Cell Banks Preparation In Biopharmaceuticals Production

On the pharmaceutical market, we can distinguish traditional medications, obtained as a result of chemical processes, and those which are produced using genetic engineering techniques by living cells, such as bacteria, fungi, plants or mammalian cells [37]. The emergence of recombinant DNA technology (rDNA) and its application in the pharmaceutical industry has resulted in the rapid development of many biotechnological companies and the increase in the availability of biopharmaceuticals, which are recombinant proteins with therapeutic properties. This made the mass production of safe and effective medications possible. Currently, many diseases are treated with biopharmaceuticals produced by the DNA recombination technology [7, 65].

Manufacturing biopharmaceuticals is strictly regulated by international guidelines defining the proper conduct during production process. The entire technological process, such as: selection of the expression system, cell culture, creation of cell banks, production, isolation and purification of protein, characterization of the substance, final formulation of the medicinal product and its authorisation are subject to rigorous controls by international regulatory authorities, such as: The United States Food and Drug Administration – FDA, European Medicines Agency – EMA (European Medicines Agency), European Directorate for the Quality of Medicines and HealthCare – EDQM, and World Health Organization – WHO. These organisation ensure the coordination of the assessment and supervision over granting market authorisation to medicinal products for human and veterinary use on the entire territory of the European Union (EMA) and the United States (FDA). These regulations require manufacturers to comply with numerous procedures throughout the production process and to inform patients about existing risks associated with the use of a given medicinal product [11]. EDQM deals with the quality control of medicinal and veterinary products already authorized for market in the European Union.

The first biopharmaceutical produced with the rDNA technology in the prokaryotic system, authorized for marketing in 1982, was human insulin [32]. Other natural proteins, i.e. hormones and cytokines, belong to the next nine types of medications authorised for use by the FDA in the 1980s. Currently, many recombinant biopharmaceuticals are used in the treatment of diseases such as: metabolic disorders (e.g. type 1 diabetes, type 2 diabetes, obesity or hypoglycaemia), haematological disorders (e.g. anaemia and chronic kidney disease, haemophilia A, haemorrhagic diathesis associated with blood coagulation disorders), and in oncology (e.g., melanoma, breast cancer or colorectal cancer treatment). In 2010–2014, the biopharmaceuticals authorised for marketing were mainly monoclonal antibodies (31% of the total obtained number) and medicines with anticancer properties (16%) [61].

The key step in the production of biopharmaceuticals is the selection of a suitable expression system, i.e. a host strain and an expression vector. According to the Regulation of the Polish Minister of Health on the requirements of Good Manufacturing Practice from 2008 (Journal of Laws, No. 184, item 1143), the production of medicinal products obtained from microbial cultures should be based on a system of cell banks ensuring the prevention of undesirable changes in bacterial properties that may result from repeated cell passaging [60]. In the production of recombinant proteins in various expression systems, continuous culture is not maintained for a longer period of time. Cell banks are designed to provide the same source material for the entire manufacturing process [10, 21].

Basic assumptions and guidelines of Good Manufacturing Practice aiming at preparation of cell banks ensuring stable and reproducible production of biopharmaceuticals will be outlined in the paper. A potential Producer has to choose an appropriate set of methods necessary to develop and characterize the banks and develop appropriate procedures for their own production process. This is an area of individual “know-how” which may be part of proprietary and protected technology. Examples of basic prokaryotic expression systems and general principles for the production of Microbial Cell Banks will be discussed.

The majority of recombinant proteins is currently produced by genetic engineering, using a variety of expression systems: recombinant prokaryotic and eukaryotic strains (e.g. cell lines and yeasts), plants or transgenic organisms. These systems are readily available, safe and provide the possibility of changing the amino acid sequence of proteins in order to better adapt the final product to its function in the body. Choosing the right expression system (host and vector) is the key step in the production of biopharmaceuticals.

The features that support the use of microorganisms for the production of recombinant proteins are their diversity, metabolic potential and the ability to adapt to different environmental conditions [12]. Bacteria are the most frequently selected microorganisms, especially Escherichia coli. The use of bacteria is cost-effective due to low production costs, easy manipulation in the genome, fast growth rate and the possibility of using many molecular methods enabling to work with them at the cellular and molecular level. Bacteria are ideal for the production of low molecular weight proteins that do not require post-translational modifications [37].

