Enzolytics Inc (ENZC)

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Production Processes for Monoclonal Antibodies

Antibodies are glycoprotein structures with immune activity. They are able to identify or induce a neutralizing immune response when they identify foreign bodies such as bacteria, viruses, or tumor cells. Immunoglobulins are produced and secreted by B lymphocytes in response to the presence of antigens. The first monoclonal antibodies (mAbs) have emerged from a survey of hybridomas, and nowadays mAbs are produced mostly from cultivations of these cells. Additionally, there are studies and patents using a range of cells and microorganisms engineered for the production of mAbs at commercial scale. For some years, new methodologies have advanced with new production processes, allowing scale-up production and market introduction. Large-scale production has revolutionized the market for monoclonal antibodies by boosting its production and becoming a more practical method of production. Production techniques have only had a sizable breakthrough due to molecular techniques. Various systems of production are used, including animal cells, microorganisms, plants, and mammary glands. All of these require the technological development of production process such as a stirrer, a wave bioreactor, and roller bottles.

With the advent of genetic engineering, it has been possible to develop new methods to obtain monoclonal antibodies, both for improvement with regard to these humanized antibodies and for production models. Advances in molecular and cell biology for the development of more efficient antibodies have allowed advances in diagnostic and therapeutic areas. Such advances have triggered improvements in production processes, allowing for the reduction of production costs and thus leading to an increase in the popularization of treatments with mAbs.
All process improvements provide a consistent and reproducible production of large quantities of mAbs at a moderate cost.
Large-scale production has revolutionized the market for monoclonal antibodies by boosting its production, making this a more practical method of production. Production techniques have only had a sizable breakthrough due to molecular techniques.

In general, a process of commercial production of mAb begins with the generation of an mAb by immunizing an animal or by molecular biology methods involving the identification and optimization of the coding DNA sequence and the construction and identification of a stable high-producing clone. Improvements in cultivation are similar to those applied in other bioproducts that rely on culturing microorganisms or cells, requiring the development of a well-designed culturing process comprising the full range of control and associated operations that will support technical evaluations.

mAbs production processes in wave or single-use bioreactor (SUBs) are characterized by flexibility and low operating costs when compared to the production processes in fixed stainless steel vats. The development of bioprocesses involving these production platforms can reap greater acceptance by the industry.
Drugs based on mAbs have been controlled by regulatory agencies around the world. Therefore, it is necessary to elaborate regulatory protocols accompanying the increase in production and the nuances of the characteristics of this class of drugs.

mAbs production techniques

Hybridoma and phage display
Milstein and Köhler described the first technique developed for stable monoclonal antibody production in 1975. This technique consists of creating a hybridoma, a stable hybrid cell capable of producing a single type of antibody against a specific epitope present in an antigen. Hybridoma construction was initially produced from murine models. The technique consists of removing a pool of activated B lymphocytes from an immunized animal spleen and combining them with immortalized myeloma cells unable to produce the enzyme hypoxanthine-guanine-phosphoribosyltransferase (HGPRT), an important enzyme present in the salvage pathway, one of the pathways responsible for nucleotide production [1]. To select hybridoma cells, the pool of cells resulting from the fusion (a mix of hybridoma cells and non-fused B lymphocytes and myeloma cells) are cultivated in a selective medium containing aminopterin, which inhibits the nucleotide de novo synthesis. Myeloma cells lack the salvage pathway for nucleotide production. When they are exposed to aminopterin present in selective medium, the de novo synthesis is also blocked, and as a result, myeloma cells are no longer viable since all major pathways for nucleotide production are blocked. In contrast, non-fused, activated B lymphocytes can survive as their salvage pathway works perfectly and they can continue nucleotide production even if the de novo pathway is blocked by aminopterin. However, these cells are not immortalized and can replicate only a limited number of times after which they eventually die. With this in mind, only cells capable of replicating indefinitely and synthesizing nucleotides through the salvage pathway can survive through selection conditions, and these cells are the hybridomas.

In spite of the fact that the primary recombinant mAbs were delivered utilizing this innovation—including the first medication approved by the Food and Drug Administration (FDA) for therapeutic proposes (Table 1)—the great contribution of this technology was mostly to elucidate immune response mechanisms and control in vitro antibody production. Therefore, mAb hybridoma production from murine sources exhibits a genuine downside in human therapeutics (Figure 1).

After a few infusions, murine antibody molecules trigger the human anti-mouse antibody (HAMA) response of the human immune system [1, 12]. To work around this issue, new methodologies have been developed to deliver antibodies similar to human molecules, so the technology evolved to less immunogenic chimeric antibodies (constant regions of human antibodies linked to the variable region of the murine source), creating a new set of therapeutic possibilities (Figure 1). Subsequently, the need for an even less immunogenic alternative boosted the production of humanized antibodies (only the region that interacts with the antigen epitope is from mouse origin) (Figure 1). Even fully human antibodies (Figure 1) can be produced from genetically modified mice [13].

