Biopharmaceutical Development

5.39

Biopharmaceutical Development

CM Smales and RJ Masterton, University of Kent, Canterbury, UK © 2011 Elsevier B.V. All rights reserved.

5.39.1 5.39.2 5.39.3 5.39.4 5.39.5 5.39.6 5.39.7 5.39.8 5.39.9 5.39.10 5.39.11 5.39.12 5.39.13 5.39.14 References

Introduction Development of Vaccines The Biopharmaceutical Development Pipeline Regulatory Requirements Selection of Biotherapeutic Protein Expression Systems Development of Mammalian Cell Lines Development of Mammalian Expression Vectors Cell Culturing and Product Generation Downstream Processing of Biopharmaceuticals Viral Inactivation of Biologics Process Analytical Technology Formulation and Drug Delivery Systems Biosimilars Conclusions

Glossary biopharmaceutical/biologic Protein-based medicine or vaccine produced by a living or cellular system. biosimilars A copy of a biopharmaceutical that has lost its patent protection. development pipeline This describes the time taken and the processes required to develop, manufacture, and launch a biopharmaceutical onto the market. downstream processing The steps involved in purifying and recovering biopharmaceuticals from contaminants and impurities to generate a pure drug fit for market. electroporation A transfection technique which administers an electric impulse to the cells of interest resulting in the transient formation of micropores in the membrane through which DNA can easily diffuse. expression systems Living organisms such as bacteria, yeast, insect, plants, and cultured mammalian cells which are used to produce recombinant therapeutic proteins. expression vectors A vehicle used to transfer genetic material to a cell to allow expression of a specific protein within this cell. genetic engineering The manipulation or alteration of genetic material, specifically in this case of the cellular systems used to produce biopharmaceuticals.

489 490 490 491 491 493 493 495 495 497 497 497 498 498 498

patent A legal protection of new inventions which prevents others from making and selling their patented products. process analytical technology (PAT) The use of analytical technology to monitor biopharmaceuticals throughout the production process to validate the product reproducibly reaches specific specifications. recombinant DNA technology Biological systems express foreign-imported genes to generate products which are not normally expressed in the chosen system. recombinant mammalian cell lines Mammalian cells genetically modified to produce pharmaceuticals. transfection The process of introducing foreign DNA into a mammalian cell. transient/stable expression Transient expression occurs when a gene is successfully transported into the nucleus of the cell but is not incorporated into the cell’s genome. The expression of this gene is therefore transient. Stable delivery integrates the gene of interest into the genome of the target cell resulting in long-term gene expression. vaccine Biological sample which provides immunity to a particular disease.

5.39.1 Introduction Biopharmaceutical development encompasses a wide range of disciplines and techniques that are utilized during the development of bioscience-based medicines known as biopharmaceuticals or biologics. These drugs are composed of molecules which are produced in a living or cellular system rather than being chemically synthesized, and the production of biopharmaceuticals requires sophisticated manufacturing procedures. As such, there are a number of important issues to be considered during biopharmaceu­ tical development. Initially, development is centered on candidate identification and bioactivity optimization. Once a particular drug has been developed sufficiently, a process for the generation of enough material for clinical trials must then be put in place. The

489

490

Biopharmaceuticals, In Vitro Drug Testing and Drug Delivery

safety of these biotherapeutic proteins must also be demonstrated alongside the reproducibility and robustness of the processes used to produce these. If the trials are successful, in order to take the biotherapeutic beyond this level, a scalable manufacturing process must be set up. There is a real need to minimize the time it takes to generate such products at all stages of the development process, from taking candidate drugs through research and development to their manufacture and on to market. Processes must also be developed to deliver efficient drugs in the quantity required to meet market demands at an acceptable price. There is thus a perceived need for accelerated development of cheaper, safer, and more efficient biopharmaceuticals for effective therapy to treat many serious diseases such as cancer, immune disorders, and infectious diseases [3]. As of 2006, 165 biopharmaceutical drugs had gained approval for use, and a more detailed summary of the products which have been approved for use in the United States and Europe is provided in Reference [10]. Together, these biopharmaceuticals generated a market size [7] estimated at US$33 billion in 2004 [7]. The demand and market size seem to be set to increase for the foreseeable future [10], thus confirming the importance of these biomolecules to industry, health professionals, and the general public. In addition, new biosimilars are now being developed and are likely to be an increasingly important section of the biopharmaceutical portfolio. Finally, vaccines are also currently receiving much attention in terms of development and use with the outbreak of a number of viruses across the globe. This article largely focuses upon the development of processes for the production of such protein-based biotherapeutics using mammalian cell expression systems.

