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The major difference between biotechnology-derived products and other pharmaceutical products is the means of production used to generate the product. Biotechnology makes use of genetically modified living organisms to produce protein or peptidyl products. This statement is true for both rDNA-derived products as well as monoclonal antibody products. Biotechnology-derived products are therefore readily differentiated from proteins or peptides that have been obtained by isolation from natural source materials such as plasma, serum, or tissue, or by chemical synthesis.
Biotechnology-derived products are not significantly different from other protein pharmaceuticals after the protein purification process. Thus, the basic requirements for process validation, environmental control, aseptic manufacturing, and quality control/quality assurance systems are fundamentally the same for all pharmaceutical products. However, the complexity of these systems is often greater for biotechnology-derived products because the production of such bio-molecules generally requires highly developed cell propagation processes, complicated purification methods, and analytical control to ensure their homogeneity, lot-to-lot consistency, and safety.
This section describes in some detail only those significant factors that are unique to the processing of biotechnology-derived products. This includes descriptions of the various biological production systems now in use, and a discussion of purification issues.
rDNA Production
rDNA products are presently produced in prokaryotic (bacteria) or eukaryotic systems (e.g., yeast, mammalian cell culture). The choice of the production organism is generally a direct function of the molecular complexity of the protein that is to be produced as well as the economics and efficiency of the fermentation or cell culture process. The earliest biotechnology-derived products were produced in E. coli based on the high degree of understanding of its molecular biology. Within the last few years, however, the use of large-scale eukaryotic cell culture has become relatively commonplace.
Bacterial production of biotechnology-derived products offers a number of distinct advantages as well as certain disadvantages. As previously stated, the biology of bacteria is quite well understood and the safe and effective use of E. coli as the host organism for production has been well documented. Thus, the expression of a new protein in E. coli, if possible, is often easier to accomplish than in other, more theoretically suitable, expression systems. This may be offset, however, by the fact that E. coli produces proteins usually in a chemically reduced state. For proper folding, such proteins require the production of intramolecular disulfide bonds by oxidation. A second disadvantage is that all E. coli proteins begin their sequence with an N-formyl methionine residue that may not always be removed by E. coli proteolytic systems, thus possibly yielding a methionyl derivative of the desired natural protein. A third disadvantage of expression in E. coli is the potential for product degradation because of trace protease impurities. A fourth disadvantage is the requirement for endotoxin removal during purification. These limitations aside, the ease of use of E. coli and their generally high-expression yields for most proteins often have resulted in the continued preferential use of these bacteria, where feasible.
As previously described, the key element in rDNA technology is the recombinant plasmid, which contains the gene that codes for the protein of interest. Plasmids are simple and small circular extrachromosomal segments of bacterial DNA that are isolated from a bacterium and are self-replicating. The basic technology involves the specific enzymatic cleavage of a plasmid using endonucleases followed by the insertion of a new piece of DNA that contains the gene of interest. The resultant recombinant plasmid is considered the key raw material of rDNA technology. The recombinant plasmid is introduced into the host organism through a process called transformation, where it passes on its new genetic information and results in the production of the protein product. The large-scale growth of recombinant organisms can be conducted in commercial fermenters at scales in excess of 100,000 L, making these types of production systems extremely economical. There are, however, a number of issues that complicate E. coli fermentation systems. In some cases, the expressed protein product may cause cellular toxicity, and/or be extremely difficult to recover or purify because it may be sequestered into bacterial inclusion bodies as large semisoluble aggregates. Recent advances in E. coli molecular biology have led to the ability to express proteins into the periplasmic space, allowing for the removal of unwanted N-terminal methionine groups and leading to more readily purified proteins.
The development of eukaryotic cell culture for the production of vaccines has long been established in the pharmaceutical industry and an extensive database has been developed to ensure the suitability of such protein products in humans. The extension of this technology to rDNA products was primarily a response to the limitations in the use of E. coli. Particularly with respect to large proteins or glycoproteins, eukaryotic cell expression is an attractive alternative to a bacterial system because eukaryotic cells can secrete proteins that are properly folded and identical in primary, secondary, and tertiary structure to the natural human protein. Concerns about the economics of this production system orginally hindered its development. Recent advances, however, in improved expression levels, in large-scale cell culture using Chinese Hamster Ovary (CHO) cells, and in the formulation of more highly defined growth media have combined to dramatically improve the economic feasibility of eukaryotic cell substrates. The number of cell passages required for cloning, selection, amplification, and cell banking prior to production generally necessitates the use of immortal cell lines because nonimmortalized strains (i.e., diploid cultures) cannot be propagated long enough to provide an economically useful time in the production stage. Initial questions regarding the safety of such immortal cell lines were based on concerns over potential oncogenes and potential viral and retroviral contamination. These concerns have been minimized by the exhaustive analysis and characterization of master cell banks for adventitious (accidentally introduced) agents, by effective process validation studies, and by the safety data gathered to date for products produced by this method. The resultant thoroughly characterized master cell bank is used for full-scale production. Other eukaryotic cell lines, such as those derived from insect cells, may be useful in achieving many of the conformational and post-translational advantages that have been described for mammalian cell culture.
