3. Production and Purification of Recombinant Proteins

In order to achieve optimal growth of cells and optimal production of recombinant proteins, it is of great importance not only that conditions such as stirring, pH, oxygen pressure, and temperature are chosen and controlled appropriately but also that a cell growth and protein production medium with the proper nutrients are provided for each stage of the production process.

The media used for mammalian cell culture are complex and consist of a mixture of diverse components, such as sugars, amino acids, electrolytes, vitamins, fetal calf serum, and/or a mixture of peptones, growth factors, hormones, and other proteins (see Table 3.2). Many of these ingredients are pre-blended either as concentrate or as homogeneous mixtures of powders. To prepare the final medium, components are dissolved in purified water before filtration. The final medium is filtrated through 0.2 μm filters or through 0.1 μm filters to prevent possible mycoplasma contamination. Some supplements, especially fetal calf serum, contribute considerably to the presence of contaminating proteins and may seriously complicate purification procedures. Moreover, the composition of serum is variable. It depends on the individual animal, season of the year, suppliers’ treatment, etc. The use of serum may introduce adventitious material such as viruses, mycoplasma, bacteria, and fungi into the culture system (Berthold and Walter 1994). Furthermore, the possible presence of prions that can cause transmissible spongiform encephalitis almost precludes the use of materials from animal origin. However, if use of this material is inevitable, one must follow the relevant guidelines in which selective sourcing of the material is the key measure to safety (EMA 2011). Many of these potential problems when using serum in cell culture media led to the development of fully defined, free from animal-derived material. These medium formulations were not only developed by the suppliers, there is the trend that the key players in the biotech industry develop their own fully defined medium for their specific production platforms. The advantage of this is that the industry is less dependent on medium suppliers. The fully defined media have been shown to give satisfactory results in large-scale production settings for monoclonal antibody processes. However, hydrolysates from nonanimal origin, like yeast and plant sources, are more and more used for optimal cell growth and product secretion (reviewed by Shukla and Thömmes 2010).

Recovering a biological reagent from a cell culture supernatant is one of the critical parts of the manufacturing procedure for biotech products, and purification costs typically outweigh those of the upstream part of the production process. For the production of monoclonal antibodies, protein A resin accounts for some 10 % of the cost, while virus removal by filtration can account for 40 % of the cost (Gottschalk 2006).

More than a decade ago, the protein product was available in a very dilute form, e.g., 10–200 mg/L. At the most concentrations, up to 500–800 mg/L could be reached (Berthold and Walter 1994). Developments in cell culture technology through application of genetics and proteomics resulted in product titers well above 1 g/L. Product titers above 20 g/L are also reported (Monteclaro 2010). These high product titers pose a challenge to the downstream processing unit operations (Shukla and Thömmes 2010). With the low-yield processes, a concentration step is often required to reduce handling volumes for further purification. Usually, the product subsequently undergoes a series of purification steps. The first step in a purification process is to remove cells and cell debris from the process fluids. This process step is normally performed using centrifugation and/or depth filters. Depth filters are often used in combination with filter aid or diatomaceous earth. Often the clarification step is regarded as a part of the upstream process. Therefore, the first actual step in the purification process is a capture step. Subsequent steps remove the residual bulk contaminants, and a final step removes trace contaminants and sometimes variant forms of the molecule. Alternatively, the reverse strategy, where the main contaminants are captured and the product is purified in subsequent steps, might result in a more economic process, especially if the product is not excreted from the cells. In the case where the product is excreted into the cell culture medium, the product will not represent more than 1–5 % of total cellular protein, and a specific binding of the cellular proteins in a product-specific capture step will have a high impact on the efficiency of that step. If the bulk of the contaminants can be removed first, the specific capture step will be more efficient and smaller in size and therefore more economic. Furthermore, smaller subsequent unit operation steps (e.g., chromatography columns) could be used.

After purification, additional steps are performed to bring the desired product into a formulation buffer in which the product is stabilized and can be stored for the desired time until further process steps are performed. Before storage of the final bulk drug substance, the product will be sterilized. Normally this will be performed by a 0.2 μm filtration step. Formulation aspects will be dealt with in Chap.​ 4.

