Purification culture systems

In this review, we focus on the use of plant tissue culture to produce foreign proteins that have direct commercial or medical applications. The development of large-scale plant tissue culture systems for the production of biopharmaceutical proteins requires efficient, high-level expression of stable, biologically active products. To minimize the cost of protein recovery and purification, it is preferable that the expression system releases the product in a form that can be harvested from the culture medium. In addition, the relevant bioprocessing issues associated with bioreactor culture of plant cells and tissues must be addressed. 

Proteins produced in plant cells can remain within the cell or are secreted into the apoplast via the bulk transport (secretory) pathway. In whole plants, because levels of protein accumulated intracellularly, e. g. using the KDEL sequence to ensure retention in the endoplasmic reticulum, are often higher than when the product is secreted [58], foreign proteins are generally not directed for secretion. However, as protein purification from plant biomass is potentially much more difficult and expensive than protein recovery from culture medium, protein secretion is considered an advantage in tissue culture systems. For economic harvesting from the medium, the protein should be stable once secreted and should accumulate to high levels in the extracellular environment. 

Rhizosecretion is easy to scale up and very cost effective with respect to isolation and purification. However, the bioreactor systems used for hairy root cultures differ from those used for plant cell suspensions. Traditional bioreactor systems have recently been adapted for root culture, and this technology is now being taken to commercial scales. The most traditional system is the airlift bioreactor used for microorganisms or plant cells. This system is adapted for the culturing of roots in liquid medium. Mist culture systems have also been developed. For this technology, the volume of the culture medium is reduced and the concentration of the secreted therapeutic protein is increased. If the protein to be produced is known to be quite stable, then a less expensive hydroponic culture can be designed in a manner suitable for scale-up. 

In summary, advances in our understanding of the nutritional and hormonal requirements of cells in culture and of the role played by attachment factors and transport proteins have led to the possibility that any cell can be cultured in the absence of serum. The type of serum substitute used and the means of achieving a serum-free culture system should be determined by the ultimate purpose that the culture system is to serve. The advantages of serum-free culture are manifest both in the practical realm of providing an inexpensive and simple starting material for the purification of cell-secreted proteins, such as antibodies, and for providing a more precise definition of the in vitro environment in order to model more... 

Finally, bioprocess improvements such as solid-state fermentation (SSF), continuous and two-stage culture systems, down-stream processing (DSP) and purification were studied. 

The basic process technology in vaccine production consists of fermentation for the production of antigen, purification of antigen, and formulation of the final vaccine. In bacterial fermentation, technology is weU estabHshed. For viral vaccines, ceU culture is the standard procedure. Different variations of ceU line and process system are in use. For most of the Hve viral vaccine and other subunit vaccines, production is by direct infection of a ceU substrate with the vims. 

Directed evolution relies on the analysis of large numbers of clones to enable the discovery of rare variants with unproved function. In order to analyze these large libraries, methods of screening or selection have been developed, many of which use specialized equipment or automation. These range from the use of multichannel pipettes, all the way up to robotics, depending on the level of investment [59]. Specialized robotic systems are available to perform tasks such as colony picking, cell culture, protein purification, and cell-based assays. 

Metal complexation — One of the most insidious and widely occurrent sources of analytical variation in IEC is product complexation with metal ions. Most proteins can form complexes with metals, regardless of whether or not they are metalloproteins.1 Participant metal ions can derive from the cell culture production process, purification process buffers, or even stainless steel chromatography systems. Complexation can alter retention times, create aberrant peaks, and substantially increase peak width. To the extent that metal contamination of your sample is uncontrolled, so too will be the performance of your assay. 

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