The use of yeast strains such as Saccharomyces cerevisiae for production has been extensively explored. The production of proteins in yeast offers many theoretical advantages over E. coli while raising certain new concerns. Like E. coli, yeast can maintain stable plasmids extrachromosomally; however, unlike E. coli, yeast possesses the ability to produce glycoproteins.
Monoclonal antibodies can be produced in two major ways, depending on whether they are of human or murine (mouse) origin. For antibodies of murine origin, appropriate lymphocytes are selected from the spleens of previously inoculated mice or rats. The cell is then fused with a transformed cell line such as a myeloma cell line, producing a hybridoma cell. The hybridoma cells are then clonally selected and used to produce the monoclonal antibody products. For antibodies of human origin, human B lymphocytes can be clonally selected for the hapten binding specificity of their product antibodies; these selected cells can then be immortalized by infection with a virus. The resultant fused or transformed cell can proliferate indefinitely in a bioreactor/cell culture environment or can be injected into mice from whose ascites fluid the protein can be obtained. Antibody is produced as directed by the chromosomal information that resides in the cell or was acquired during fusion and is secreted into the medium from which it can be readily purified. The hybridoma cells must be thoroughly analyzed and characterized in the same general way as an rDNA cell bank. The resultant cell bank is used for production of product either by large-scale cell culture or by harvesting ascites fluid from mice inoculated with transformed cells.
Control of Fermentation and Cell Culture Processes
Because the production process using a living system is the fundamental cornerstone of biotechnology, the issues that relate directly to the control of biotechnology processes need to be examined. Concerns over the production of proteins in bacteria, for example, primarily involve systems for ensuring genetic stability, consistent product yield, and evidence of the lack of contamination by adventitious organisms. These same concerns apply to large-scale eukaryotic cell culture, where, as stated above, there are also significant issues relating to the use of immortalized cell lines such as the putative presence of oncogenic DNA/RNA and impurities from media proteins.
A considerable amount of knowledge has been obtained for the production of recombinant proteins in bacteria and yeast; therefore, the major fermentation issues typically are resolved by the demonstration of consistency in fermentation conditions. Fermentations with bacteria and yeast usually are performed over short, well-defined time periods to monitor and control growth rate and product expression conditions. The presence of contaminating foreign organisms may be detected by effects on growth rate, culture purity, fatty acid profile, etc., and is cause for termination of the fermentation. The genetic stability of the production plasmid for bacteria may be addressed by isolation and nucleotide sequence analysis or by DNA restriction mapping. These results may be confirmed by peptide mapping of the expressed protein for each product lot manufactured. It is very important to optimize the fermentation conditions so that the amount of proteolytic processing of the target protein that may occur can be either limited or avoided completely. Proteolytic processing is often a problem in E. coli fermentations and may lead to recovery difficulties and low product yields. Finally, the conformation of the protein and its effects on potency must be addressed by the fermentation process.
The origin of large-scale cell culture techniques for the production of biotechnology-derived products can be traced back to the vaccine industry. Developments such as large-scale cell suspension cultures using recombinant organisms that secrete the desired protein into the media have had a significant impact on biotechnology. Large glycosylated proteins in quantities sufficient for the marketplace can now be produced. The use of eukaryotic cell cultures, however, is complicated by issues such as genetic stability, protein folding, and culture conditions, including cell viability and growth rates. For example, the genetic stability of cell cultures cannot be addressed as readily as E. coli fermentations by techniques such as plasmid sequence analysis because the gene that codes for the product is incorporated into the cell genome and is not easily recovered. One alternative is peptide mapping of the expressed protein, which requires a resolution and sensitivity adequate to detect subtle mutations.
The absence of adventitious organisms in cell cultures is critical. In addition to demonstrating that bacteria, yeast, and molds are not present in cell cultures, the manufacturer must provide for each culture evidence that mycoplasmas and adventitious viruses are not present. It is important to recognize that certain hybridomas used for monoclonal antibody production may contain endogenous retroviruses. However, it must be demonstrated that any viruses present in the culture are removed from the final product. This requires the development of suitable analytical techniques to ensure the absence of contamination by mycoplasmas or human and animal adventitious viruses.
The degree and type of glycosylation may be important in the design of cell culture conditions for the production of glycosylated proteins. The degree of glycosylation present may affect the half-life of the product in vivo as well as its potency and antigenicity. Although the glycosylation status of a cell culture product is difficult to determine, it can be verified to be consistent if the culture conditions are highly reproducible.