5.39.2 Development of Vaccines The largest and fastest growing groups within the biotherapeutics are the recombinant monoclonal antibodies (MAbs) and vaccines which provide immunity to a number of diseases. Vaccines are a therapeutic protective inactive or attenuated microorganism or purified product, which stimulates the body’s immune response to produce antibodies which recognize the agent as foreign. The vaccine is then destroyed by the body; however, the immune system remembers this and is then ready to recognize and destroy active pathogens that resemble the vaccine if it were to infect the body at a later date. Monovalent vaccines immunize the body against a single pathogen, whereas multivalent vaccines immunize the body against more than one strain of the same pathogen or against different organisms. The number of vaccines administered has increased significantly over the last 20 years and they are used for the protection against a vast array of diseases such as measles, mumps, influenza, tetanus, diphtheria, and bacterial meningitis. The power of vaccines and vaccination was demonstrated by the worldwide eradication of the smallpox disease [8] and there is now much effort and research being undertaken into developing vaccines against human immunodeficiency virus (HIV) and cancer, particularly in the prevention of relapse in cancer patients. Originally, vaccines were inactivated or dead microorganism strains were, such as used those vaccines administered for influenza and hepatitis A. Inactivation was achieved by the addition of the chemical formaldehyde or β-propiolactone [8]. Some vaccines actually contain live microorganisms, but their pathogenicity is attenuated due to abnormal culturing leading to inhibition of virulent properties. An example is the tuberculosis vaccine which is developed from an attenuated strain of Mycobacterium bovis [8]. In some infections, it is not the microorganism which brings about the disease state but rather the toxins they produce. Therefore, toxoid-based vaccines are inactivated toxins which bring about immunity against the toxic compounds and work against diseases such as diphtheria and tetanus [8]. Instead of injecting an inactive or attenuated microorganism, other vaccines, termed subunit vaccines, work by introducing only a fragment of the microorganism, such as an external surface protein of the pathogen. An example of such a subunit vaccine is the vaccine against Haemophilus influenza type b [8]. Genetic engineering and the development of recombinant DNA technology now allow a single protein of the microorganism to be produced to generate a vaccine to invoke immunity against diseases such as hepatitis B [8]. The advantage of this latter approach is significantly improved safety with no risk of recovery of virulence of the microorganism within the human body. Considerable research has been undertaken into improving recombinant vaccine devel­ opment and a method of production could follow a similar process to the other biologics as discussed in the sections below.

5.39.3 The Biopharmaceutical Development Pipeline It takes about 10–15 years to develop and launch a biopharmaceutical, this time covering from initial discovery of a new drug candidate to the time when it is finally approved and available for treating patients (see Figure 1). The process of biopharmaceutical production is a long and complicated procedure which usually follows a well-trodden and defined research and development pipeline. The pipeline usually begins with a prediscovery step where time is spent on understanding the disease to be treated and investigating ways in which to potentially treat the disease. This phase of development may be extremely long and usually builds upon work by many academic and industrial laboratories. Biomolecules involved in the disease of interest are identified during this stage and either eliminated as potential targets or forwarded as targets for potential medicines. These targets are then further validated to confirm that they are actually involved in the disease of interest and that they can be influenced by a drug. A drug molecule, also known as a lead compound, that can act upon the target of interest is then sought. Many promising compounds are initially selected which must then go through early safety tests. One of the key early tests is how specific a drug target is and the identification or eradication of off-target effects. Lead compounds which successfully pass the safety studies are then altered structurally to make them safer and more effective.

Biopharmaceutical Development

Development

Discovery

Pre Drug Preclinical discovery discovery trials

491

Clinical trials

Health regulatory Large-scale Phase 4 authority manufacturing studies approval

Phase 1 Phase 2 Phase 3

10–15 years Figure 1 Biopharmaceutical drug discovery and development follows a well-defined research and development pipeline. The discovery and development of new biologics is a long and complicated process which takes about 10–15 years. The overall process is termed the research and development pipeline and the majority of original candidates fail to reach the end of this line. Initially, a disease state is chosen and target molecules are selected during the prediscovery phase. Drug candidates are then chosen and preclinical testing is undertaken to ensure that the drug is safe to use in the following human clinical trials. Safe candidates then require approval for patient use, before large-scale production of the new medicine can occur. The drug is studied further during ongoing trials in the fourth phase.

During preclinical testing further controlled trials are performed on optimized compounds in the laboratory and in animal studies. Often, a process will initially start with many drug candidates (e.g., can be as high as 5000–10000+ for small molecules or, for example, phage display-generated libraries), but during testing and development attrition occurs to lead to the identification of one to five candidate molecules which can be assessed by clinical trials in humans. Testing is undertaken in three trials and successful candidates must be shown to be safe and effective. In phase 1, the drug is tested on healthy volunteers (20–100) to investigate as to how the product affects the human body. In phase 2, the drug is tested on a small group (100–500) of patients suffering from the disease of interest to study the safety and efficacy against the disease. In phase 3, the trial is completed on a large group of patients (1000–5000) to investigate efficacy and side effects of the drug. This trial is the most expensive and extensive of all three. Failure at any one of these steps means that the drug will not be approved. Candidates that successfully pass through the clinical trial phases must then be approved by the health regulatory authority in order to allow the new medicine to be sold to patients. Upon successful registration, the candidate can be manufactured at a large scale and then distributed for use in patients. However, before this a process for the manufacture of the product must be in place for the generation of clinical trial material. Postmarketing studies make up phase 4, the final trial; these studies monitor the long-term effects of the medicine. As described above, this article focuses upon the development and manufacturing stages of biopharma­ ceutical production. A more complete description of the early discovery stages and clinical trials phase is described by Jain [6].

5.39.4 Regulatory Requirements Before a biopharmaceutical can enter large-scale manufacturing, the government agencies (Food and Drug Administration (FDA) in the United States and the European Medicine Agency in Europe) must approve that the drug is safe and suitable to be put on the market and to be used by patients. One condition of the approval is that the biopharmaceutical company must conduct further monitoring and safety checks once the product is out in the market and being used on patients through phase 4 clinical trials and postmarket safety surveillance. Besides regulating the safety of biopharmaceuticals, there is a legal requirement for biopharmaceutical companies to constantly check the quality assurance of each stage of the development process and clearly document all information at each stage. Compliance of regulatory requirements must occur for facility designing, at testing, within production systems, during trials, at scale-up and manufacturing, and during labeling, advertising, and marketing of the product. Constant assurance of the efficacy of the biologic is also required throughout the development pipeline.