A great improvement in mAb production has come with the development of phage display libraries. This methodology helps to investigate interactions between molecules (protein-protein, protein-peptide, and protein-DNA) and consists, basically, in cloning Fab-region-coding genes amplified from B lymphocytes into bacteriophage plasmid vectors. Then the bacterium can be transformed with these vectors, going on to express the heterologous genes from a viral capsid. This capsid contains viral proteins and proteins encoded by the Fab sequence received by that specific cell. Once the library is complete, the affinity between proteins produced from different Fab regions can be tested against the antigen of interest and the cell transformed with the plasmid that contains those genes can be readily sequenced. The advantages of this methodology are the following: the same library has the potential to generate a great number of new antibodies, it is an in vitro process, so it does not require animal immunizations steps, and because of that, toxic antigens can be tested. Also, a greater variety of antigens can be tested, and antibody molecules can be rapidly obtained [13].

Culture production factors

Cell lines
One of the most critical steps in developing an mAb production system is to choose the cell line. The cells must be stable and secrete the desired protein with the correct conformation at high levels. Based on these requirements, the mammalian cell is the most commonly chosen expression system for mAb production. The main advantage of a mammalian expression system is that the cellular machinery is adapted for the production, processing, and secretion of highly complex molecules. The great majority of commercial mAbs are produced in Chinese hamster ovary (CHO) and NS0 cells, originating from plasmacytoma cells that were modified until IgG generation in nonsecreting B cells. Genetic modifications in CHO cells have generated cell lines capable of producing a high quantity of humanized mAbs. These cell lines were able to secrete up to 100 pg/cell/day [14]. Other modifications led to a high production of a chimeric mAb, ranging from 80 to 110 pg/cell/day [15]. NS0 modifications also have been made, leading to higher mAb production rates, ranging from 20 to 50 pg/cell/day [16]. In smaller quantities, hybridoma cell lines are also used in industrial mAb production. Some hybridoma strains are reported to have a production rate up to 80 pg/cell/day [16]. In spite of this, different mammalian cell lines and even more peculiar expression systems such as genetically modified plant cells, genetically modified insect cells, and genetically modified microorganism cells have also been used in mAb production and have gained space in the biopharmaceutical industry [1, 8]

Microorganisms modified by genetic engineering techniques have attracted much focus in industry, because these cells are simpler to handle and to modify when compared to animal cells. Other advantages of production methods using genetically modified microorganisms are that these cells have well-defined expression systems, and the production methodology is reproducible and easy to validate. Modified yeast cells, such as Pichia pastoris have a great potential for usage since these cells are known to achieve high secretion levels of heterologous proteins. Yeast cultivation systems for mAb production are easier scale-up and are cheaper when compared to mammalian cell cultivation systems. They can be cultivated in regular stirred tank bioreactors, in batch, or in feed-batch modes of operation. Generally, microorganisms do not have physicochemical and biological characteristics for the appropriate expression and posttranslational processing of mAbs [4].

Modified plants have also gained attention since plants are easy to cultivate and propagate. Other cultivation advantages such as cheap medium, low maintenance cost, and high production yields make plant production a cheaper alternative when compared to mammalian cell cultures [17]. However, there are some limitations—different glycosylation patterns and post-translational processing can also make plant cell utilization difficult [17].

Production platforms
The cell culture for mAb production can follow three different types of processes. The simplest of them is batch production, which consists of a closed system where a bioreactor is sterilized and prepared with a medium containing all the nutrients needed for cellular growth and product manufacturing and then, cells are inoculated. There is no feeding system with fresh medium or withdrawal of spent medium. As the process runs, nutrient concentration decreases and waste metabolites are produced, lowering cell viability. In spite of being a simple process, batch is not the most suitable type of production platform for mammalian cell cultures, as the environment inside the reactor quickly becomes unfavorable for cell growth and, at the same time, waste product concentration increases. Cultivation factors such as initial nutrient concentration and waste metabolite production directly determine the maximum concentration that cells can reach in a bath culture. Generally, this type of cultivation reaches a maximum density of 1–2 × 106 cells/mL, and then the cell viability drops rapidly [1]. The production process lasts for 4–7 days, when productivity reaches certain concentration of interest [1]. Supernatant is collected and the product is recovered by downstream processes. The time that each batch takes to finish also depends on the production kinetics. If the production is growth dependent (production occurs concomitantly with cellular growth), batch processes can be stopped as soon as cells reach the stationary phase. But if the product is not associated with growth (production only starts when the growth rate decreases), the culture needs to be carried for a longer period of time since production only starts at stationary phase.

