Pharmaceutical biotechnology
The Pharmaceutical Biotechnology is an application-oriented science, scientific methods and techniques for development, testing, production and approval of drugs includes. It is therefore a sub-area of red biotechnology . Pharmaceutical biotechnology is closely related to bioprocess engineering , genetic engineering , analytics , pharmaceutical technology and drug approval .
Manufacture of biotechnological drugs
The biotechnological production of recombinant drugs ( biopharmaceuticals ) has taken on a broad role in modern pharmacy. In addition to the creation of genetically modified organisms (GMOs) for the production of recombinant therapeutic proteins , the historical fermentation of bacteria and fungi should be mentioned, which allowed the industrial production of low-molecular drugs such as antibiotics , HMG-CoA reductase inhibitors and immunosuppressants . The main difference to GMOs is that the natural biochemical metabolism of the production organism is used. Breeding improvements in product yield, as has been impressively demonstrated for Penicillium since the Second World War , must be distinguished from the conscious isolation and modification of genomic DNA by the producer, who has new, non-species-specific biosynthetic capabilities.
30 years after the first successful cloning attempts to introduce non-species-specific genetic information into Escherichia coli , the genetic modification of various production organisms and the extraction of any recombinant proteins including physiological glycosylation variants has become routine. Today 115 drugs with 84 therapeutic proteins in drugs are approved for use on humans (as of February 2006). Optimistic estimates indicate that by 2015 half of the new innovative drugs will be proteins or oligopeptides , and an increase in pharmacy sales from € 220 million (1996) to over € 1 billion by the end of the decade is expected. At the same time, the marketing of drugs as biosimilars (also sometimes referred to as biogenerics) is to be expected.
All therapeutic products manufactured recombinantly must comply with the monograph “Products manufactured using recombinant DNA technology” of the European Pharmacopoeia . The following definition of the European Pharmacopoeia is given, which is consistent with the guidelines of the European Medicines Agency and the Food and Drug Administration (FDA): "Products manufactured using recombinant DNA technology are manufactured through genetic modification, in which the DNA coding for the required product is also made Using a plasmid or viral vector is introduced into a suitable microorganism or a suitable cell line , in which this DNA is expressed and translated . The desired product is then obtained by extraction and purification. The cell or microorganism present before the vector is taken up is called the host cell, the stable connection between the two used in the manufacturing process is called the host-vector system. "
The monograph shows that the biotechnologically produced product must be characterized by the entire manufacturing process. Not only the question of chemical identity and purity is crucial, but also those of the biological production system are relevant for the identity of the therapeutic protein. In contrast to classic substances that are not produced recombinantly, the extension of the definition to include questions of the vector, the producer line and the extraction and purification is an additional safety aspect, since changing the production line and changing the working method can result in end products with unknown or different metabolites that are toxicologically and are pharmacokinetically important to the patient.
Like all production processes in pharmacy, the manufacture of biotechnological drugs must also be carried out in accordance with the rules of Good Manufacturing Practice (GMP).
Production of biotechnological products
The manufacture of biotechnological therapeutics can be divided into a manufacturing or upstream process and a purification or downstream process. As part of the upstream process, the production strain is grown from cell banks and the successive cultivation and biomass propagation via Erlenmeyer flasks or laboratory fermenters to the industrial production fermenter. Once the desired protein has been produced in sufficient quantities, the downstream process is followed by the depletion, inactivation, extraction and purification of the desired product, as well as the bioanalytical tests and galenic formulation of the product.
Production lines and vector systems
Microorganisms
The majority of the recombinant active ingredients are produced in microorganisms, with Escherichia coli being the most important producer. The use of microorganisms is advantageous because of the simple and undemanding cultivation. Further advantages are the usually shorter fermentation times compared to mammalian cells and the higher chemical and physical protein stability during processing. Microorganisms are often more easily accessible for genetic manipulation than animal or plant cells.
Industrially used production organisms must have the GRAS status (" Generally recognized as safe " or in German "Generally viewed as safe"), i. H. they must not be pathogenic or form toxic or antibiotic substances. Typical examples are E. coli K12, Bacillus subtillis , Lactobacilli and some Streptomyces species as representatives of bacteria, and Aspergillus , Penicillium , Pichia , Mucor , Rhizopus and especially Saccharomyces cerevisae as typical representatives of filamentous fungi. It should be noted that low molecular weight natural substances are usually released into the medium. High molecular substances such as the recombinant proteins discussed here (e.g. insulin, interferons) are accumulated intracellularly and can precipitate out as so-called inclusion bodies .
