Marco A. Cacciuttolo and Alahari Arunakumari Medarex Inc., Bloomsbury, New Jersey, U.S.A.
INTRODUCTION
This chapter covers the general principles involved in the scale-up of biotechnology-derived products obtained from cell culture. The first two sections focus on technologies currently used in the manufacture of commercial products. The subsequent sections include a practical guide to process design and scale-up strategies typically used to translate process development into large-scale production of biological products.
Advantages of Biologics as Therapeutic Agents
The main advantage of biologics over traditional small molecule drugs is that biologics are usually proteins that can be normally found in the body. If the bio-logy of these proteins is well understood, the regulatory approval is facilitated, as toxicology and immunogenicity could be demonstrated at a much faster pace than in traditional small drug product approval cycles. In addition, biologics offer the advantage of multiple sites of interaction between the drug and the tar-get, which is not usually possible to achieve with the use of small molecule drugs.
The introduction of biologics as credible therapies is evidenced by the increase in the approval rate of biologics over the last two decades. This is in contrast to the decline in the introduction of new small molecules (Fig. 1).
The market potential of biologics is also projected to grow exponentially over the next few years (Fig. 2). These are some of the many drivers for
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the development of biologics, and the production methods of these molecules from cell culture will be covered in this chapter.
With increasing market demand for biotechnology-derived products, the global manufacturing capacity for cell culture at one point was estimated not to be able to meet the projected needs. However, within a few years, significant technology advancements in the areas of expression vectors, host cell lines, and media development have been made. For instance, expression levels for antibodies have gone up from less than 500 mg/L to over 5 g/L in cell culture. This improvement has made it possible not only to meet the demand with existing capacity but also to make biopharmaceutical produc-tion much more cost-effective.
In the case of protein separation technologies, further scale-up or multiple cycles will be needed until these challenges of increased throughput from cell culture are successfully met, for instance with, the use of improved resins having much higher binding capacities combined with good resolution.
General Considerations in the Development and Scale-Up of Cell Culture Processes
One obvious reason for scale-up in biologics is to meet market demand.
Usually, small lots of product are produced during early evaluation of the Figure 1 Trends in regulatory approvals of small molecule therapeutics versus biologics. Source: Adapted from Ref. 1.
drug, as the cost of manufacturing can be quite onerous. As the product can-didate advances in clinical trials, more material is required and increases in scale of production or yield in the process, or both, are usually implemented.
Another powerful reason to scale-up is to decrease the cost of manufacturing.
Both the scale of manufacturing, and process improvement, regardless of the scale of manufacturing, have a profound effect on the direct cost of manufac-turing, as shown in Figure 3.
If the product candidate is considered to be promising, then the next phase of planning is perhaps the most challenging one: when to scale-up and to what scale. Figure 4 can be used to make an estimation of pro-duction scale for a batch-based process, depending upon estimated product demand and process yield.
This decision to scale-up is usually made two to three years before the projected regulatory filing date for the approval of the product, which in turn means about three to four years before the launch of the product. This is why the decision to scale-up is in direct opposition to the process development timeline, and special care has to be taken when developing a process for bio-logics in order for manufacturing not to be limitation on product approval.
In addition to basic engineering design principles, the scale-up of biotechnology products requires an understanding of the cellular and regulatory mechanisms that govern cell physiology and the biophysical Figure 2 The biotech industry revenue projections. Source: 2002 data from Ref. 2.
Figure 3 The impact of process scale and yield on direct cost of manufacturing.
Figure 4 Scale of manufacturing as a function of product demand and cell culture yield.
and biochemical characteristics of products. A thorough understanding of process operations and process limitations is essential for successful technol-ogy transfer from development to manufacturing. The design and operation of the facility, including appropriate segregation of products, personnel and equipment at each stage of manufacturing, must comply with current regu-latory guidelines. The true measure of successful scale-up is validation of the process at the manufacturing scale and ultimate approval of the biopharma-ceutical product.
Due to the complexity of biological systems and the physical and biochemical characteristics of the protein products, the design and scale-up of biological processes can be challenging. Batch sizes for the production of biotechnology-derived products can reach 10,000 L (3), 12,500 L (4,5), and up to 20,000 L (6). Although these scales of operation are often smaller than conventional bacterial or yeast fermentation, the high value of indivi-dual production lots requires careful planning and process control. For this reason, laboratory and pilot scale data together with actual experience are essential for the effective selection of scale-up strategies, equipment, and process parameters (7).
