Cell banking systems

Recombinant biopharmaceutical production cell lines are most often initially constructed by the introduction into these cells of a plasmid housing a nucleotide sequence coding for the protein of interest (Chapter 3). After culture, the resultant product-producing cell line is generally aliquoted into small amounts, which are placed in ampoules and subsequently immersed in liquid nitrogen. Therefore, the content of all the ampoules is identical, and the cells are effectively preserved for indefinite periods when stored under liquid nitrogen. This batch of cryopreserved ampoules forms a 'cell bank' system, whereby one ampoule is thawed and the cell therein cultured in order to seed, for example, a single production run. This concept is applied to both prokaryotic and eukaryotic biopharmaceutical-producing cells.

The cell bank's construction design is normally two tiered, consisting of a 'master cell bank' and a 'working cell bank' (Figure 5.6). The master cell bank is constructed first, directly from a culture of the newly constructed production cell line. It can consist of several hundred individually stored ampoules.

Culture of the newly constructed production cell line

Culture of the newly constructed production cell line

V, Master cell bank

Working cell bank Number 1

Figure 5.6 The master cell bank/working cell bank system. For simplicity, each bank shown above contains only five ampoules. In reality, each bank would likely consist of several hundred ampoules. Working cell bank number 2 will be generated from master cell bank vial number 2 only when working cell bank number 1 is exhausted and so on

These ampoules are not used directly to seed a production batch. Instead, they are used, as required, to generate a working cell bank. The generation of a single working cell bank normally entails thawing a single master cell bank ampoule, culturing of the cells therein and their subsequent aliquoting into multiple ampoules. These ampoules are then cryopreserved and form the working cell bank. When a single batch of new product is required, one ampoule from the working cell bank is thawed and used to seed that batch. When all the vials that compose the first working cell bank are exhausted, a second vial of the master cell bank is used to generate a second working cell bank, and so on.

The rationale behind this master cell bank/working cell bank system is to ensure an essentially indefinite supply of the originally developed production cells for manufacturing purposes. This is more easily understood by example. If only a single-tier cell bank system existed, containing 250 ampoules, and 10 ampoules were used per year to manufacture 10 batches of product, the cell bank would be exhausted after 25 years. However, if a two-tier system exists, where a single master cell bank ampoule is expanded as required, to generate a further 250 ampoule working cell bank, the entire master cell bank would not be exhausted for 6250 years

The upstream processing element of the manufacture of a batch of biopharmaceutical product begins with the removal of a single ampoule of the working cell bank. This vial is used to inoculate a small volume of sterile media, with subsequent incubation under appropriate conditions. This describes the growth of laboratory-scale starter cultures of the producer cell line. This starter culture is, in turn, used to inoculate a production-scale starter culture that is used to inoculate the production-scale bioreactor (Figure 5.7). The media composition and fermentation conditions required to

Figure 5.7 Outline of the upstream processing stages involved in the production of a single batch of product. Initially, the contents of a single ampoule of the working cell bank (a) are used to inoculate a few hundred millilitres of media (b). After growth, this laboratory-scale starter culture is used to inoculate several litres/tens of litres of media present in a small bioreactor (c). This production-scale starter culture is used to inoculate the production-scale bioreactor (d), which often contains several thousands/tens of thousands litres of media. This process is equally applicable to prokaryotic or eukaryotic-based producer cell lines, although the bioreactor design, conditions of growth, etc., will differ in these two instances

Figure 5.7 Outline of the upstream processing stages involved in the production of a single batch of product. Initially, the contents of a single ampoule of the working cell bank (a) are used to inoculate a few hundred millilitres of media (b). After growth, this laboratory-scale starter culture is used to inoculate several litres/tens of litres of media present in a small bioreactor (c). This production-scale starter culture is used to inoculate the production-scale bioreactor (d), which often contains several thousands/tens of thousands litres of media. This process is equally applicable to prokaryotic or eukaryotic-based producer cell lines, although the bioreactor design, conditions of growth, etc., will differ in these two instances promote optimal cell growth/product production will have been established during initial product development, and routine batch production is a highly repetitive, highly automated process. Bioreactors are generally manufactured from high-grade stainless steel and can vary in size from a few tens of litres to several tens of thousands of litres (Figure 5.8). At the end of the production-scale fermentation process, the crude product is harvested, which signals commencement of downstream processing.

Figure 5.8 Typical industrial-scale fermentation equipment as employed in the biopharmaceutical sector (a). Control of the fermentation process is highly automated, with all fermentation parameters being adjusted by computer (b). Photographs (a) and (b) courtesy of SmithKline Beecham Biological Services, s.a., Belgium. Photograph (c) illustrates the inoculation of a laboratory-scale fermenter with recombinant microorganisms used in the production of a commercial interferon preparation. Photograph (c) courtesy of Pall Life Sciences, Dublin, Ireland

Figure 5.8 Typical industrial-scale fermentation equipment as employed in the biopharmaceutical sector (a). Control of the fermentation process is highly automated, with all fermentation parameters being adjusted by computer (b). Photographs (a) and (b) courtesy of SmithKline Beecham Biological Services, s.a., Belgium. Photograph (c) illustrates the inoculation of a laboratory-scale fermenter with recombinant microorganisms used in the production of a commercial interferon preparation. Photograph (c) courtesy of Pall Life Sciences, Dublin, Ireland

Figure 5.8 (Continued)
Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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