5.39.5 Selection of Biotherapeutic Protein Expression Systems Today the majority of biopharmaceuticals are produced by recombinant DNA technology through expression of gene products that would not normally be made in these systems. This in vitro production of recombinant therapeutic proteins can be achieved from a variety of expression systems, including bacteria, yeast, insect, plant, transgenic animal, and cultured mammalian cell systems. Table 1 describes examples of approved biotherapeutics expressed using different expression systems. A comparison of expression

492

Table 1

Biopharmaceuticals, In Vitro Drug Testing and Drug Delivery Example biopharmaceutical products and expression systems used to produce these

Protein

Therapeutic use

Company

Date approved

Expression system

Human insulin Human growth hormone Hepatitis B surface antigen Human growth hormone HER2 receptor

Diabetes Growth hormone deficiencies Vaccine hepatitis A and B Growth disturbance Breast cancer

Eli Lilly Pharmacia now Pfizer GlaxoSmithKline Biopartners Genentech

Bacterial E. coli Bacterial E. coli Yeast S.cerevisiae Yeast S. cerevisiae Mammalian CHO

Follicle-stimulating hormone Factor VIIa

Infertility Hemophilia

Organon Novo Nordisk

Human growth hormone IL-2 receptor

AIDS-associated wasting Transplant rejection

Serono Hoffman La Roche

1982 (US) 1995 (US) 2002 (EU) 2006 (EU) 1998 (US) 2000 (EU) 1997 (US) 1996 (EU) 1999 (US) 1996 (US) 1997 (US) 1999 (EU)

Mammalian CHO Mammalian BHK Mammalian Mouse C127 Mammalian NS0

This table provides a number of examples of biopharmaceuticals which have been approved and are on the market with details of the expression systems used to generate them (details obtained from [3, 10], please see this reference for a complete summary of approved biopharmaceuticals in the United States (US) and Europe (EU) up until 2006). HER, human epidermal growth factor; IL, interleukin; AIDS, acquired immune deficiency syndrome; CHO, Chinese hamster ovary; BHK, baby hamster kidney; NS0, mouse murine myeloma.

Table 2

Comparison of the characteristics of different biotherapeutic expression systems

Characteristics Cell growth rate Cell doubling time (hours) Cell density Complexity and cost of culture medium Expression yield Protein folding Posttranslational modifications Process costs

Bacteria

Yeast

Insect

Mammalian

High 0.5 High Low High No No Low

High 1.5 High Low Medium No Low Low

Low 18-24 High High Medium Yes Medium Medium

Low 24 Low High Low Yes High High

A general comparison of cultured expression hosts describing the advantages and disadvantages of each system.

systems is shown in Table 2. Currently, bacteria and mammalian cells are regarded as the key expression systems for the production of biologics. In 2006, Walsh [10] reported that since 2003, 31 therapeutic proteins were approved, of which nine were produced in Escherichia coli and 17 in mammalian cells. The advantage of using a bacterial system is that high cell concentrations can be achieved rapidly and relatively inexpensively to produce high yields of recombinant protein. However, bacteria are incapable of performing many of the essential posttranslational modifications required to synthesize complex recombinant human proteins in a biologically active state. For such complex therapeutic proteins, it is imperative to have these correct alterations to be clinically effective, so this often limits the choice of expression host to mammalian cells, since these cell systems contain the organelles and enzymes required to perform the correct posttranslational modifications [12]. These cells, however, have a slow growth rate and doubling time compared to bacterial systems, and the process of recombinant protein production in such systems is relatively expensive and timeconsuming. Yeast, insect, and plant cells all grow to higher cell densities than mammalian cells and they have shorter fermentation cycles and are thus less expensive. They are also able to undertake some posttranslational modifications such as glycosylation but the modifications are not as advanced as in mammalian cells. Mammalian cell systems are therefore currently the most effective producers of large complex therapeutic proteins which have specific folding and posttranslational modification requirements. However, as shown in Table 2 and described above, the culturing of these cells is complex, expensive, and relatively slow; there is thus significant interest in developing alternative systems for generating less expensive biologics more quickly. There are now studies describing antibody production in yeast [5] and filamentous fungi [11] systems, and progress is being made in the production of whole and fragments of antibodies in bacteria [3]. Further, there are many approved simple therapeutic proteins which have been produced in E. coli, such as the first recombinant protein, insulin (Humulin, produced by Eli Lilly in 1982) [10]. Aside from yeast and microbial systems, investigations are also being undertaken into expressing therapeutic protein in transgenic systems such as in plants, in the milk of transgenic animals, and in the whites of eggs produced by transgenic chicken [10]. These systems work by modifying the genome of organisms by introducing recombinant DNA which forces the organism to produce recombinant proteins. Despite the developing promise of these alternative systems, mammalian cells remain the key

Biopharmaceutical Development

493

workhorse in biopharmaceutical production and considerable research is currently being performed to further enhance the efficiency of mammalian cell culturing. Indeed, productivity levels continue to improve dramatically with recombinant protein yields reaching in excess of 5 g l−1 [10].

