Harvesting and clarification

The culture fluid contains cells and also cell debris, especially in cultures with protracted stationary phases for maximization of mAb production ftnm dying cells. This cellular material needs removing not only because the proteins will interfere with subsequent purification but also cell proteolytic enzymes will be released which may destroy the product during purification. Thus the first downstream processing step, supernatant clarification, must be carried out immediately to minimize product degradation. With cultures of free-suspension cells the choice of clarification is usually between centrifugation and filtration. 

Filtration systems used for sterilization (membrane filters) can be used to separate cells from the supernatant but only for relatively small volumes as they do rapidly become clogged, and for this sort of volume centrifugation is usually easier but not as efficient at removing small cell debris. Tangential flow filtration is used to reduce problems of filter blockage by continuous recirculation of the. suspension across the membrane, and is recommended for larger volumes (several litres). Low volume systems are available from recognized filtration suppliers (e.g. Millipore. Sarstadt. Pall). Filtration systems have been reviewed (43). 

The classical concentration method for culture supernatants is precipitation, ammonium sulfate being the commonest used material. Other agents used are sodium sulfate and PEG (44). A method using ammonium sulfate is give in Proterai (11. 45). 

This concentration method is used to precipitate gammaglobulins and remove albumins 

Prepare a saturated stock solution of (NH )2S04 by adding 100 g to 100 ml distilled water, stir for 12-24 h. and filter (Whatman No.l paper), 

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Fig. 11. Flow chart of a typical pDNA production process. After fermentation cells are harvested and lysed by addition of alkaline solution. Clarification by filtration is followed by a series of chromatographic steps. After a final 0.22 pm filtration step the purified plasmid is aliquoted and stored...

Fig. 2.2. Clinical manufacturing processes. (A) Upstream processes include raw materials qualification, cell expansion, transfection, and harvesting. (B) Downstream processes include cell lysis and clarification, chromatography, and filtering, vialing, and storage. Recoveries are indicated for each step.

The first step following fermentation is the separation of solids from the liquid growth media, a step generally referred to as cell separation. More specifically, when the desired products are contained within the cells (intracellular) this step is called cell harvesting and when the products are extracellular it is known as broth clarification. The list of antibiotics with their producer organisms, molecular weight, and whether they are extracellular or intracellular is shown in Table 14.6. 

Cell harvesting, broth clarification for recovery of antibiotics and hormones fi om the culture medium, for example, mycelia... 

Manufacture Scale-Up. The adenovirus harvest process consisted of three general steps concentration, lysis, and clarification. In the small-scale process, the intact adenovirus-infected cells in culture medium were aliquoted and batch centrifuged to achieve a 30-fold concentration, and the supernatant was manually removed and replaced with a smaller volume of the freeze buffer. Although sufficing for harvest volumes of no more than lOL, such a manual process would prove to be inadequate on a larger manufacturing scale. 

B. cinerea development, alone or associated with other microorganisms, lowers potential grape quality. The enological consequences are serious in wines made from altered grapes oxidations, degradation of color and aromas, and fermentation and clarification difficulties. The objective measurement of the sanitary state of the harvest therefore presents an obvious interest. 

Downstream Processing Microfiltration plays a significant role in downstream processing of fermentation products in the pharmaceutical and bioprocessing industry. Examples are clarification of fermentation broths, sterile filtration, cell recycle in continuous fermentation, harvesting mammahan cells, cell washing, mycelia recovery, lysate recovery, enzyme purification, vaccines, and so forth. 

Clarification steps must be validated to yield product with a given specification (e.g., no viable cells and a defined particulate level, if any). The specifications should enable production of feedstream for the purification steps. Varying amounts of cell fragmentation during processing can lead to out-of-specification material in the next step. A recent FDA form 483 noted that for a 3.0-pm filter used to clarify the fermenter harvest, no study had been conducted to evaluate the effect of operating the filter at the specified maximum pressure limit on cell fragmentation. 

In this article, this new energy crop has been introduced and its benefits extolled, in particular, the yield data of Table 1. However, the specifics were done within the economic and time constraints of the grant and are left open for more detailed scrutiny. In particular, the botany and plant physiology of the SSPs need clarification by experts, as do the physical characteristics of Table 1. Further studies are needed to establish heating values for the SSPs and to determine combustion characteristics. Knowledge of the cultivation and harvest of these plants must be expanded. The hope is, however, that the set of plants discussed here makes direct burnable biomass an economically feasible alternative on a broad scale. 

Microfil- tration Symmetric micro-porous polymer membrane. Pore size 0.05-10 ym Hydrostatic pressure 1-5 bar Sieving mechanism, pore si2e and particle diameter determine separation characteristics Sterile filtration, clarification, cell harvesting bacteria, viruses separation. 

Clarification, or harvest, is typically the first step in downstream processing. During clarification, the cells, cell debris, and other particulates are removed from the cell culture broth, which contains the product. Clarification can be accomplished by depth filtration or by centrifugation followed by some form of filtration. Tangential flow filtration (ITT), sometimes also referred to as cross-flow filtration (CFF) is also used for clarification. Tangential flow filters are designed either in a flat sheet mode or hollow fiber mode. 

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