Bacteria of the species E. coli strains are the most frequently the microbes of choice used for a large-scale production of proteins due to the well-known genome sequence, the best-defined transcription and translation system, the large selection of promoters described, the ease of genetic manipulation and the well-known metabolic and regulation pathways [23, 36]. About 30% of proteins with therapeutic properties, approved for use by the FDA, are produced in this host [3]. A high level of protein expression can be obtained in E. coli strains, by applying strong promoters. Moreover, E. coli can accumulate recombinant proteins in an amount of up to 80% of its dry matter, and is characterized by the ability to survive in various environmental conditions [15]. These bacteria multiply quickly on relatively inexpensive substrates, and the large-scale recombinant protein synthesis process in bioreactors has been well studies and described [59]. Currently, the K-12 E. coli strain and its derivatives [49, 75] are routinely used in production. Despite the use of E. coli strains as the system of choice, these microorganisms are not free from disadvantages affecting production processes. E. coli contains lipopolysaccharide (LPS endotoxin), for which reason the proteins obtained in this system need to be specially purified, so that they could be used in the production of biopharmaceuticals applied in human and animal therapy [78]. After entering the bloodstream, bacterial endotoxins can cause fever, hypotension, respiratory failure, endotoxic shock and ultimately sepsis. Therefore, many methods have been developed to remove endotoxins from recombinant protein preparations [45]. In addition, the overexpression of recombinant proteins in E. coli can lead to the formation of inactive, insoluble aggregates called inclusion bodies (IBs). One of the methods to avoid or reduce the production of IB is to lower the temperature or change the culture conditions, which will cause both an increase in protein expression and will affect its solubility [23].

The first biopharmaceutical produced by means of genetic engineering in E. coli was human insulin. It was achieved by David Goeddel’s team (Genentech) in 1978. Next, Genentech and Lilly signed an agreement for the commercialization of recombinant insulin, which was authorized by the FDA in 1982. These were Humulin R (rapid-acting insulin) and Humulin N (NPH, indirect-acting insulin) preparations [32, 56]. Most recombinant proteins with therapeutic properties produced in the E. coli system are used in the treatment of infectious diseases, the group of endocrine diseases or metabolic disorders [20]. In the cells of E. coli, interferons (alpha-1, alpha-2a, alpha-2b and gamma-1b) are successfully produced, as well as human albumin, hormones (insulin analogues, calcitonin, parathyroid hormone, human growth hormone, glucagon, somatropin), interleukins 11 and 2, TNF-alpha (tumour necrosis factor), G-CSF (granulocyte macrophage colony stimulating factor, granulocyte and macrophage colony stimulating factor), plasminogen activator [20] and many recombinant enzymes which are applied in diagnostics and molecular biology [37].

Apart from E. coli strains, other microorganisms display application potential as “cell factories” for the production of recombinant proteins. One of them is Lactobacillus lactis from the group of probiotic bacteria with the status of GRAS (Generally Regarded as Safe), exerting a positive impact on human health. These |bacteria do not produce endotoxins, thanks to which they can be used to manufacture medicines and food [12]. In addition, L. lactis has the ability to secrete produced proteins into the culture substrate through Sec-type secretion system [35]. For this strain various promoters are used, induced by environmental stress conditions, i.e.: changes in pH or culture temperature. Probably the best-known example is the nisin-induced promoter, which, together with regulatory elements from the nis operon, derived from L. lactis creates an expression system called NICE (the Nisin-Inducible Controlled Gene Expression System), used to produce lysostaphin and membrane proteins [51]. Another example is the P170 promoter activated by a drop in pH (below the value of 6) during the passage of cells from the exponential to stationary growth phase in the culture cultivated with the addition of glucose [51]. This system has a significant advantage. It undergoes self-induction by lactic acid accumulating in the substrate during bacterial growth. In this way, the system based on the P170 promoter can be easily used in production [41]. In 2003, bacteria of the species L. lactis were used for the first time to overproduce eukaryotic membrane protein – the human KDEL receptor (endoplasmic reticulum protein retention receptor) without forming inclusion bodies [42].

Other microbes used in the production of recombinant proteins are bacteria of the Pseudomonas genus, characterized by a fast growth rate and the ability to secrete proteins. Several strains like P. fluorescens, P. aeruginosa or P. putida are a good alternative to expression systems based on E. coli strains. The culture of bacteria from the Pseudomonas genus in bioreactors does not require such a strict control of the aeration parameters and sugar concentration in the medium, as in the case of E. coli, while maintaining high production of biomass and high expression of recombinant protein [12]. In P. aeruginosa the so-called type III secretion system is used for the secretion of proteins from the cytoplasm outside the cell. This bacterium was used to develop an expression system in which a 54 amino acid signal peptide derived from endotoxin S (Exo S) was combined with the produced protein under the control of the inducible P_tac promoter. This system was activated in the cell by calcium deficiency in the bacterial growth environment obtained by the addition” of EGTA (chelating compound – ethyleneglycol-OO’-bis (2-aminoethyl)-N, N, N’, N’-tetraacetic acid) to the culture substrate [16].