5.39.6 Development of Mammalian Cell Lines Here we use the development of mammalian cell lines as an example of development of a biopharmaceutical production process. As described above, the majority of biopharmaceuticals now used in the clinic are produced by recombinant DNA technology through expression of gene products that are not normally expressed in the chosen system. Further, as described above, in vitro production of recombinant therapeutic proteins can be achieved from a variety of expression systems, although many biopharmaceutical drugs currently on the market or in development are produced from in vitro cultured mammalian cell lines, particularly Chinese hamster ovary (CHO) cells. The original source of the CHO cell line was a female Chinese hamster by T. Puck in 1957 and this has been further adapted and modified to generate the industrially relevant CHO derivatives used today. A survey of the literature indicates that all approved biopharmaceuticals produced in mammalian cell expression systems to date are secreted proteins because they generate therapeutic protein by expressing genes cloned into cells and then secrete the desired protein into the surrounding media. Cell lines selected for high productivity in the manufacturing of biomolecules need to be capable of consistently producing high concentrations of a uniform product and have optimal growth characteristics, to be able to grow to high viable cell concentrations and then remain viable for a reasonable length of time. When choosing a cell line, there is a need to consider the speed at which a high yield can be achieved and the importance of protein modifications. CHO, hybridoma, and nonsecreting murine myeloma (NS0) cells are the most commonly used model mammalian systems for the production of recombinant proteins [3]. Over time, cells have been modified and selected to optimize cell growth conditions, which in turn lead to increased recombinant protein production. Cell lines have been improved to show enhanced stability, development of more robust host cell lines, and adaptation to growth in specific environments. Cell line development has directly led to an increase in the amount of recombinant protein produced, although this approach, along with media development, has largely been driven by trial-and-error approaches rather than a knowledge-based strategy for the enhancement of recombinant protein production. In 2004, Wurm reported that production processes have improved greatly since the mid-1980s to generate a near 100-fold yield improvement in product titer (50 mg l−1 in 1986 to 4.7 g l−1 in 2004) [12]. Most of this improvement was attributed to media optimization by modifying media formulations, generating different media for different stages of the growth phase of the cells, and supplementing vital nutrients that have been depleted during culture to eliminate nutrient deprivation (feeding strategies). This approach reduces the stress on cells and results in higher cell concentrations. As a direct result of this, more cells exist in the culture and thus more recombinant protein is produced. The enhanced selection of highly productive suspen­ sion-adapted host cells has also contributed to this significant improvement, the topic of which is discussed in further detail in the next section.

5.39.7 Development of Mammalian Expression Vectors In order for the host cell to produce the foreign protein product of interest, appropriate expression vectors need to be engineered and designed so that efficient transcription of the recombinant gene is achieved. This vector must then be transfected into the cell. The vectors with the gene of interest contain a promoter located at the 5′-end of the DNA sequence that drives the gene expression. The type of promoter present determines how efficiently expression is driven. Some vectors use a relatively weak promoter (e.g., Simian virus 40 (SV40)), whereas others contain a strong promoter (e.g., hCMV (Cytomegalovirus)). In the case of recombinant biologic production, strong promoters are used. Prior to transfection, the vector is usually linearized to improve the efficiency of DNA uptake. Various methods of introducing the foreign DNA of interest into mammalian cells exist. These include DNA transformation, direct microinjection, electroporation, liposome encapsulation, and the use of viral vectors. The advantages and disadvantages of each of these methods are detailed in Table 3. The most common method of DNA transformation (encapsulation via the endosomal pathway route) involves mixing DNA with calcium chloride and then incubation in a buffered saline and phosphate solution to generate a precipitate. Cultured cells are mixed with this DNA calcium phosphate co-precipitate, and following adherence to the cell surface enters the cell via endocytosis. Alternatively, DNA can be injected into the cytoplasm or directly into the nuclei of cultured cells using a glass capillary micropipette powered by a syringe in a process known as microinjection. The technique of electroporation involves administration of an electric impulse to the cells of interest, which results in the transient formation of micropores in the membrane through which the DNA can easily diffuse. DNA can also be encapsulated into lipid micels known as liposomes. The cationic lipids mask the DNA’s negative charge generating an overall net positive charge. This liposome/nucleic acid complex fuses with the negatively charged cell membrane and is readily taken up by the cells. Finally, viruses containing an inserted nonviral gene in their genome can infect cultured cells and carry the introduced DNA into cells. However, this approach is not used for biopharmaceutical production due to fears of disease from the virus.

494

Table 3

Biopharmaceuticals, In Vitro Drug Testing and Drug Delivery Comparison of methods for gene transfer into cultured mammalian cells

Gene Transfer Method DNA transformation Direct microinjection Electroporation Liposome encapsulation Viral vectors

Advantages

Disadvantages

• Components are cheap and easily available • Both transient and stable transfection • DNA can be injected directly into the nucleus or into the cytoplasm • High efficiency of DNA uptake • Does not limit size of DNA carried

• Reaction is dependent on pH so protocol can be difficult to optimise • Requires expensive equipment

• Most efficient method of DNA transfection

• Requires operator expertise • Can be very cytotoxic (50% viability) • Low efficiency of gene transfer • Liposome may be immunogenic • Vectors may be immunogenic • There is a limit to the DNA size delivered • Fear of disease from virus

Five methods of gene transfer that have a range of advantages and disadvantages in terms of efficiency, cost, ease of use, and safety. Viral infection is the most effective method but it also has the most disadvantages and therefore a nonviral transfection method is preferred for biopharmaceutical manufacturing.