In contrast to batch, a second type of production process utilized is continuous fermentation. There are two types of continuous production: chemostat cultures and perfusion cultures. Concerning chemostat cultures, fresh medium is added to the bioreactor and fermented medium is removed along with cells at a constant flow rate so that the culture volume remains unchanged. The flow rate (dilution rate) controls cellular growth and when these two variables are equal, the bioreactor reaches equilibrium—cell concentration, nutrient concentration, and product concentration are held constant. In this context, the culture can be kept in equilibrium for several months reaching a cell density of 10–30 × 106 cells/mL [1]. To avoid viable cell loss along with the constant outflow of the by-products of cell metabolism, many manufacturing plants have developed a cell-recycling system and thus, the perfusion culture method was developed where cells are kept inside the bioreactor. The disadvantages of continuous fermentation are the use of a large amount of expensive culture media and the difficulty in recovering the product, which comes out fairly diluted. These two disadvantages are consequences of the constant medium flow rate. To work around the product dilution problem, some production manufacturing plants have ultrafiltration systems which retain the product inside the bioreactor [18]. Another obstacle of this type of process is that the establishment of culture conditions for a stable industrial production plant can take months. For this to occur, the strain used must be very stable and have its physiological aspects clearly elucidated, such as growth rate, productivity, and response to certain stress conditions. It is not uncommon to hear that numerous attempts are made before the settlement of a stable production plant is achieved, but, once settled, this production process can bring many advantages, since it can be operated in smaller-volume bioreactors, and therefore have greater production flexibility.

The third type of process for producing monoclonal antibodies is by far the most utilized at industrial scale, which is fed-batch process. In this process, the cell density reaches 8–12 × 106 cells/mL, and cell viability in the bioreactor is enhanced by controlled nutrient addition at specified intervals [1]. The production process can take 12–20 days [1]. Usually, the same medium used in the initial culture is also used for feeding, but in a more concentrated version. The feeding solution composition can be designed to supply the cells based on their metabolic state at different culture phases by analyzing and identifying the spent medium nutrients that are being more consumed. Furthermore, the medium used in feeding can be modified to promote cell growth or to stimulate molecule production, since different components may modify the behavior of cells, changing the metabolism for different purposes. The feed solution can also be designed to minimize the production of waste metabolites that cause cell stress when in excess. However, their production is not completely avoidable as they eventually reach harmful concentrations. It is relatively easy to scale up and operate this system. More summarized data about the advantages and disadvantages of each process for mAb production can be seen in Table 2.
A lot of effort has been made to increase cell longevity in batch and feed-batch modes of operation. It is expected that the longer the cells are maintained viable, the greater the antibodies’ production will be. So, in order to maintain cell viability, some culture parameters can be optimized, such as culture media, feed solution, and mAb secretion rates and by-product production. To improve mAb titers in the batch platform, the start medium can be supplemented with glucose and amino acids, increasing mAb production up to eightfold when compared with regular media [9, 26]. Improvements for the fed-batch platform can be achieved by adjustments in feed solution, as mentioned before. Feed solutions containing glucose and aminoacids/glutamine have been reported to increase mAb titers from two to fourfold, reaching production of up to 2 g/L, when compared with the batch production platform [19].
The optimization of the antibody secretion rate can be achieved by high-density cell cultivation. On a fed-batch platform, a high cell cultivation culture can reach an mAb productivity rate of 0.94 g/L/day and a final titration of 17 g/L, while a continuous culture performed at high density conditions can reach final titration and productivity rates of 0.8 and 1.6 g/L/day, respectively [20]. Optimizing mAb secretion highly depends on the cell line chosen for production. Each cell strain can be influenced by the manufacturing conditions and respond differently to increasing or decreasing mAb production and secretion [19]. The accumulation of toxic by-products is a great bottleneck in manufacturing processes since they can inhibit cell growth and then directly affect mAb production. Although a few strategies to minimize this by-product accumulation have shown to be promising, some are not applicable for a large-scale production. Optimizing medium composition and feed solutions with substrates that reduce toxic compound production is the most common strategy used at industrial scales of production [19].

Although most mAbs are produced by fed-batch process, there are tendencies indicating that in the future many bioprocesses will be operated in continuous platforms, especially for the production of biopharmaceuticals. On these platforms, the production system will be coupled to upstream and downstream processes [21]. However, for this to actually happen, a great improvement in technological development still needs to be achieved.

Conclusions and perspectives
Actually, the trade of monoclonal antibodies makes up half of marketed biopharmaceuticals, reaching $ 75 billion. For some years, new development methodologies of antibodies have advanced with new production processes, allowing scale-up production and market introduction, and demands for high-quality biologics will continue to increase in the coming decades. Generally, processes are similar to those applied in the scheduling for other bioproducts/biosimilars that rely on culturing microorganisms or cells, requiring the development of a well-designed culturing process comprising the full range of control and associated operations that will support technical evaluations.

In combination with increasing pressure from regulatory agencies for enhanced quality and lower process costs from the health care systems, we are facing an important challenge. It will be necessary to make changes in plant design aiming for highly flexible multi-purpose facilities for small production volumes.
https://www.intechopen.com/books/fermentation-processes/production-processes-for-monoclonal-antibodies