Gram-positive bacteria from the Bacillus genus are also used as hosts for the production of recombinant proteins. The advantage of this species is the lack of LPS in the structure of their cellular shields, belonging to the GRAS family and secretion of the protein directly into the culture substrate, as well as the lack of the creation of inclusion bodies. The last two features greatly facilitate the purification process of the protein product. On the other hand, a system based on the bacteria of the genus Bacillus has a number of disadvantages, i.e.: (i) the production of large amounts of cellular proteases which can degrade the structure of the produced protein; (ii) instability of plasmids in cells; (iii) the difficulty of culturing cells to achieve high density [78]. Nevertheless, due to the numerous above-mentioned advantages, Bacillus bacteria are readily used in biotechnology. The most commonly used species are: B. megaterium, B. subtilis and B. brevis, in which, for example, amylase, EGF (epidermal growth factor), interferons, lipase A, penicillin acylase, staphylokinase, streptavidin were produced [15, 75]. In order to use the bacteria of the genus Bacillus for the production of recombinant proteins the strains having a relatively low protease activity, carrying stable plasmids and the ability to grow under different nutrient conditions, are constructed using genetic engineering methods. The first such strain was the B. subtilis, constructed in 1984 with the deletion of aprA and nprE genes, encoding the proteases [39]. In contrast, in another strain B. megaterium, a stable plasmid was used with a promoter derived from the operon associated with the use of xylose, which made high expression of heterologous proteins possible. This system was induced with 0.5% xylose and blocked by the presence of glucose in the substrate [40].

Many other microorganisms such as Streptomyces, Corynebacterium [12], Caulobacter, Methylobacterium, Anabaena or Staphylococcus carnosus [75] are also used for the production of recombinant proteins in prokaryotic systems.

However, being prokaryotes, the above-described microorganisms are not used for the synthesis of proteins with high molecular weights or ones derived from higher organisms. These proteins often have a complicated structure, requiring the presence of disulphide bridges or cell chaperones to obtain appropriate conformation [20]. In addition, many of these proteins require additional post-translational modifications after synthesis, which cannot be performed in prokaryotic organisms, for example glycosylation or phosphorylation, and which are the essential feature of many eukaryotic proteins [57]. And yet the low cost and ease of bacterial culture is an unparalleled advantage over any other expression system, so despite the limitations described, microorganisms are always the preferred choice both on a laboratory scale and on an industrial scale [38]. Currently, biopharmaceuticals produced with recombinant DNA technology derived from prokaryotic expression systems constitute 1/3 of available medicines sold on the market [37].

During the production of biopharmaceuticals, the bacterial culture is not continuously maintained (strain passaging). Continuous passaging is disadvantageous, due to the risk of contamination of the production strain with other biological material present in the laboratory and, above all, due to the possibility of genetic drift (possible spontaneous mutagenesis), which may result in the loss of genotypic/phenotypic stability of the strain, and thus its features characteristic [21, 66]. In order to avoid this phenomenon, the so-called cell bank system, that enables the same, unchanged starting material for the entire manufacturing process is developed [10, 21]. The creation of cell banks reduces the costs of continuous culturing and allows the storage of cellular material in an unchanged and intact form, making it a good alternative to continuous culturing. In the further part of the paper, the method of establishing and conducting Microbial Cell Banks (MCB) will be discussed.

Currently, many different expression systems are available: expression vectors and host strains – described in Subsection 2., allowing for the efficient production of recombinant proteins. The choice of the system depends on the way the cell culture is conducted, the type and level of protein expression, the location of the protein produced, and the required post-translational modifications, as well as biological activity. The bacterial host strain should be, first and foremost, genetically compatible with the vector used, for the production of heterologous protein to be as effective as possible [80]. The most important aspect is the stability of plasmid DNA (pDNA) in bacterial cells (structural and segregative stability), because the whole process of obtaining recombinant protein in the production strain takes place without the use of antibiotics. A plasmid without antibiotic pressure should remain in the host strain for the minimum of 80 generations for such a “pair” to be classified as a stable expression system. International organizations like WHO (in the report on the standardization of biological products) [79] and EMA (in standards referring to medicinal products in the preclinical and clinical phase) [18] stipulate careful and thoughtful use of selection markers, which antibiotic resistance genes are in technological processes. A manufacturer should consider the possibility of not using antibiotics during the recombinant protein production. In addition, Zaleski P. et al. indicate that due to the spread of antibiotic resistance in the gene environment through horizontal gene transfer, it is not acceptable to use them in clinical tests. The residue of antibiotics in medicinal products may also negatively affect the quality of the final product and human health [81]. In addition, the bacterial strain used, should be free of as many endogenous proteases as possible, which can degrade synthesized polypeptides [71].

