions of biopharmaceuticals will take longer and cost more than small-molecule generics. Sales and marketing costs are likely to be incurred, as most biosimilars will not be approved as interchangeable with the innovators’ product. Howev- er, even with these costs, it is likely that the price of biosimilars may be lower than innovators’ biopharmaceuticals and the savings may be significant enough for healthcare payers to exert pressure or offer incentives to switch to the lower cost alternative. Safety issues such as immunogenicity, which are difficult to predict from animal models, remain a concern and may potentially affect the widespread use of biosimilars.

7.9

Conclusion

Biopharmaceuticals have become well established in the treatment of serious diseases. Hundreds of molecules, targeting a range of diseases, are being evaluated in the academic research community and in the pipelines of the pharmaceutical industry. In parallel, the technology for manufacturing these molecules is advancing. Higher capacity processes with better yields and reliability are beginning to make an impact on the high cost of producing these medicines, allowing more efficient use of costly manufacturing plants. Fur- ther progress in the technologies of biopharmaceutical discovery, development, and manufacturing is likely to increase the supply of important medicines to the patients who need them.

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