In large-scale manufacturing, stable transfection is required (although transient expression is now being used for the rapid expression of early phase material); this involves integrating the gene of interest into the genome of the target cell, resulting in long-term expression and, as the genetic information, including the recombinant gene, is distributed equally to the daughter cells, expression continues into the next generation. Through stable expression, grams per liter of recombinant protein can be generated with high producing cell lines; however, the generation of stably transfected cell lines for manufacturing processes is labor-intensive and time-consuming, and generally takes from 3 to 9 months. In contrast, transient expression which occurs when the gene is successfully transported into the nucleus of the cell but is not incorporated into the cells genome can occur within days rather than months. The drawback of transient expression is that the production of the target protein generally lasts only days and the yields are much lower than in stable expression (tens to hundreds of mg per liter). Consequently, transient expression systems are sometimes used for small-scale development studies for rapid expression technology before generating stable systems for large-scale manu­ facturing. Further, research is being undertaken into generating large-scale transient expression systems to speedup the product generation stage of the biopharmaceutical development process [12]. Through transfection the DNA is randomly integrated into the genome and the location of the insertion affects the stability and transcription rate of the gene of interest. In order to prevent negative positional effects that can occur, such as due to integration near the telomere of the chromosome, technology such as retrotargeting has been developed to allow the insertion of the gene into specific loci in the genome. The targeting of gene integration into a transcriptionally active site enhances gene expression and avoids the random approach, which the technologies described above result in. Another approach to further improve expression levels involves modifying and optimizing coding regions of the gene of interest. An example of useful codon optimization is when the gene contains rare transfer RNA (tRNA) codons which can restrict or limit the expression levels–these tRNAs can be replaced with more common and abundant codons to allow high expression of the gene [12]. As a general rule, the amount of protein product produced from recombinant DNA is roughly proportional to the number of functional gene copies present [2], although this is not always the case. For example, it is generally accepted that messenger RNA (mRNA) levels reach a saturation level beyond which additional transcription and generation of mRNA do not result in further recombinant protein production. Sometimes, as described above, cells are high producers because the gene is integrated at a favorable site rather than multiple copies of the gene being present. There are, however, potential methods to improve expression systems, thereby increasing the levels of recombinant proteins by increasing the copy number of the integrated gene by gene amplification. One method used to achieve this is to use a selectable marker such as dihydrofolate reductase (DHFR) incorporated into the same plasmid as the recombinant gene of interest to allow selection of cells containing the engineered recombinant vector [12]. Over several rounds of selection, the additional integrated gene of interest is amplified. The DHFR system protects the cells containing the recombinant vector and selection gene by providing resistance to methotrexate (MTX) [12] which is present in the culture. Cells without the DHFR enzyme become poisoned by MTX and die, whereas cells containing DHFR survive due to the enzymes ability to inhibit MTX. A different approach is to use the glutamine synthetase (GS) system where cells are grown in a glutamine-free media, and in order for them to survive they must produce glutamine. Cells containing the GS system selection gene, with the recombinant gene, are able to produce their own glutamine as GS synthesizes glutamine from glutamate and ammonium. In contrast, cells without the GS vector are unable to synthesize glutamine and are therefore unable to survive in a glutamine-free environment. Once transfection has successfully been completed, limited dilution can be performed whereby single cells are transferred to a second cultivation vessel and then allowed to grow and expand to produce clonal populations. The resulting individual clones are then screened, and the highest producing cell clones that stably express the gene of interest are selected and isolated and cultured further before being subjected to analysis and screening to select a final cell line for manufacturing. This screening and selection of optimum high producing cell clones is time-consuming, monotonous, and labor-intensive in the development of mammalian cell lines engineered to express biotherapeutic proteins [3]. This more traditional approach of screening candidates has now often been replaced by automated cell sorting.

Biopharmaceutical Development

495

5.39.8 Cell Culturing and Product Generation For large-scale manufacturing of biopharmaceuticals, the host cells of choice expressing the desired protein are resurrected from virus-free frozen stocks of a working cell bank. As the cells grow, the culture volume expands and the cells are transferred into larger reactors or inoculum vessels. A choice of two culture systems is then usually used. The most commonly used is fed-batch culturing; This involves feeding the culture with key nutrients during the fermentation process. As described earlier, by feeding cells and developing and optimizing media, the growth and productivity of a culture can be enhanced. Upon feeding with nutrients such as glucose and glutamine, the maximum viable cell concentration is achieved and duration of culture can be increased due to the cells remaining viable for longer time periods because nutrients have not become limiting. A second approach, continuous perfusion culturing, also involves feeding the culture by continuously adding fresh media; however, spent medium is also continuously removed. This approach therefore prevents starvation of nutrient and also, unlike in the fed batch, prevents the buildup of toxic byproducts such as lactate and ammonia which eventually kill the cells by inducing apoptosis and the culture is then terminated. The key disadvantages of the perfusion system are its complexity and the extra time it takes in comparison to feeding cultures. Whichever system is selected, the cultures have traditionally been grown in suspension with cells being adapted to such growth, in stirred stainless steel tanks with volumes of 20 000 l now being used with cell numbers reaching in excess of 107 ml−1 [3]. In recent years, there has been an increased demand for disposable culturing approaches, particularly during small-scale or at the inoculum stage of large-scale manufacturing, because the tank cleaning and sterilizing steps are eliminated, saving significant time and, in turn, money. The cultures are mixed in the culture vessel by either rocking or stirring to allow successful mass transfer of oxygen and carbon dioxide; there are strict controls of temperature, pH, and dissolved oxygen content within the reactors [3] to optimize cell growth and productivity. Antifoam can also be used to prevent foam formation. In academic laboratories, another approach used to optimize culture growth is the addition of serum to the media as it contains molecules which aid in cell growth, such as growth factors. There is, however, concern with using media containing serum in manufacturing because of the possible introduction of adventitious agents into the culture system. Further, serum is relatively expensive and introduces many additional undefined molecules which must be removed during purification. These concerns have driven the development of chemically defined proteinfree media which are now routinely used in the manufacturing process. Such media does not contain any animal-derived material and as every component is well characterized and necessary for cell growth it is easily reproducible. This is in direct contrast with serum where the quality and components can change from batch to batch, potentially bringing variations into culturing. Cultured mammalian cells follow a typical growth curve with four key stages: lag, exponential, stationary, and finally death or the decline phase. During the lag phase, the cell numbers increase slowly as the cells adjust to the new environment. Once adjusted, the cells grow and replicate rapidly at a rate characteristic of the selected cell type in the chosen conditions. During rapid cell growth (exponential phase), usually less recombinant protein is produced and secreted. Instead, larger quantities of protein are generated when cells are in the stationary and death phase; these stages develop when the environment becomes hostile and cells become stressed due to nutrient depletion and the accumulation of toxic byproducts. The stationary phase occurs when the rate of cell death is equivalent to cell replication and this constant level of cell numbers remains until the culture cell number reduces to a low, but nonzero number in the death phase. The stationary and death stages are therefore more important in terms of protein accumulation in the biotechnology industry [13]. This two-stage growth then production phenomenon has been termed the ‘biphasic production concept’ and is thought to reflect the energy requirements of the cell during the different growth phases. Presumably, when cells are rapidly growing and dividing, large amounts of energy are required for protein and DNA synthesis, while in the stationary phase less energy is required for these cellular processes, thus freeing up energy which can be diverted toward recombinant protein production.