The efficient and timely completion of scale-up to commercial manufac-turing is critical to biotechnology companies. In some cases, novel unit opera-tions or techniques are required to achieve adequate expression, recovery, quality, or integrity of the product, which may not be feasible with more conventional techniques. However, this may cause costly delays in product approval because the use of new technologies may be associated with a greater degree of uncertainty as the scale of the operation increases. In addition, the ease of process validation may be an important factor influencing the selection between novel and conventional process techniques (4,8). For example, cell cul-ture processes can be conducted either as a batch or as a continuous process.
However, the time required to validate a continuous process may be longer than that for a batch process. As a consequence, this may impact the time required for preparation and submission of documents to regulatory agencies, as well as the time needed for review and approval. For many companies, the duration of clinical development and the strategy for efficacy studies may deter-mine the difference between success in the marketplace and total failure.
The timelines needed to complete technology transfer may vary with the complexity of the process. A team composed of manufacturing and develop-ment personnel should be responsible for facility design or integration of a process into an existing facility. The team is also responsible for equipment specifications and defining the physical relationship of process operations in order to comply with regulatory standards. The team must be aware of the relevant scale-up criteria to be used because their misapplication can lead to significant performance differences between bench top and manufactur-ing plant scales (9). For this reason, stepwise scale-up is recommended. In addition, successful scale-up requires that manufacturing personnel be
properly trained on process requirements and good manufacturing practices to provide an efficient and seamless transition to commercial production within the shortest time possible.
Recent advances in safety, selectivity, quality, and integrity of mole-cules obtained from recombinant microorganisms and immortalized cell lines have provided a wide range of products used as therapeutic agents. Marketed biotechnology products can be classified into five categories (3): coagulation factors, enzymes, hormones and growth factors, molecular inhibitors/
antagonists, and vaccines. Examples of marketed biotechnology products are presented in Table 1. This table illustrates the diversity of cell lines (bacteria, yeast, and mammalian cells) used to produce licensed products.
In addition to the expression systems listed below, other expression systems, such as insect cells, plant cells, and transgenic animals and plants, are currently being evaluated at preclinical and clinical stages.
As seen in Table 1, most of the cell lines used to manufacture biologics employ recombinant cells, in particular CHO and myeloma cell lines, that can be optimized to express complex proteins at high yields and are amen-able to scale-up. Current trends in the industry show that in addition to these cell lines, human cells lines such as HEK293 and PER.C6 (11), yeasts (12), and molds (13) could also be alternatives to express recombinant proteins. The incentive to use a human cell line is to mimic human proteins and to express recombinant proteins which otherwise could not be expressed in other cell lines. The use of yeast and fungi is intended to primarily decrease the cost of manufacturing as the manufacturing technology for these expression systems uses traditional fermentation techniques and state-of-the-art know-how to generate high cell density fermentations. In addition, significant advances have been made recently in yeast and fungi to express glycosylated proteins (12). It is expected that more data will be generated using these expression systems in the near future.
FUNDAMENTALS: TYPICAL UNIT OPERATIONS
Comprehensive descriptions of the basic unit operations commonly used in the production of biotechnology products are available in the literature (14). This section focuses on the typical unit operations currently used for production of biological molecules in cell culture and the technologies used for the purification of pharmaceutical proteins. For each of these opera-tions, laboratory and pilot scale experiments provide the basis for scale-up, particularly to define the expected range of process operating parameters.
Bioreactor Operation
Commercial manufacturing operations in biotechnology usually employ bioreactors or fermentors for product expression. In this discussion, the term fermentor will refer to bacterial or fungal processes and the term bior-eactor to animal cell cultures. While extensive description of the operation
Table 1 Examples of Biotechnology-Derived Products
Protein Clinical application Production process
Coagulation factors
Recombinate (F VIII)a Hemophilia rCHO, bleed-feed
Kogenate (F VIII)a Hemophilia rBHK-21, bleed-feed
Novo Seven (F VIIa)a Hemophilia A rBHK
Bene Fix (FIX)a Hemophilia B rCHO
Enzymes
Pulmozyme (Dnase I)a Cystic fibrosis rCHO, suspension
Cerezyma Gaucher’s disease rCHO, microcarriers
Activase (tPA)a Thrombolytic agent rCHO, suspension Abbokinase (Urokinase) Pulmonary embolism Human kidney cells Aldurazyme (Laronidase) Mucopolysaccharidosis
Welferon (IFN alfa)a Hepatitis C treatment Namalva Roferon (IFN alfa-b) Hepatitis C treatment rE.coli Infergen (IFN alfa) Hepatitis C treatment rE.coli Intron A (IFN alfa) Hairy cell lymphoma rE.coli
Epogen (Epo)a Stimulation of erithropoiesis rCHO, Roller bottles Avonex (IFN beta)a Multiple sclerosis rCHO
Betaseron (IFN beta) Multiple sclerosis rE.coli Proleukin (IL) Metastatic renal carcinoma rE.coli Gonal F (FSH)a Induction of ovulation rCHO
Saizen (hGH)a Growth hormone deficiency rC127, Roller bottles PEGASYS
Synagis (Mab) Prevention of RSV disease rNS/0, suspension
Herceptin (Mab) Breast cancer rCHO, suspension
OKT3 (Mab)a Rescue of acute renal rejection/GVHD
Mouse ascites Zenapax (Mab) Prevention of acute
renal rejection
rNS/0, suspension Reopro (Mab) Prevention of cardiac
ischemic complications
rSP2/0
(Continued)
of fermentors and bioreactors is available elsewhere (9,14), this chapter will focus on bioreactors used in the manufacture of complex proteins.