When designing an upstream and purification protocol, the possibility for scaling up should be considered carefully. A process that has been designed for small quantities is most often not suitable for large quantities for technical, economic, and safety reasons. Developing a purification process, i.e., the isolation and purification of the desired product also called the downstream process (DSP), to recover a recombinant protein in large quantities occurs in two stages: design and scale-up.

Separating the impurities from the product protein requires a series of purification steps (process design), each removing some of the impurities and bringing the product closer to its final specification. In general, the starting feedstock contains cell debris and/or whole-cell particulate material that must be removed. Defining the major contaminants in the starting material is helpful in the downstream process design. This includes detailed information on the source of the material (e.g., bacterial or mammalian cell culture) and major contaminants that are used or produced in the upstream process (e.g., albumin, serum, or product analogs). Moreover, the physical characteristics of the product versus the known contaminants (thermal stability, isoelectric point, molecular weight, hydrophobicity, density, specific binding properties) largely determine the process design. Processes used for production of therapeutics in humans should be safe, reproducible, robust, and produced at the desired cost of goods. The DSP steps may expose the protein molecules to high physical stress (e.g., high temperatures and extreme pH) which can alter the protein properties possibly leading to loss in efficacy. Any substance that is used by injection must be sterile. Furthermore, the endotoxin concentration must be below a certain level depending on the product. Limits are stated in the individual monographs which are to be consulted (e.g., European Pharmacopoeia: less than 0.2 endotoxin units per kg body mass for intrathecal application). Aseptic techniques have to be used wherever possible and necessitate procedures throughout with clean air and microbial control of all materials and equipment used. During validation of the purification process, one must also demonstrate that potential viral contaminants are inactivated and removed (Walter et al. 1992). The purification matrices should be at least sanitizable or, if possible, steam-sterilizable. For depyrogenation, the purification material must withstand either extended dry heat at ≥180 °C or treatment with 1–2 M sodium hydroxide (for further information, see Chap.​ 4). If any material in contact with the product inadvertently releases compounds, these leachables must be analyzed and their removal by subsequent purification steps must be demonstrated during process validation, or it must be demonstrated that the leachables are below a toxic level. The increased use of plastic film-based single-use production technology (e.g., sterile single-use bioreactor bags, bags to store liquids and filter housings) has made these aspects more significant in the last decade. Suppliers have reacted by providing a significant body of information regarding leachables and biocompatibility for typical solutions used during processing. The problem of leachables is especially hampering the use of affinity chromatography (see below) in the production of pharmaceuticals for human use. On small-scale affinity, chromatography is an important tool for purification and the resulting product might be used for (animal) toxicity studies, but for human use the removal of any leached ligands below a toxic level has to be demonstrated. Because free affinity ligands will bind to the product, the removal might be cumbersome.

Scale-up is the term used to describe a number of processes employed in converting a laboratory procedure into an economical, industrial process. During the scale-up phase, the process moves from the laboratory scale to the pilot plant and finally to the production plant. The objective of scale-up is to produce a product of high quality at a competitive price. Since the costs of downstream processing can be as high as 50–80 % of the total cost of the bulk product, practical and economical ways of purifying the product should be used. Superior protein purification methods hold the key to a strong market position (Wheelwright 1993).

Basic operations required for a downstream purification process used for macromolecules from biological sources are shown in Fig. 3.3.

As mentioned before, the design of downstream processing is highly product dependent. Therefore, each product requires a specific multistage purification procedure (Sadana 1989). The basic scheme as represented in Fig. 3.3 becomes complex. A typical example of a process flow for the downstream processing is shown in Fig. 3.4. This scheme represents the processing of a glycosylated recombinant interferon (about 28 kDa) produced in mammalian cells. The aims of the individual unit operations are described.

Once the harvest volume and product concentration can be managed, the main purification phase can start. A number of purification methods are available to separate proteins on the basis of a wide variety of different physicochemical criteria such as size, charge, hydrophobicity, and solubility. Detailed information about some separation and purification methods commonly used in purification schemes is provided below.