5.39.9 Downstream Processing of Biopharmaceuticals Once the recombinant product is secreted into the culture medium, there is a requirement to recover and purify the target biologic from other biological contaminants and impurities to generate a pure drug fit for market. Any impurities could stimulate an adverse immune response in human beings. Such impurities are either process-generated contaminants produced during the process of manufacturing or product-related contaminants, such as aggregated, degraded, and inactive protein product. The clearance and removal of these involve a variety of processes in a system known as downstream processing. This stage of manufacturing is expensive, often contributing over 40% of the manufacturing cost, and a bottleneck can form at this stage of the production of biopharmaceuticals at a large scale [4]. With the increases in product yields observed over the years, there is now tremendous pressure on this process and much interest in developments that can improve downstream processing. Through every step of downstream processing, product quantity is reduced due to loss and incomplete recovery so there is a need for efficient, cost-effective processing with as few steps as possible. It is also beneficial to combine recovery steps and plan the processes so products from the first step are compatible for the second, otherwise additional processing is required (e.g., to change the buffer or concentrate the sample before the next step can be undertaken). Further, it is necessary to continually validate the authenticity and integrity of the product throughout downstream processing. There are many strategies that can be used to continually monitor the target protein such as checking absorbance readings, undertaking gel electrophoresis and performing high-performance liquid chromatography on the product throughout the processes. For downstream processing, one of the initial considerations is the source of the biologic and how the molecule is presented for processing. Proteins produced by mammalian cell cultures are secreted into the media, and so are ready for purification straight

496

Biopharmaceuticals, In Vitro Drug Testing and Drug Delivery

from culturing. In contrast, when the proteins are expressed intracellularly as in bacterial cells, an extra step is required to break the cell walls open to release the protein of interest. Initially, centrifugation is often used to either (1) remove the cell debris, when the product of interest is in the supernatant or (2) collect cells when the product is produced intracellularly. In a centrifuge, the culture is revolved around a fixed point generating a gravitational field. This forces the cells to form a solid phase at the bottom called the pellet and the secreted product is found in the liquid supernatant allowing easy separation of the two states. Any intracellular proteins of interest are released from the cell pellet through lysis. This can be achieved enzymatically, chemically, or physically, although generally the latter mechanical approach is selected for large-scale applications through disruption of cells by grinding, high pressure or osmotic shock using high-speed agitator bead mills or high-pressure industrial homogenizers. Filtration is then used to refine cell extracts or culture supernatants, through either depth filters or tangential flow filtration modules. This step also reduces batch volumes and exchanges the buffer for a solution more suitable for further processing. High levels of recombinant gene expression can result in aggregation of recombinant proteins; this is especially common in E. coli hosts. These misfolded proteins can form inclusion bodies and the protein present is biologically inactive. However, if these inclusion bodies are isolated and purified, the biological activity of the protein of interest can be restored. In order to achieve this, the inclusion bodies initially need to be isolated from the host cells; then the protein within the inclusion body is resolubilized using chaotrophic agents such as urea, and the protein is slowly encouraged to refold into its biologically active form by gradually replacing the resolubilizing solution with an appropriate buffer. The processes and choices for purification are, from this stage forward, identical for both intracellular and secreted recombinant proteins. Typical downstream processing will contain an initial purification stage (using filtration, centrifugation, precipitation, or adsorption chromatography), followed by intermediate proces­ sing (using a form of adsorption chromatography, where binding and elution occur), and finishing with final polishing steps (chromatography), usually with one or two approaches being used per stage. During precipitation, the protein is forced to form an insoluble aggregate by adding a precipitating agent such as ammonium sulfate, often the preferred choice due to being inexpensive and highly soluble. This precipitating solution interrupts the interaction between water and the polar side chains of amino acids, causing the proteins to interact with themselves instead of the water; thus, aggregates form which fall out of solution. The insoluble precipitate can then be removed and separated from the precipitating reagent by centrifugation. The aim of precipitation is to concentrate the target protein, to fractionate the sample to separate the protein of interest from contaminants and to allow a buffer change to prepare the material for the next processing step. With mammalian cell cultures, it is now not often that a precipitation step is used and filtration and chromatographic steps are the usual steps in purification. Further, even if precipitation is used, the subsequent purification still heavily rely upon adsorptive chromato­ graphic procedures, the key aims of which are to remove other molecules/proteins which are chemically and physically different from the target protein and then to capture the protein of interest in a more concentrated form by reducing the liquid volume. There are four traditionally used chromatographic purification approaches: (1) ion exchange, (2) affinity, (3) hydrophobic interaction, and (4) gel filtration; and the selection of which approach to use and at what stage of the purification procedure varies between biomolecules. Ion exchange is the most commonly used chromatographic approach for downstream processing; this uses a charged resin to form the stationary phase in a column. Identically charged compounds pass through the column and are discarded, whereas the desired oppositely charged compounds (which will include the target protein) bind to the column allowing separation on charge. The biomolecules interacting with the column are eluted using a second buffer which is usually high in salt concentration. Occasionally, the eluted sample from the ion-exchange column is directly applied to a hydrophobic interaction chromatography column. This compatible combination of the two processes can be achieved because the effluent from the ion-exchange column usually has a high salt content and this second approach uses hydrophobic interactions, which are strongest at high ionic strength as in a high salt solution, to immobilize the biologic, and allow other components to be discarded [4]. A third choice for processing is affinity chromatography and this is reported to be the most efficient chromatographic purification approach [4]. This works by the binding of the protein of interest to a column which consists of immobilized natural ligands known to interact specifically with the desired protein. The column is washed to remove all unbound molecules and then the protein of interest is eluted using a second, usually low, pH buffer, which is not optimal for the binding of the target protein to the column. This process is used for the purification of therapeutic antibodies as protein A and G columns have a strong affinity for MABs. The limiting factor for this approach is an appropriate ligand is required and, for the case of antibody purification, protein A resin, is generally used but is expensive. Consequently, research is being undertaken toward finding a replacement or alternative to protein A resin for the purification of antibodies. After purification by affinity chromatography, the protein is purer but it is usually diluted as it is in a larger volume of solution. In order to reduce the solution volume, an ultrafiltration step is often added here. This involves passing the solution through a membrane while the protein of interest is retained by the filter. The collected, more concentrated, protein may then pass through further purification steps. Gel filtration chromatography, the last chromatographic approach to be discussed here, is not an absorptive approach; instead, proteins are separated through porous beads on the basis of size. A matrix is formed containing beads carefully selected for their pore structure. Upon addition of the material to the matrix, smaller molecules fit into the pores of the beads and their passage through the column is slowed down because the molecules pass through the beads. In comparison, larger molecules are unable to diffuse into the porus beads so they quickly pass between the beads and are eluted first from the column. The other molecules are then eluted in order of decreasing size. With this approach, it is also important to consider the shape of the molecule as well as the size; certain shapes fit more favorably into the pores than others, so molecules with the same molecular weight, but different shapes, would not necessarily elute at the same time. This approach is quite a mild purification process and it is often used as the final