Mammalian cells
The production of proteins by mammalian cells takes place preferentially when a glycosylated product is desired (e.g. follitropin , erythropoietin ) or a therapeutic agent is required which must correspond to the human conformation. Animal cells and microorganisms differ significantly in these requirements. Bacteria, for example, are not capable of post-translational glycosylation and are only partially capable of correctly folding human proteins. When these products are used therapeutically, high demands are placed on the quality in terms of purity and their structural properties. Correct folding and glycosylation are crucial factors for the biological and pharmacological activity of the target proteins.
If mammalian cells are used, the production facility must be equipped with a higher technical standard. Of the cell lines from molecular biology research used in pharmaceutical biotechnology, the following lines are of particular interest as expression systems: for example the Chinese hamster ovary (CHO) cell lines, baby hamster kidney (BHK) cell lines, monkey kidney cell lines of the Vero type in the Vaccine production and mouse myeloma cells (NSO-GS) as gene expression systems for recombinant proteins.
plants
Genetically modified plants , so-called pharmaceutical plants , are increasingly being used as an alternative to the established production systems. For example, duckweed such as Lemna minor or the moss Physcomitrella patens are used in the production of biopharmaceuticals. Like plant cell cultures , these plants can be cultivated in closed systems ( photobioreactors , e.g. moss bioreactors ) and thus enable production conditions according to GMP guidelines . In the optimal case, the protein can be purified directly from the culture medium, which simplifies the downstream process and lowers production costs.
Cell banks
The sustainable quality of a manufactured active ingredient depends crucially on the producer used. The care and maintenance of a high quality producer line for batch production over a longer period of time is of great interest to the pharmaceutical company, as the approved product may only be manufactured in the cell line approved by the approval authority. The validation of cell banks and mammalian cell lines must therefore be carried out in order to document the reliability of a production process and to reproduce high product quality. For this reason, the genetically engineered cell line for production is cultivated as a so-called master seed in order to provide a basis for aliquoting working cell lines (working seeds) with several hundred ampoules that are stored in liquid nitrogen and removed when required. A loss of the displayed seed system or the consumption of the master seed kept in reserve can only be replaced by re-validated and approved cell banks.
According to the Genetic Engineering Act (GenTG), information on the clear taxonomic characterization and information on the genetic modification are required from the host or producer. Further mandatory information is whether plasmids or endogenous viruses occur naturally and whether toxic, mutagenic, carcinogenic or allergenic effects of the host are to be expected. In order to protect people and the environment, the GenTG also requires that information be given about the risk to animals or plants in the event of an unintended release. As part of the operating and production process, it must be described how the host organism can possibly be transferred, how high the minimum infectious dose is if the route of administration is known, which emergency therapeutic agents or vaccines must be stored, and how decontamination or disinfection is to be carried out.
After removal and cultivation into a working culture on solid agar (microorganisms) or in liquid culture (microorganisms and mammalian cells), successive scaling up takes place from the shake flask via the laboratory fermenter to the industrial bioreactor . Scaling up takes place in decadic steps based on empirical values, for example from an inoculum with a volume of 30 ml via 300 ml Erlenmeyer flasks and 3 or 30 l laboratory fermenters to 300 l industrial fermenters.
Vector systems
The creation of a genetically modified organism means the introduction of an additional, mostly alien DNA into the host genome. This technical process is called a cloning strategy, in which a complementary DNA ( cDNA ) is usually integrated into expression vectors by insertion . This cDNA is an exact copy of the mRNA that no longer contains the informationless intron areas . The juxtaposed expression sequences can be transferred into the host for controlled biosynthesis and clearly differentiated from the original gene.
When describing the vector used for cloning, information must be given to the licensing authority regarding the origin of the control element (s ) responsible for replication ( replicons ), which describe promoters or enhancers as expression-regulating information units, and information about the origin of the genes for Provide information for selection purposes. Further important information is data on the stability of the expression vector in the host cells as well as the assessment of the infection and tumor potential (e.g. prooncogenes).
In addition to the question of transformation and the introduction of the vector, the characterization of the recombinant DNA in the host cell is of interest. These tests are not only to be tested for seed cells, but also for production cells after one or more fermentation steps. With the help of restriction enzymes , Southern blot analysis techniques and PCR needs to be determined whether the status and stability of the expression construct is correct. Of interest are the extrachromosomal location of the recombinant DNA in prokaryotes , the location and type of integration into the host genome in eukaryotes, the number of copies in the cell and the genetic and possible phenotypic expression after cell division. The importance of the genetic stability studies lies in the increased information content on the copy number in relation to the productivity of the culture, indications of deletions and insertions of the expression vector, and they allow statements to be made about the protein identity.