Products from biotechnological industry must be separated from biological systems that contain suspended particulate material, including whole cells, lysed cell material, and fragments of broken cells generated when cell breakage has been necessary to release intracellular products. Most downstream processing flow sheets will, therefore, include at least one unit operation for the removal (“clarification”) or concentration, just the opposite, of particulates. Most frequently used methods are centrifugation and filtration techniques (e.g., ultrafiltration, diafiltration, and microfiltration). However, the expense and effectiveness of such methods is highly dependent on the physical nature of the particulate material and of the product.

The solubility of a particular protein depends on the physicochemical environment, for example, pH, ionic species, and ionic strength of the solution (see also Chap.​ 4). A slow continuous increase of the ionic strength (of a protein mixture) will selectively drive proteins out of solution. This phenomenon is known as “salting out.” A wide variety of agents, with different “salting-out” potencies are available. Chaotropic series with increasing “salting-out” effects of negatively (I) and positively (II) charged molecules are given below:

Ammonium sulfate is highly soluble in cold aqueous solutions and is frequently used in “salting-out” purification.

Another method to precipitate proteins is to use water-miscible organic solvents (change in the dielectric constant). Examples of precipitating agents are polyethylene glycol and trichloroacetic acid. Under certain conditions, chitosan and nonionic polyoxyethylene detergents also induce precipitation (Cartwright 1987; Homma et al. 1993; Terstappen et al. 1993). Cationic detergents have been used to selectively precipitate DNA.

Precipitation is a scalable, simple, and relatively economical procedure for the recovery of a product from a dilute feedstock. It has been widely used for the isolation of proteins from culture supernatants. Unfortunately, with most bulk precipitation methods, the gain in purity is generally limited and product recovery can be low. Moreover, extraneous components are introduced which must be eliminated later. Finally, large quantities of precipitates may be difficult to handle. Despite these limitations, recovery by precipitation has been used with considerable success for some products.

Endogenous and adventitious viruses, which require the presence of living cells to propagate, are potential contaminants of animal cell cultures and, therefore, of the final drug product. If present, their concentration in the purified product will be very low and it will be difficult to detect them. Viruses such as retrovirus can be visualized by (nonsensitive) electron microscopy. For retroviruses, a highly sensitive RT-PCR (reverse-transcriptase polymerase chain reaction) assay is available, but for other viruses, a sensitive in vitro assay might be lacking. The risks of some viruses (e.g., hepatitis virus) are known (Walter et al. 1991; Marcus-Sekura 1991), but there are other viruses whose risks cannot be properly judged because of lack of solid experimental data. Some virus infections, such as parvovirus, can have long latent periods before their clinical effects show up. Long-term effects of introducing viruses into a patient treated with a recombinant protein should not be overlooked. Therefore, it is required that products used parenterally are free from viruses. The specific virus testing regime required will depend on the cell type used for production (Löwer 1990; Minor 1994).

Viruses can be introduced by nutrients, by an infected production cell line, or they are introduced (by human handling) during the production process. The most frequent source of virus introduction is animal serum. In addition, animal serum can introduce other unwanted agents such as bacteria, mycoplasmas, prions, fungi, and endotoxins. It should be clear that appropriate screening of cell banks and growth medium constituents for viruses and other adventitious agents should be strictly regulated and supervised (Walter et al. 1991; FDA 1993; ICH 1999b; WHO 2010). Validated, orthogonal methods to inactivate and remove possible viral contaminants are mandatory for licensing of therapeutics derived from mammalian cells or transgenic animals (Minor 1994). Viruses can be inactivated by physical and chemical treatment of the product. Heat, irradiation, sonication, extreme pH, detergents, solvents, and certain disinfectants can inactivate viruses. These procedures can be harmful to the product as well and should therefore be carefully evaluated and validated (Walter et al. 1992; Minor 1994; ICH 1999b). Removal of viruses by nanofiltration is an elegant and effective technique and the validation aspects of this technology are well described (PDA 2005). Filtration through 15 nm membranes can remove even the smallest non-enveloped viruses like bovine parvovirus (Maerz et al. 1996). Another common, although less robust, method to remove viruses in antibody processes is by ion-exchange chromatography. A number of methods for reducing or inactivating viral contaminants are mentioned in Table 3.5 (Horowitz et al. 1991).