After selecting an appropriate expression system which enables high and stable expression of the recombinant protein, the next step is to create fully described and characterized cell banks. The general requirements for the production of biopharmaceuticals, starting from the creation of cell banks and ending with the registration of medicinal products used in the treatment of humans and animals, are contained in the Polish Pharmacopoeia and ICH standards developed by The International Council on Harmonization of Technical Requirements for Registration of Pharma ceuticals for Human Use. ICH provides manufacturers (e.g. biopharmaceutical companies) with guidelines for the production of safe and well-characterized medicinal products (Fig. 1).

Fig. 1.

Recombinant proteins are produced by genetic engineering, in which the DNA encoding a particular product is introduced using an expression vector into a suitable microorganism (bacterial cells), where the gene for the assumed product is expressed and the protein is produced in strictly controlled growth conditions [7]. Bacterial cells are referred to as a host cell prior to the introduction of a vector, and a stable host cell and vector system is called a host-vector system. In accordance with the guidelines contained in ICH Q5B [27] and the Polish Pharmacopoeia [19], the characterisation of the host-vector system is the first required stage in the entire technological process. ICH Q5B provides guidance on the construction and characteristics of the expression vector and host strain. The manufacturer should demonstrate the suitability of the selected host-vector system by:

After selecting a single colony producing a recombinant protein, the formation of cell banks follows. The cell bank system ensures that an identical population of cells is always preserved, which is the key element allowing for the creation of homogeneous biotechnology products. Properly prepared, described, stored and characterized cell bank, additionally processed in safe, controlled and monitored conditions, always ensures the same origin of source material used in the entire technological process. Cell banks should be created in accordance with the principles of GMP (Good Manufacturing Practice). The GMP principles ensure high quality and purity of materials used for production, as well as full control over the method and place of manufacture. The application of these rules increases the level of safety for the manufactured biotechnological products, as it allows for the full reproduction of each stage of the technological process [17, 25].

The bacterial cell bank used for the production of plasmid vectors was described as a homogeneous (fully defined) suspension of starting bacterial cells, separated into individual containers in a single operation, co-processed, in such a way that their stability, purity and identicality are ensured. It should be stored under strictly defined conditions, e.g. at –70°C or below. Depending on the type of organism used, various methods of cell storage are acceptable, but they should always ensure the adequate level of cell viability over a long period of storage [19].

Manufacturers can prepare their own cell banks in accordance with the GMP requirements, or obtain them from external sources. Then they are responsible for ensuring the quality of each cell bank, by conducting examination of each of them, regardless of their source.

According to the guidelines contained in ICH Q5D [28], it is possible to create a two- or one-level cell bank system. Before the production of a biopharmaceutical, two types of cell banks are established, referred to as respectively: the Master Cell Bank (MCB), and the Working Cell Bank (WCB), also referred to by manufacturers as MWCB (Manufacturer’s Working Cell Bank) [25, 64, 66]. The master cell bank is established first, directly from a single well-isolated bacterial colony, obtained after transformation of the bacterial strain with an expression vector selected on a medium supplemented with an antibiotic. The resulting bacterial culture is mixed with a protective agent (e.g. a cryoprotectant), it is portioned and frozen in vials for deep-freezing. The MCB should contain in appropriate number of vials with frozen cultures, so as to ensure a sufficient amount of homogeneous starting material for the entire production process. Every precaution should be taken during the formation of the MCB to avoid cross-contamination with other biological material [74]. It should also be noted that the MCB should first be fully characterized before it can be used to create a WCB.

Next, basing on one or multiple vials from the MCB, the WCB, constituting direct material used for protein production in bioreactors, is established [10]. If more than one vial from the WCB is used for fermentation, the cell suspension (so-called inoculum) for inoculating the production bioreactor should be prepared in such a way that allows for obtaining a homogeneous biological material.

ICH Q5D guidelines indicate that MCB and WCB cell banks may differ from each other in certain factors, such as the components of culture substrates and conditions of culture cultivation. Similarly, the conditions used in the preparation of MCB and WCB may differ from those used directly during protein production in bioreactors. However, as long as these changes do not affect the expression level of the recombinant protein, they are acceptable.