Production and bioprocess technology
The production of recombinant proteins takes place by fermentation in the bioreactor, which offers optimal conditions for growth and active ingredient formation for cultivation. A distinction must be made between discontinuous, so-called batch-wise production, and continuous production, as already explained above. As part of the active ingredient production, the entire process must be described by documenting the production parameters such as temperature, pH value, oxygen and carbon dioxide saturation, process duration and auxiliary materials used. Ideally, you should only work with one cell line in the production area in order to avoid foreign or cross-contamination. Additional in-process controls must prove the actual quality. A frequent problem is possible external contamination or the activation of retroviruses that are introduced with the production line.
Depending on the requirements of the organism to be cultivated, different bioreactor systems are to be selected. Prokaryotes with a stable cell wall can be cultivated in bioreactors with stirrers, as they are not very sensitive to the resulting shear movements. Eukaryotic cells do not have a cell wall and are very sensitive to physical influences, which is why airlift bioreactors are often used for their cultivation in order to avoid cell damage from rotating blades. In the case of airlift bioreactors, the air / CO 2 mixture is blown in on the ground , which leads to convection of the liquid medium through the baffle plates used.
A third type of bioreactor that is often found is the so-called membrane bioreactor or hollow fiber reactor . Technically, it is a combination of an ultrafiltration unit and a bioreactor. The semipermeable membrane of the hollow fibers, which often consist of polysulfone or microporous polypropylene, holds back cells or separates them from the medium flowing through them, so that mostly low molecular weight products or metabolites can only pass through. Another advantage is that undesirable polymeric products such as polysaccharides , foreign proteins and enzymes are retained, thus facilitating product processing. In the pharmaceutical industry, the reactor type is used technically for the biosynthesis of the blood coagulation factor VIII , which is expressed in BHK cells (baby hamster kidney). The production is characterized by a high cell density in the perfusion reactor and a correspondingly high yield.
The qualitative composition of the nutrient medium depends on the requirements of the organism to be cultivated. For animal cells these are often much more complex than for microorganisms. Typical media for mammalian cells are, for example, composed of mineral salts, antibiotics, vitamins and physiological proteins such as growth factors, insulin or transferrin . Often fetal calf serum is added to the medium , the use of which is not without risk due to the possible prion contamination. More recent developments will presumably allow FCS to be dispensed with in the future, since genetically engineered albumin or human platelet lysate offer alternatives.
Mammalian cells can be differentiated into adherent and non-adherent cell types according to their growth behavior. Adhering cells only grow on solid media, they form a cell monolayer and stop growing upon vicinal contact. The majority of the production lines used show adherent behavior, which is why they can be grown on glass, zirconium or polystyrene balls. Successful techniques use immobilization techniques in the cultivation of mammalian cells with a high cell density . Their advantage is that cells in open-pore microcarriers and when used in fluidized bed reactors can be cultivated significantly longer and more efficiently, thus increasing the space-time yield. This is the process used to produce follitropin , for example .
In addition to the detailed presentation of the production in the bioreactor, for the sake of completeness, the chemical synthesis of short-chain peptides with less than 70 amino acids, which are obtained in routine operation with the help of synthesis machines, should be briefly discussed. Examples are oxytocin , gonadoliberin and desmopressin , but the antisense RNA fomivirsen , which was approved by the FDA in 2002, is also produced chemically.
Extraction and enrichment
Obtaining the desired end product from a culture approach is referred to as the downstream process in the entirety of all work steps. In the pharmaceutical industry, these work steps include the extraction, isolation, purification, formulation and packaging of a fermentation product into the finished end product. All processing steps must also be validated here and must be documented for the approval authorities and for the pharmaceutical company's own safety. A generally valid description of the downstream process is not possible. as it must be based on the requirements and physico-chemical properties of the respective product.
In contrast to low molecular weight substances and metabolites, the desired proteins are present intracellularly as so-called single cell proteins. Prokaryotic producers such as E. coli do not secrete this into the medium, but rather accumulate the protein in the cytosol, where it precipitates or aggregates as an inclusion body after the solubility product has been exceeded. Inclusion bodies show problems with the renaturation of the precipitated protein. Using various renaturation reactions (use of urea , guanidine hydrochloride , EDTA , dithiothreitol ), proteins are dissolved again and ideally regain their native conformation, which is important for the biological or pharmacological effect. The subsequent in vitro folding of the protein conformation is a complicated, mostly empirical process, which depends heavily on factors such as temperature, ionic strength, pH , properties of the renaturation medium and also the viscosity .