There are a variety of types of bioreactors described in the literature.
Among them, the stirred tank bioreactor is the most commonly employed Table 1 Examples of Biotechnology-Derived Products (Continued )
Protein Clinical application Production process
Leukine (GMCSF) Induction chemotherapy for acute leukemia
ryeast Neupogen (GCSF) Treatment of neutropenia rE.coli Remicade (Mab) Rheumatoid arthritis rSP2/0
Enbrel Rheumatoid arthritis rCHO
Avastin (Mab) Metastatic colorectal cancer rCHO Bexxar Botox (Toxin) Muscle relaxation activity,
cervical dystonia
Botulinum sp.
Campath (Mab) B-cell chronic lymphocytic leukemia
rCHO Erbitux (Mab) Metastatic colorectal cancer SP2/0
Humira (Mab) Rheumathoid arthritis rCHO
Kinerect (Anakinra) Rheumarthoid arthritis rE.coli MYOBLOC (Botulinum
toxin type B)
Cervical dystonia Botulinum sp.
Ontek (denileukin diftox) Cutaneous T-cell lymphoma rE.coli Xoliar (Mab) Metastatic colorectal cancer rCHO Vaccines
Vaqta Hepatitis A vaccine MRC5 cells
Recombivax (HbsAg) Hepatitis B vaccine ryeast Engerix-B (HbsAg) Hepatitis B vaccine ryeast
GenHevac B (HbsAg)a Hepatitis B vaccine rCHO, microcarriers HB Gamma (HbsAg)a Hepatitis B vaccine rCHO
Comvax (HbsAg) Combination of PedvaxHIB and Recombivax HB LYMErix (OspA) Lyme disease vaccine rE.coli
RotaShield Rotavirus vaccine FRhk2
Varivax Varicella vaccine MRC5 cells
FluMist Influenza virus vaccine Eggs
aSource: Adapted from Ref. 10.
due to its performance record and ease of operation. Cells growing in bioreactors take up nutrients from the culture medium and release products, byproducts, and waste metabolites. Mass transport phenomena required for adequate supply of nutrients and removal of waste metabolites are greatly influenced by mixing and aeration rates. Agitation is used to maintain cells in suspension, to provide a homogeneous mix of nutrients, and to prevent the accumulation of toxic gases (15).
Aeration is also an essential requirement for aerobic cell lines. The design of aeration devices includes single-orifice tubes, sparger rings, and diffuser membranes. Bubble sizes may vary with each device and optimiza-tion is required to achieve the maximum ratio of surface area to gas volume transfer rate which generates a minimal of foaming to prevent damaging effects on cell viability (16,17). The effect of aeration on cell productivity is complex and depends on cell line, medium components (including cell proteins), and characteristics of foam formation and collapse. The optimal aeration rate then is determined empirically at each scale.
In the case of airlift bioreactors, air flowing upwards in a column-shaped bioreactor vessel is used to generate sufficient mixing of gases and cells simultaneously thereby replacing the need for conventional impellers of stirred tank bioreactors (18). High volume of airflow can result in foam-ing in this type of bioreactors, which can be suppressed with the addition of appropriate antifoam agents. The existing production scales in air-lift bioreactors are 2000 L and 5000 L.
Bioreactor technology also involves the application of single-use or
‘‘disposable’’ bioreactors, such as hollow fiber bioreactors, and more recently the concept of disposable stirred tank bioreactors up to the 2000 L scale has been introduced (19). This type of single use or disposable technology could make current stainless steel bioreactor equipment and facility design obsolete and may facilitate introduction of clinical stage manufacturing in a far more flexible format and faster than conventional hard-piped designs. This is an important innovation for minimizing capital expenditure, turn-around time from product campaigns, time to commissioning, and for facilitating concurrent product manufacturing.