Approximately 100 MCB vials and 100–500 WCB vials are routinely prepared in the production process [66] (Fig. 2). The ICH guidelines also allow the possibility of creating a production system based only on a newly established MCB, in the case when a small number of frozen portions (vials) is necessary to produce the desired product in each production cycle [9]. In some cases, apart from MCB/WCB, establishment of separate banks is also required: one for the host cells themselves (bacterial strain without plasmid) and the other one for the DNA of the expression vector alone (pDNA frozen in a buffer allowing long-term storage of the material without degradation of its structure, e.g. in TE buffer). For research purposes, in order to develop individual stages of “their own” technology and its optimization, the manufacturer can also set up a research cell bank (RCB).

Fig. 2.

Each manufacturer, after establishing a system of cell banks, must develop full documentation confirming each stage of production. It should contain the history of the host strain culture, the method of isolating the strain, description of all genetic manipulations, the method of bacterial vector introduction, the method of selecting the chosen clone, reagents used for cell culturing and bank creation, the method of freezing and the number of cell passages. Next, the following must be described in detail: type of bank system applied (one or two levels), specification of the bank size (number of prepared vials), method of vial closure (whether vial caps with internal or external thread were used), method used to prepare a bank/banks, including the type of means used to freeze cells and the conditions of vial storage. The manufacturer must implement procedures developed to avoid microbial contamination and cross-contamination of the prepared bank with other cell types present in the laboratory and have a register system for each individual vial of the bank. The documentation should include a description of the labelling system and the type of labels used, which must withstand the storage process without losing the information contained. Each bank vial should be labelled with the strain name, date of production, its code and number [74].

In order to ensure continuous, uninterrupted production of biopharmaceuticals, the manufacturer should carefully consider all measures which ought to be taken to protect production from unplanned events such as fires, power outages or human error. Cell banks should be stored under strict conditions which allow long-term stability of the strain producing the recombinant protein (for example: at –70°C or at ultra-low temperature – liquid nitrogen). Stability of the strain under storage conditions should be checked by determining its survivability (details – point 4.1). It is also recommended that the cell banks used in production be stored in two or more sites within a given facility or in centres distanced from each other, in order to avoid possible losses in the case of a fortuitous event, natural disaster or failure within the factory. All stored containers should be treated identically, and, when removed from the frozen bank once, a vial is not allowed to be redeposited again. According to ICH guidelines, access to cell banks should be limited and fully controlled. A register of the location, identity and inventory of each individual vial with the production strain should be kept [66]. Both banks (MCB and WCB) should be kept under identical conditions. 4. Cell banks characterization

According to the guidelines contained in ICH Q5D [28] and the Polish Pharmacopoeia [19], each new MCB and WCB batch should first be fully characterized before being allowed to enter further stages of the production of recombinant proteins [7]. Due to the fact that all WCB banks are established with well-characterized MCBs, they can be tested in a more limited way, focusing mainly on the study of microbial contaminants that could have taken place during the preparation of vials with the production strain. The newly formed MCB is usually tested only once [10, 66]. Each of the cell banks, prior to conducting all the tests, holds the status – “in quarantine”. Only after obtaining appropriate results is it allowed to enter further stages of the manufacturing process.

The selected testing strategy (the type of methods used to test banks) depends on the type of bank, type of strain and the Manufacturer themselves. One or more of the cell banks (MCB or WCB) should always be fully characterized. If it is impossible to accurately characterize the output bank, one can perform detailed characterisation of each newly created batch of the WCBs. The scheme of basic parameters which should be determined for individual MCB and WCB cell banks in accordance with the Polish Pharmacopoeia is presented in Table I. However, these are only general guidelines. The manufacturer must develop individual analytical methods for the characterization of cell banks. The Polish Pharmacopoeia indicates that the host strain should also be characterized.

One of the reasons for the rapid development of the pharmaceutical market is the growing number of developed technologies for the production of biopharmaceuticals. A large part of the produced proteins with therapeutic properties is currently derived from bacterial expression systems. In order to ensure a uniform starting material for the entire technological process, it is necessary to establish cell banks with appropriate quality standards. The Master Cell Bank (MCB) is created first under well-defined conditions based on the procedures described in detail, from a single well-selected and characterized bacterial colony. The Working Cell Bank (WCB) is prepared as a second step from one or several MCB vials. These banks must be characterized with regard to the properties of the strain and must be free of any cross-contamination that may have occurred during their manufacture. A well-developed system for the establishment and characterisation of MCB/WCB is crucial for subsequent phases of developing a biotechnological product, and then for its approval by appropriate authorities responsible for the registration of medicinal product, such as EMA or FDA. For the needs of the manufacturing process (proprietary “know-how”), a potential Manufacturer themselves must appropriately choose a set of methods and procedures necessary to operate the banks.