The response to his opposition was that the President of NIH another iron lady, by the way simply fired him from the project. The big pharmacological industry really came into play when the therapeutic use of mAbs appeared to become feasible. The reason for this was that the research and developmental costs of new drugs were so high that only big, capital-strong companies could afford to produce them. The product costs were also relatively insignificant. Although mAbs belonged to the most expensive drugs, one could get kilogram amounts, enough for a set of clinical trials, from a contract manufacturer for a few million dollars.
The remaining budget, usually in excess of million, was spent on clinical trials. The high costs of clinical research are not really explained by technical and organizational complexity. They rather have to do with the fact that clinical trials provide the most important efficacy and tolerability data, on which the registration of new drugs depends. But the way of mAbs to the clinic was not as easy as anticipated: a number of hurdles had to be overcome before their therapeutic use became reality. The first and most serious concern was that rodent antibodies proved to be strongly immunogenic in humans.
This was first observed in patients who received mAbs for diagnostic purposes, and was manifested in the production of human anti-murine antibodies HAMA. Additionally, adverse effects ranging from allergic reactions to anaphylactic shock can occur.
Although immunogenicity of xenogeneic antibodies per se was not a surprise, the observations in the praxis made the industry realize that only human mAbs could be reasonably used for therapeutic purposes. This then led to the next problems, namely, a human cell line to be used as a fusion partner for hybridoma could not be identified, and alternative methods, for example, transformation of antibody-producing human B cells with Epstein-Barr virus, 17 did not yield cell lines appropriate for industry-scale culturing.
All these difficulties finally led to the recognition that the problem of immunogenicity could not be solved by means of cell biology, and the emphasis was shifted toward molecular biological approaches. Reiji Kannagi, in Methods in Enzymology , Monoclonal antibodies directed to glycolipid determinants have been widely utilized for the study of expression, distribution, and function of carbohydrate determinants in a variety of cells and tissues.
Microanalysis of glycolipids and glycoproteins is greatly facilitated by the application of monoclonal antibodies whose specificity for specific carbohydrate se-quences and structures is well established. Some antibodies directed to carcinoma-associated carbohydrate determinants are clinically applied for diagnosis of cancers. Anti-glycolipid antibodies are applied for analysis of carbohydrate-mediated cell adhesion and for cloning of glycosyltransferases and related genes. This chapter describes the methods for preparation of monoclonal antibodies against carbohydrate antigens and for determination of the specificity of established monoclonal antibodies.
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- Making monoclonal antibodies.
- Cellular imaging system.
- Multiplication by 7 (Math Club).
Monoclonal antibodies are single antibodies made by fusing antibody-producing cells with an immortalized cell line, resulting in a cell line called a hybridoma. New technology has made rabbit monoclonal antibodies possible, and rabbits have much better immune responses than mice. I have had great experiences with rabbit monoclonal antibodies.
This technique gives rise to many hybridoma clones, each secreting a unique monoclonal antibody against TRAIL. Antibodies in the growth media can further be purified and used in different immunological techniques. The diversity of monoclonal antibodies make them suitable in different immunologic techniques such as enzyme-linked immunosorbent assays ELISAs , flow cytometric analysis, Western blot analysis, immunoprecipitation, immunohistochemistry, and biologic analysis.
Antibodies with defined binding characterisics are important, especially in ELISA and flow cytometric analysis. In addition, antibodies that give the best signal and lowest background are preferred, especially in immunohistochemistry. In other immunologic techniques, like in vivo experiments and cytotoxicity assays, antibodies that neutralize bioactivity of TRAIL are needed. During past years, the availability of monoclonal antibodies against TRAIL has increased considerably. Table I also includes information about applications, references, and commercial availability. References are based on use of the actual antibody and the origin, if possible.
The antibodies are listed randomly. Table I. Thomas S. A good way to monitor the growth stage of a cell culture for controlling changes in cultivation is watching the DO and pCO 2 levels, which can also be adjusted to maximize the production of proteins such as mAbs [ 1 ]. 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.
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Cultivation factors such as initial nutrient concentration and waste metabolite production directly determine the maximum concentration that cells can reach in a bath culture. 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. 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. 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. Adapted from [ 1 , 21 ] colocar referencias. A lot of effort has been made to increase cell longevity in batch and feed-batch modes of operation. 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. The optimization of the antibody secretion rate can be achieved by high-density cell cultivation.
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On a fed-batch platform, a high cell cultivation culture can reach an mAb productivity rate of 0. 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.
Various production systems have been developed and have evolved, while new alternatives are emerging. The production of mAbs at commercial scale can be performed with adherent cells or suspension cells, although the latter is by far the most used and is better established with more efficient production methods available for cells cultivation. Thus, scale-up using suspension cells is easier. Some cultivation issues and worries have arisen regarding the production scale increase, maintenance of product quality, contamination control, demand for oxygen supply, and control over DO and CO 2 removal, among others.
Regarding suspension cell cultures, aeration is in part dependent on the agitation of the culture inside the bioreactor, which can lead to cell shear stress.
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