Process for Recovery and Purification
The recovery of protein products obtained from either fermentation or cell culture is generally based on efficient protein separation techniques such as those listed in Table 1. The recovery process begins with isolation of the desired protein from the fermentation or cell culture medium, often in a very impure form. The advantage of cell culture and yeast-derived products is that many of these proteins are secreted directly into the medium, thus requiring only cell separation to obtain a significant purification. For E. coli-derived products, lysis of the bacteria is often necessary to recover the desired protein. It is important in each case to achieve rapid purification of the desired protein because proteases released by the lysed organisms may cleave the desired product. Such trace proteases are a major concern in the purification of biotechnology-derived products because they can be very difficult to remove, may complicate the recovery process, and can significantly affect final product stability.
Table 1. Chromatographic Purification Methods Used for
Biotechnology-derived Products
Reversed-phase chromatography
Hydrophobic interaction chromatography
Charge-transfer chromatography
Size-exclusion chromatography (molecular sizing)
Ion-exchange chromatography
Affinity chromatography
Monoclonal antibodies
Cellular receptors
Metal chelate
The recovery process is usually designed to purify the final product to a high level. The purity requirement for a product depends on many factors, although chronic use products may be required to have much higher purity than those intended for single-use purposes. Biotechnology products contain certain impurities that the recovery processes are specifically designed to eliminate or minimize. These impurities include trace amounts of DNA, growth factors, residual host proteins, endotoxins, and residual cellular proteins from the media. The most common impurities of concern and suitable assay methods to detect them are presented in Table 2.
Table 2. Potential Impurities and Contaminants in Biotechnology-derived Products
Impurities or Contaminants Detection Method
Endotoxin Bacterial Endotoxins Test 85, Pyrogen Test 151
Host cell proteins SDS-PAGEa, Immunoassays
Other protein impurities (media) SDS-PAGE, HPLCb, Immunoassays
DNA DNA hybridization, UV spectrophotometry,
Protein binding
Protein mutants Peptide mapping, HPLC, IEFc, MSd
Formyl methionine Peptide mapping, HPLC, MS
Oxidized methionines Peptide mapping, amino acid analysis, HPLC, Edman
degradation analysis, MS
Proteolytic Cleavage IEF, SDS-PAGE (reduced), HPLC,
Edman degradation analysis
Aggregated proteins SDS-PAGE, HPSECe
Deamidation IEF, HPLC, MS, Edman degradation
Monoclonal antibodies SDS-PAGE, immunoassays
Amino acid substitutions Amino acid analysis, peptide mapping, MS, Edman
degradation analysis
Microbial (bacteria, yeast, fungi) Microbial Limit Tests 61, Sterility Tests 71,
microbiological testing
Mycoplasma Modified 21 CFR Methodf, DNAFg
Viruses (endogenous and adventitious) CPEh and HAdi (exogenous virus only), reverse
transcriptase activity, MAPj
a  Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
b  High-performance liquid chromatography.
c  Isoelectric focusing.
d  Mass spectrometry.
e  High-performance size-exclusion chromatography.
f  Draft guidelines relating to Code of Federal Regulations, Title 21.
g  DNA-binding fluorochrome.
h  Cytopathic effect.
i  Hemadsorption.
j  Murine antibody production.
Chromatofocusing and reversed-phase chromatography are purification methods that use chemicals, either in the stationary (bonded) phase or in the mobile phase, that may become impurities in the final product. As in any new technology, the burden of validation (i.e., demonstrating removal of potentially harmful chemicals) is incumbent on the manufacturer. Validation is necessary when isolating end product monoclonal antibodies or using a technique that contains a monoclonal antibody purification step. The process must demonstrate removal of leaching antibody or antibody fragments. It is necessary to ensure the absence of adventitious agents such as viruses and mycoplasmas in the cell line that is the source of the monoclonal antibodies. The main concern is the possibility of contamination of the product with an antigenic substance whose administration could be detrimental to patients. Continuous monitoring of the process is necessary to avoid or limit such contamination. The problem of antigenicity related to the active as well as host proteins is one that is unique to biotechnology-derived products in contrast to traditional pharmaceuticals. Manufacturing methods that use certain solvents should be monitored if these solvents are able to cause chemical rearrangements that could alter the antigenic profile of the drug substance. The manufacturer is also obligated to produce evidence regarding performance consistency of novel chromatographic columns. Considerations for single-use products such as vaccines may differ because they are not administered continuously and, in this case, antigenicity is desirable. On the other hand, validating the removal of ligand or extraneous protein contamination is necessary. Unlike drugs derived from natural sources, manufacturers of biotechnology-derived products have been required to provide validation of the removal of nucleic acids during purification. Vaccines may again be different in this regard because of the accumulated clinical history on these products.