Biopharmaceutical Development

497

chromatography step and for polishing the product. However, other chromatographic approaches may be chosen instead for the final step of the downstream processing. During the polishing stage, impurities with very similar characteristics to the desired protein are removed. The aforementioned packed bed chromatographic approaches are consistently and widely used during large-scale manufacturing as they successfully achieve efficient purification. However, this occurs at an extremely high cost and throughput is low, with yields reducing throughout each purification step. Further, the process of packing the chromatographic bed is time-consuming and labor intensive, and validation of the process is necessary. Yet currently there are no alternate approaches that can match those with regard to similar purification and resolution; however, the limitations, expense, and low yields drive the need to research into new and improved alternatives. Examples of suggested alternatives include expanded bed absorption, aqueous two-phase extraction, membrane chromatography, crystallization, and monolith columns; [4] however, even though many of these approaches are being used at small laboratory scale, these are yet to be developed for industrial large-scale use due to being less efficient than the traditional chromatographic approaches and, in some cases, scale-up issues exist. The effective removal of impurities during downstream processing is essential for the production of safe and efficient proteinbased biopharmaceuticals and there is regulatory control and guidelines that require the clearance of impurities to a consistent and acceptably low level. The removal of host cell proteins is particularly paramount due to their antigenicity and the possible resulting adverse effects such as inducing an immune response. Due to the fact that these impurities come from the cells generating the protein product of interest, there is fear that the immune response triggered in humans by host cell proteins may react against the desired biologic. Immunoassays are used to detect the level of host cell proteins and to support this approach two-dimensional gel electrophoresis is performed. There is no specific limit or guide set by the regulatory body regarding the acceptable level of host cell proteins, although a general standard of 1–100 ppm levels are recorded for the majority of biologics [9].

5.39.10 Viral Inactivation of Biologics When considering mammalian cell expression systems, further purification or downstream processing steps are required to remove potential harmful viruses which may be introduced to the system through various routes such as infected cell lines/host or through poor culture technique, including unsterile conditions or contaminated reagents and equipment [1]. To ensure product safety and sufficient viral clearance at least two extra processing steps are added to the workflow to complete good working practice and to satisfy the regulatory body. Each viral-inactivation step should achieve four to five log reduction in the virus load. The most widely used approaches to removing viruses are low pH holds/treatment, heating the protein solution to 60 °C for 10 h (liquids), 90 °C for 10 h or 80 °C for 72 h (lyophilized), UV irradiation and sometimes solvents and detergents are used [4]. In addition, filtration and chromatography can be used to remove viral load.

5.39.11 Process Analytical Technology As mentioned earlier, biologics must be manufactured in a safe manner to produce a consistent product which is efficient and has high efficacy against the original disease target. Validation of this is achieved through use of appropriate analytical techniques and documentation providing assurance that continual use of the process setup will constantly generate a product reaching specific specifications with a reproducible product. This requires monitoring of the biotherapeutic throughout the production process. Traditionally, approaches such as gel electrophoresis, high-performance liquid chromatography, and absorbance measurements were used to assess purity and integrity of the protein product. However, more advanced methods of validation are being supported by the regulatory bodies and the FDA is driving this with their process analytical technology (PAT) initiative which encourages the use of such approaches. For example, matrix-assisted laser desorption/ionization mass spectrometry and other methods are now being utilized to ascertain the identity of the protein product, to assess purity, record aggregation levels, and confirm glycosylation patterns.