Unwanted bacterial contamination may be a problem for cells in culture or during pharmaceutical purification. Usually the size of bacteria allows simple filtration over 0.2 μm (or smaller) filters for adequate removal. In order to further prevent bacterial contamination during production, the raw materials used have to be sterilized, preferably at 121 °C or higher, and the products are manufactured under strict aseptic conditions wherever possible. Production most often takes place in so-called clean rooms in which the chances of environmental contamination is reduced through careful control of the environment, for example, filtration of air. Additionally, antibiotic agents can be added to the culture media in some cases but have to be removed further downstream in the purification process. However, the use of beta-lactam antibiotics such as penicillin is strictly prohibited due to oversensitivity of some individuals to these compounds. Because of the persistence of antibiotic residues, which are difficult to eliminate from the product, appropriately designed manufacturing plants and extensive quality control systems for added reagents (medium, serum, enzymes, etc.) permitting antibiotic-free operation are preferable.

Pyrogens (usually endotoxins of gram-negative bacteria) are potentially hazardous substances (see Chap.​ 4). Humans are sensitive to pyrogen contamination at very low concentrations (picograms per mL). Pyrogens may elicit a strong fever response and can even be fatal. Simple 0.2 μm filtration does not remove pyrogens. Removal is complicated further because pyrogens vary in size and chemical composition. However, sensitive tests to detect and quantify pyrogens are commercially available. Purification schemes usually contain at least one step of ion-exchange chromatography (anionic-exchange material) to remove the negatively charged endotoxins (Berthold and Walter 1994). Furthermore, materials used in process such as glassware are typically subjected to a depyrogenation step prior to use often by the use of elevated temperatures (≥180 °C) in a dry heat oven.

The application of continuous mammalian cell lines for the production of recombinant proteins might result in the presence of oncogene-bearing DNA fragments in the final protein product (Walter and Werner 1993; Löwer 1990). A stringent purification protocol that is capable of reducing the DNA content and fragment size to a safe level is therefore necessary (Berthold and Walter 1994; WHO 2010). There are a number of approaches available to validate that the purification method removes cellular DNA and RNA. One such approach involves incubating the cell line with radiolabeled nucleotides and determining radioactivity in the purified product obtained through the purification protocol. Other methods are dye-binding fluorescence-enhancement assays for nucleotides and PCR-based methods. If the presence of nucleic acids persists in a final preparation, then additional steps must be introduced in the purification process. The question about a safe level of nucleic acids in biotech products is difficult to answer because of minimal relevant know-how. Transfection with so-called naked DNA is very difficult and a high concentration of DNA is needed. Nevertheless, it is agreed for safety reasons that final product contamination by nucleic acids should not exceed 100 pg or 10 ng per dose depending on the type of cells used to produce the pharmaceutical (WHO 2010; European Pharmacopoeia 2011).

As mentioned before, minor amounts of host-, process-, and product-related proteins will likely appear in biotech products. These types of contaminants are a potential health hazard because, if present, they may be recognized as antigens by the patient receiving the recombinant protein product. On repeated use the patient may show an immune reaction caused by the contaminant, while the protein of interest is performing its beneficial function. In such cases the immunogenicity may be misinterpreted as being due to the recombinant protein itself. Therefore, one must be very cautious in interpreting safety data of a given recombinant therapeutic protein.

Generally, the sources of host- and process-related protein contaminants are the growth medium used and the host proteins of the cells. Among the host-derived contaminants, the host species’ version of the recombinant protein could be present (WHO 2010). As these proteins are similar in structure, it is possible that undesired proteins are co-purified with the desired product. For example, urokinase is known to be present in many continuous cell lines. The synthesis of highly active biological molecules such as cytokines by hybridoma cells might be another concern (FDA 1990). Depending upon their nature and concentration, these cytokines might enhance the antigenicity of the product.

“Known” or expected contaminants should be monitored at the successive stages in a purification process by suitable in-process controls, e.g., sensitive immunoassay(s). Tracing of the many “unknown” cell-derived proteins is more difficult. When developing a purification process, other less-specific analyses such as SDS-PAGE are usually used in combination with various staining techniques.

Product-related contaminants/variants may pose a potential safety issue for patients. These contaminants can be aggregated forms of the product, a product with heterogeneity in the disulfide bridges, N- and C-terminal variants, glycosylation variants, deamidated product, etc. Some of these contaminants/variants are described in the following paragraphs.