Immunotherapy for Infectious Diseases
Immunotherapy for Infectious Diseases Immunotherapy for Infectious Diseases
Production of Igs and MAbs Targeting Infectious Diseases 87 behind the major process improvements in mammalian cell technologies in the last few years. The manufacture of antibodies in transgenic animals or plants might be expected in the future, but quality considerations exist at the present time. Large-Scale Production In Vitro Figure 8 shows a typical flow diagram of the process of large-scale MAb manufacture. The manufacturing process attempts to obtain the highest possible product quality and safety, consistently produced at low costs in high amounts. A few manufacturing facilities have been established that allow production of several hundred kilograms of purified antibody a year. Very important for the success of the entire manufacturing process is the availability of cloned cells that express the antibody stably at high levels. Commercial manufacturers usually do not publish detailed data describing levels of expression. According to the experience of the authors, cell clones, either hybridomas or recombinant cells such as CHO, can be established displaying specific expression rates in the range of 10–50 �g of antibody per 10 6 cells per day. As described previously, the establishment of these cell clones requires several subcloning and/or gene amplification steps. The first logical step is the adaptation of the cloned cells to serum-free growth media. Again, commercial manufacturers do not publish the exact composition of media. Nevertheless, serum-free and even protein-free growth media have been empirically optimized that are also free of raw materials from animal origin, to avoid potential risks of contamination. Often enzymatically digested plant extracts are used as supplements to improve the growth-promoting quality of serum-free media (131). These growth media are generally considered safe and are relatively inexpensive (approximately $1/L) when produced in large scale. Compounding of media is usually done by specialized companies that also certify the quality. Once the cloned cells are adapted to a certain medium, an extensive program of stability testing is necessary to ensure that the cells are expressing the antibody in consistent quality and quantity over a certain number of passages. The number of passages in stability testing depends on the final production scale envisaged. If we assume that X cell passages are needed to reach the final production harvest, extension of stability testing to approximately 1.5 � X cell passages is recommended, to ensure process consistency. It is therefore also recommended to perform the adaptation and stability testing of the cloned cells in a bioreactor that most closely simulates the physical environment of the bioreactor that will be used for the manufacturing process. Once fully adapted and stable cell clones are obtained, a master cell bank (MCB) is established in accordance with the principles of good laboratory practice and good manufacturing practice. Usually, a working cell bank (WCB) is also established concomitantly from the master cells. The combination of MCB and WCB ensures a practically unlimited source of cells of identical quality for the manufacturing process. Each new production lot of product is then inoculated from the WCB and used for production up to passage level X. Full characterization of the MCB and the WCB is mandatory. The characterization includes a series of investigations that establish and define cell identity and safety in a clearly traceable and reproducible manner. These cells and the manufacturing process define the final drug with respect to all its characteristics (132). In addition, various quality control tests are conducted on each batch of the biologic drug before
88 Kunert and Katinger Fig. 8. Scheme of large-scale manufacture of antibodies. QC, quality control; QA, quality assurance. release for sale or clinical testing. Manufacturing consistency must be proved in so-called consecutive lots, and changes in the established manufacturing process are only allowed under strict change control, validation, and approval by the licensing authority (depending on the status of the drug and the nature of the change). To establish maximum safety, controls are introduced at various process steps. For example, to control potential contaminations (such as viruses), samples from the crude culture harvest are tested since the chance finding of contaminants, if any, is highest at this particular step of the process. Production in a Bioreactor A great variety of devices for in vitro cultivation of animal cells have been developed. The choice of a proper bioreactor depends on the following criteria: 1. Cell type: in suspension or adherent to growth-supporting solid matrices 2. Cell culture method: batch, batch-fed, semicontinuous, or re-fed batch, continuously perfused 3. Scale of production. Some common characteristics of animal cells that determine the design of in vitro cultivation systems (i.e., design of bioreactor and culture method) are low growth rates, sensitivity to shear stress, direct sparging, and ammonia. Designing large-scale culti-
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88 Kunert and Katinger<br />
Fig. 8. Scheme of large-scale manufacture of antibodies. QC, quality control; QA, quality<br />
assurance.<br />
release <strong>for</strong> sale or clinical testing. Manufacturing consistency must be proved in so-called<br />
consecutive lots, and changes in the established manufacturing process are only allowed<br />
under strict change control, validation, and approval by the licensing authority (depending<br />
on the status of the drug and the nature of the change). To establish maximum safety,<br />
controls are introduced at various process steps. For example, to control potential contaminations<br />
(such as viruses), samples from the crude culture harvest are tested since the<br />
chance finding of contaminants, if any, is highest at this particular step of the process.<br />
Production in a Bioreactor<br />
A great variety of devices <strong>for</strong> in vitro cultivation of animal cells have been developed.<br />
The choice of a proper bioreactor depends on the following criteria:<br />
1. Cell type: in suspension or adherent to growth-supporting solid matrices<br />
2. Cell culture method: batch, batch-fed, semicontinuous, or re-fed batch, continuously perfused<br />
3. Scale of production.<br />
Some common characteristics of animal cells that determine the design of in vitro<br />
cultivation systems (i.e., design of bioreactor and culture method) are low growth rates,<br />
sensitivity to shear stress, direct sparging, and ammonia. Designing large-scale culti-
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