5.39.12 Formulation and Drug Delivery Systems The protein product generated from the manufacturing process is usually in a liquid solution. A decision must be made as to the state of the final biologic; it can either be in a liquid or solid form. When making this choice, certain factors of the product must be considered including, the cost, its stability, dose quantities, the route of administration, and the storage conditions of the drug. Most therapeutic proteins are injected directly into the body; [1] therefore, for this administration a liquid form is required. However, in this liquid state, the biopharmaceutical must be refrigerated. For easier storage, the product can be dried to form a solid-state formation using various approaches including lyophilization, crystallization, or spray (freeze) drying. In this state, the product can be stored at ambient temperatures. Aside from administration via injection, other options include oral (in the form of a solid tablet/ capsule, or as a liquid) or diffusion through the skin from either skin patches or topical delivery in a cream. All forms have their own advantages and disadvantage and usually the decision as to which approach to use is dependent on the required site of action, whether it is systemic or local.

498

Biopharmaceuticals, In Vitro Drug Testing and Drug Delivery

Further in order to maintain stability and for delivery, the formulation of the drug must also be considered. Often additives are included to protect the product from degradation and to keep the product in its active form during storage. However, the current method for determining the optimum formulation is very much via trial and error, [4] although research is being undertaken to try to develop predictive, knowledge-based approaches to formulation development. A final consideration is how the drug should be packaged for efficient delivery. After manufacturing and formulating of a biopharmaceutical, a stable compound which can be transported to the market, then stored effectively to minimize degradation before being successfully delivered to the biological site of action in a reasonable time frame, and can act as an effective treatment should be produced.

5.39.13 Biosimilars Upon identification of a successful candidate, biopharmaceutical companies usually apply to the appropriate authorities to be granted a patent. This provides the company with exclusive rights to the drug and prevents others from making and selling their patented product, allowing companies to recoup the large investment of costs incurred during the research and development of the product. The exclusive time period is, however, limited, usually lasting 20 years from the date of filing. Once this period is over, the rights granted are annulled and other companies are legally allowed to bring identical or similar products onto the market. Biosimilars, also known as follow-on biologics, are a copy of the biopharmaceutical that has lost its patent protection. They differ from generic molecules which are identical to their branded reference product, as biosimilars, as the name suggests, are only similar to the counterpart product. Due to the similarity, these drugs incur relatively low cost for production as they are able to follow an abbreviated pathway for approval, although they must be shown to be equal to the reference product in terms of safety and efficacy. In 2006 the first biosimilar product, Omnitrope (Sandoz, Holzkirchen, Germany), was approved in Europe and the United States, this being a recombinant growth factor used for the treatment of growth hormone deficiencies. The reference product in this case was Genotropin (Pfizer) [10]. Currently, the United States have yet to set up legal and regulatory pathways for approving and bringing biosimilars to the market, whereas the European Union has recently established and issued a specific regulatory pathway providing rules and guidance on this issue [10]. This area of the biopharmaceutical industry is likely to increase in the future with the number of biologic products coming off patents steadily increasing. However, second-generation, improved products will be more attractive to manufacturers and the public.

5.39.14 Conclusions In summary, the production and development of biopharmaceuticals is a complicated, time-consuming, and expensive process. It requires expansive knowledge of the technology and equipment available and expertise in the biological and cell expression systems used. One of the biggest challenges is the number of choices and key decisions which must be made at each stage of the production of biologics. Further, no two different biologics are generated in exactly the same way as the processes are all optimized around the specific product being produced. Despite this, many factors remain the same for all biopharmaceuticals through development process, with the primary goal being to generate high yields of a pure protein of high quality, in an active form, which is consistently produced from batch to batch as cheaply and quickly as possible.

References [1] Bates R (2006) Downstream processing. In: Ozturk SS, Hu WS (eds.) Cell Culture Technology for Pharmaceutical and Cell-Based Therapies, pp. 439–482. Boca Raton, FL: CRC Press. [2] Bebbington C and Hentschel C (1985) The expression of recombinant DNA products in mammalian cells. Trends in Biotechnology 3: 314–317. [3] Birch JR and Onakunle Y (2005) Biopharmaceutical proteins: Opportunities and challenges. Methods in Molecular Biology 308: 1–16. [4] Bracewell DG, Mumtaz MAS, and Smales CM (2009) Downstream processing. In: Walker J, Raplet R (eds.) Molecular Biology and Biotechnology, 5th edn., pp. 492–512. London: The Royal Society of Chemistry. [5] Curran and Bugeja V (2009) The biotechnology and molecular biology of yeast. In: Walker J, Raplet R (eds.) Molecular Biology and Biotechnology, 5th edn., pp. 159–195. London: The Royal Society of Chemistry. [6] Jain K (2009) Biotechnology-based drug discovery. In: Walker J, Raplet R (eds.) Molecular Biology and Biotechnology, 5th edn., pp. 307–336. London: The Royal Society

of Chemistry.

[7] Lawrence S (2005) Biotech drug market steadily expands. Nature Biotechnology 23: 1466. [8] Mcmullan N (2009) Vaccines. In: Walker J, Raplet R (eds.) Molecular Biology and Biotechnology, 5th edn., pp. 337–350. London: The Royal Society of Chemistry. [9] Shukla AA, Jiang C, Ma J, et al. (2008) Demonstration of robust host cell protein clearance in biopharmaceutical downstream processes. Biotechnology Progress 24: 615–622. [10] Walsh G (2006) Biopharmaceutical benchmarks 2006. Nature Biotechnology 24: 769–776. [11] Ward M (2002) Expression of antibodies in Aspergillus niger. Genetic Engineering News 22: 48. [12] Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nature Biotechnology 22: 1393–1398. [13] Zanghi JA, Fussenegger M, and Bailey JE (1999) Serum protects protein-free competent chinese hamster ovary cells against apoptosis induced by nutrient deprivation in batch culture. Biotechnology and Bioengineering 64: 108–119.