Validation of the manufacturing process
Valid guidelines, which are regularly published on the Internet by the most important approval authorities of the FDA and the European EMEA, do not have a legal and merely recommendatory character. Their creation is based on the results of the cooperation between the approval authorities and the pharmaceutical companies. For this reason, however, deviations must always be justified separately. In summary, the recommendations (guidelines, notes of guidance) describe procedures for the production and testing of recombinantly manufactured products, the documentation of results, but also the writing of approval applications. Some essential quality criteria should be briefly discussed:
Bacteria and fungus contamination
A contamination by bacteria or fungi in the biotechnological manufacturing process is seldom encountered, since all raw materials can be adequately checked by selective sterility tests. Typical contaminations are therefore viral contamination or the introduction of mycoplasmas, which cannot be retained by conventional sterile filtration with a 0.22 µm filter. To avoid this problem, 0.1 µm filters are often used, which can safely remove mycoplasmas . In addition to microorganisms themselves, microbial products such as lipopolysaccharides are a serious problem as pyrogens . There is a risk of contamination if nutrient solutions are improperly diluted or prepared. By ultrafiltration or special Zeta filter pyrogens can be separated.
Viral contamination
Viral contamination is more difficult to detect and remove in the manufacturing process and when testing the end product. Viruses cannot be detected by standard sterile tests and cannot be separated with sterile filters (0.1 µm). Contamination by retroviruses from the production line itself, such as HI, hepatitis B , Epstein-Barr , SV40 or cytomegalovirus , is often caused by the introduction of inadequately prepared animal sera that are used in culture media, or by contaminated chromatographic columns in the Downstream process. The detection of viral infections is carried out with the help of the PCR technique or with immuno- ELISA test methods, which are used intensively to check for viral infections in the production lines. Methods for separating viruses are predominantly of a physical nature, such as ultrafiltration, column chromatography or nanofiltration . The application of heat (e.g. pasteurization ) or chemical substances can lead to biological inactivation ( denaturation ) of the therapeutic protein. Therefore, these methods cannot always be used.
Foreign proteins
In every biotechnological production, unwanted proteins are found by the producer, which have to be separated off by various methods of filtration or chromatography. As shown in the past using the example of recombinant insulin, recombinant erythropoietin or follitropin , the presence of foreign proteins has a high allergic potential for the patient, which can range from slight immune reactions to anaphylactic shock . Foreign proteins not only include chemically different proteins, but also desired therapeutic proteins that are incorrectly or incompletely biosynthesized by the producer. The source of possible foreign proteins does not necessarily have to be the producer, but nutrient media or ligands from the column material are also conceivable. Detection methods for the qualitative and quantitative determination of foreign proteins and quality assurance are 2D gel electrophoresis, HPLC- MS and immunassays .
Foreign DNA
On the one hand, DNA in the end product is a host-specific contamination or has been added intentionally as marker DNA to control the downstream process. Because of a possible oncogenic effect, DNA must be removed from the end product, and according to international guidelines the maximum content is limited to 10 pg per dose. Foreign DNA, but also foreign RNA, can be effectively broken down by nucleases. One example is Benzonase, which is a genetically modified endonuclease that specifically degrades nucleic acids without destroying recombinant proteins.
Chemical contamination
Chemical impurities are mostly low molecular weight substances such as lipids , vitamins , antibiotics or high molecular weight substances such as polysaccharides that were introduced by the producer or through the nutrient medium. The majority, however, comes from exogenous sources such as nutrient media or as residues from solvents (e.g. detergents, salts, proteolytic inhibitors) that are left behind when cleaning and maintaining bioreactors and connected technical systems. The detection of residues is carried out by HPLC-MS or GC-MS .
Active substance identity
Recombinantly produced therapeutic proteins are tested according to international standards of the regulatory authorities and the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH). The pharmacologically active substance is examined for identity, purity and content, which include parameters such as relative molar mass , isoelectric point, charge, solubility and hydrophobicity. In the context of this short article and the variety of proteins on the market today, a complete listing of important analytical variables is not possible, and reference is made to the regulations and monographs published on the Internet. In addition to the physico-chemical properties, therapeutic proteins must be examined for their correct primary structure and protein folding with the help of NMR , X-ray spectroscopy or immunochemical methods. After enzymatic cleavage into short fragments, the primary structure is characterized using gel electrophoresis or HPLC. The HPLC- MALDI-TOF method, which allows a sequence analysis directly from the intact protein, is of great interest . Particular consideration is given to chemical changes such as N-terminal methionylation, the presence of N- formyl methionine or signal sequences that can be explained by bacterial protein biosynthesis . Furthermore, N- and C-terminal modifications of the therapeutic protein can be found through proteolytic processes or the formation of false or additional disulfide bridges (e.g. insulin) through oxidation.