Filtration Operations
Filtration technologies are used extensively throughout the biotechnology industry (20,21). Membranes and filters can be used for medium exchange during cell growth, cell harvest, product concentration, diafiltration, and formulation or for removal of viruses and control of bioburden. For example, micro filtration is used to replace spent medium with fresh medium (22) or to recover secreted proteins (5,22). Ultra filtration membranes with sub-micron pore sizes are used for product concentration and buffer exchange by diafiltration. Unlike in affinity capture step with Protein A where binding is very specific, for ion exchange capture steps preconditioning of cell culture
harvest by diafiltration into defined buffer composition can dramatically improve process consistency by providing more uniform load conditions.
Nanometer ultra filtration using filters with tightly controlled pore sizes can be used for virus removal (23). Filtration with 0.2 mm dead-end filters is used for removal of microorganisms (24). Sterilizing filters are validated for product-specific bubble point, product compatibility, and microbial reten-tion. Depth filtration with disposable filter modules has been extensively used to clarify mammalian cell culture or to polish the clarified supernatants, due to ease of operation, high flow rates, and good product recoveries. Currently, charged depth filters with the added advantage of viral removal are entering into biotech processing, especially in the case of purification processes with limited viral clearance capability (25). Depth filters may also contribute to the removal of process contaminants, for example, DNA and endotoxin, and could be integrated into the process at various stages of the protein purification scheme. Due to the cost of these filters, it is preferable to use them wherever process volumes are low.
The key process parameters for filtration scale-up are trans-membrane pressure, filtration area, shear rate, operating time, temperature, flux rate, protein concentration, and solution viscosity (5).
Centrifugation
Centrifugation is used in fermentation processes as well as in blood serum fractionation. Scale-up of operations for separation of product-containing cells from supernatant fluid or secreted products from host cells is well estab-lished (26). Although batch centrifugation is often used at the laboratory scale, continuous centrifugation is preferred at the production scale. When centrifugation is used for biotechnology applications, it is preferable to use high-throughput low-shear centrifuges to minimize the shear sensitivity of animal cells. The centrifugation step is typically followed by depth-filtration to remove suspended solids not completely removed by centrifugal forces in order to minimize the impact of these molecules on downstream purification.
Filtration may be the preferable unit operation for separating secreted products from host cells because of the relatively mild operating conditions.
A second advantage of filtration is that the cleaning validation is relatively simple compared to the elaborated cleaning validation required for contin-uous centrifuges. However, as the process volume increases, the economics of using filters decrease and space considerations increase in order to accom-modate large filtration units. The operating cost and the increased complexity of operation of large filter units requiring high flow rates and with low shear make them unsuitable for very large-scale operations.
Because of the above considerations, it is preferred to use filtration as a clarification step for small scales (less than 2000 L of culture harvest), whereas centrifugation might be the choice for larger scales of operation.
Chromatography
Chromatography is a commonly used unit operation for the purification of proteins in biotechnology applications. It is capable of combining relatively high throughputs with high selectivity. A major advantage of this technique is that it can be optimized to achieve high resolution of the desired product from its contaminants. The selection of the appropriate gel is very much dependent on an understanding of the physical and chemical characteristics of the target protein product. Chromatography steps can be designed to selec-tively either capture the product or remove contaminants. For ion exchange gels, contaminant removal is achieved by optimizing the pH and conductivity of the equilibration, wash, and elution buffers. Affinity chromatography is often used as an initial capture step to provide high specificity, high selectivity, and volume reduction. However, affinity chromatography gels, such as Protein A or Protein G, are costly, especially in early process steps with crude product streams. The use of crude material on affinity matrices may require extensive cleaning which contributes to the cost and can reduce the effective lifetime of the gel. Hydrophobic interaction chromatography (HIC), which
Chromatography is a commonly used unit operation for the purification of proteins in biotechnology applications. It is capable of combining relatively high throughputs with high selectivity. A major advantage of this technique is that it can be optimized to achieve high resolution of the desired product from its contaminants. The selection of the appropriate gel is very much dependent on an understanding of the physical and chemical characteristics of the target protein product. Chromatography steps can be designed to selec-tively either capture the product or remove contaminants. For ion exchange gels, contaminant removal is achieved by optimizing the pH and conductivity of the equilibration, wash, and elution buffers. Affinity chromatography is often used as an initial capture step to provide high specificity, high selectivity, and volume reduction. However, affinity chromatography gels, such as Protein A or Protein G, are costly, especially in early process steps with crude product streams. The use of crude material on affinity matrices may require extensive cleaning which contributes to the cost and can reduce the effective lifetime of the gel. Hydrophobic interaction chromatography (HIC), which