In the case of glycosylated therapeutic proteins, the pattern of the sugar chains must be checked and, if necessary, compared with the natural human form. Post-translational processes, as already described above, lead to significantly different N- and O-glycosylations but also to possible acetylations, hydroxylations, carboxylations, deamidations and undesired oxidations. Proof of the correct post-translational modification by the producer can be carried out with the help of isoelectric focusing, capillary electrophoresis and mass spectrometry .
Content and effectiveness
The content is determined using absolute methods that refer to the number of amino acids or the amount of nitrogen (micro-Kjeldahl determination; Kjeldahl nitrogen determination ) in the molecule. The protein determination according to Lowry or Bradford is used, but a validation for the absolute amount must be carried out in advance. The European Pharmacopoeia describes only a relatively small spectrum of possible bioanalytical methods that can be used to characterize the product. Often, further substance-specific examinations are required in the individual monographs.
The effectiveness test must be carried out for each batch and must be given in national or international units per mL. If this is not possible, the information is given in biological units that have been calibrated with a stored international standard. It is recommended that the biological effect be correlated with physicochemical properties.
Protein formulation
Recombinant therapeutic proteins have to be administered parenterally because of their instability when administered orally. They are subject to the monograph “Parenteral preparations” and must be free from suspended matter, they must not contain any pyrogens and, as an injection solution, should be adapted to physiological conditions with regard to tissue tonality and pH value.
Biotech companies
Virtually none of the traditional pharmaceutical companies do not do R&D activities in biotechnology these days .
“Pure” biotech companies
Amgen is the world's largest independent biotechnology company dedicated exclusively to biotechnology. Other companies include BiogenIdec , Celgene , Gilead , Genzyme and Vertex Pharmaceuticals .
Pharmaceutical / biotech company
Looking at the R&D expenditure of the 400 most research-intensive companies in the EU and the 1000 research-intensive companies in third countries, Roche and Pfizer are in first and second place in 2010 with around 7 billion euros each, followed by US-Merck (fifth place) ), Novartis (8th place) and Johnson & Johnson (10th place).
Individual evidence
- ↑ JR Gasdaska, D. Spencer, L. Dickey: Advantages of Therapeutic Protein Production in the Aquatic Plant Lemna . In: BioProcessing Journal . March 2003, p. 49-56 .
- Jump up ↑ A. Büttner-Mainik, J. Parsons, H. Jérome, A. Hartmann, S. Lamer, A. Schaaf, A. Schlosser, PF Zipfel, R. Reski, EL Decker: Production of biologically active recombinant human factor H in Physcomitrella. In: Plant Biotechnology Journal. 9, 2011, pp. 373-383. doi: 10.1111 / j.1467-7652.2010.00552.x
- ^ Eva L. Decker, Ralf Reski : Moss bioreactors producing improved biopharmaceuticals. In: Current Opinion in Biotechnology. 18, 2007, pp. 393-398. doi: 10.1016 / j.copbio.2007.07.012 .
- ↑ A. Baur, R. Reski, G. Gorr: Enhanced recovery of a secreted recombinant human growth factor using stabilizing additives and by co-expression of human serum albumin in the moss Physcomitrella patens. In: Plant Biotech. J. 3, 2005, pp. 331-340. doi: 10.1111 / j.1467-7652.2005.00127.x
- ↑ Biotechnology for Smart Drugs Via Amgen company website, accessed April 7, 2012.
- ↑ a b Pharmaceutical and biotech companies are top worldwide in R&D investments 2010 vfa-bio.de, accessed on April 7, 2012.
literature
- O. Kayser: Basic knowledge of pharmaceutical biotechnology. Teubner, Wiesbaden 2002.
- O. Kayser, H. Warzecha: Pharmaceutical Biotechnology. Wiley-VCH, Weinheim 2012.
- T. Dingermann, T. Winckler, I. Zündorf,: Genetic engineering, biotechnology. 3rd edition, Wissenschaftliche Verlagsgesellschaft, Stuttgart 2019.
- I. Krämer, W. Jelkmann: Recombinant drugs - medical progress through biotechnology. 2nd Edition. Springer-Verlag, Berlin / Heidelberg 2011.