Purification, protein recovery

There is much current interest aimed at the implementation of processes that integrate the upstream and downstream operation for protein recovery.131419 Although adsorption in fluidised beds provides a considerable saving in cost and time over conventional purification techniques, it still deploys a discrete operation with which the desired protein is captured at termination of fermentation or once a cell suspension has been disrupted. The main... 

Specifications for chromatographic packings often describe maximum loading in terms of the maximum capacity of a unit quantity of the packing to bind some analyte, often a well characterized protein such as bovine serum albumin. The static loading capacity35 is very different from functional capacity,2 which is the maximum amount of a particular feedstock that can be loaded and still achieve acceptable purification and recovery. Functional capacity is determined empirically for each type of load and associated set... 

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. 

In contrast to the protein recovery methods discussed above, protein purification is still based predominantly on laboratory-developed procedures that are often not directly scalable because of the high costs of the chemicals employed, the difficulties... 

The ELISA can be used for identification and quantitation of the protein product (biopharmaceutical) of interest throughout the development, production, and manufacturing process. For example, in the initial development phase, ELISAs can aid in the selection of the best cell line. In the early manufacturing steps, it can be used to identify the appropriate product-containing pools or fractions in process to be subjected to further purification. Because of the selectivity of ELISA, it is a suitable tool to select out the protein of interest from complex protein mixtures, such as cell culture fermentation media or product pools in early steps of protein recovery as well as downstream processing. Even complex mixtures do not require much sample preparation. It is important to determine... 

Ultrafiltration (UF) pressure difference protein recovery purification of polymer solutions... 

Integrative protein recovery operations are supposed to tolerate particle-containing biological suspensions as initial feedstock and to deliver a clarified product concentrate which can be transferred to further purification steps. Ideally, a first fractionation of the protein matrix contained in the feed is performed as well, thus combining clarification, concentration, and capture in a single process step. 

A number of applications are given in this chapter that detail the capture and recovery of intracellular proteins including recombinant proteins. The initial protein recovery steps, regardless of the source, are usually associated with large volumes and crude solutions, requiring removal of particles and reduction of volume before their purification can take place. Centrifugation, filtration, precipitation, solvent extraction, and batch adsorption are common unit operations involved in the preliminary steps of protein recovery. Expanded-bed adsorption, as described here, is an approach for the initial protein recovery that eliminates the need for clarification and volume reduction. In this process, a crude starting solution is pumped directly onto an... 

R. Shiloach (personal communication, 1999) believes that ion-exchange adsorbents are a powerful tool in the purification of proteins. They can be used in the initial steps of the protein recovery process by capturing the proteins, enrichment, or polishing. It is likely that new and improved adsorbents will continue to be introduced. 

Johansson, K.-E. (1989b) Protein recovery and blotting techniques, in Protein Purification - Principles, High-Resolution Methods and Applications (Janson, J.-C., L. Ryden, Eds.) VCH, Weinheim. 

In principle, a capture step is designed to maximise capacity and/or speed at the expense of some resolution. However, there is usually significant resolution and purification from molecules which have significant physicochemical differences compared to the target protein. Recovery will be of concern in any preparative situation, especially for production of a high value product, and it is important to assay for recovery during optimisation of the capture step. Examples of capture steps are shown on page 30. 

RPC is not recommended for protein purification if recovery of activity and return to a correct tertiary structure are required, since many proteins are denatured in the presence of organic solvents. 

Bio separation and pharmaceutical separation Recovery of antibiotics Purification and recovery of enzymes Purification of proteins Recovery of vitamins... 

A general outline of an inclusion body protein recovery/purification scheme is present in Figure 2. For a given protein, optimal conditions for each individual step have to be established and are a function of the composition of the starting material and the characteristics of the protein including protein size, the presence of inter and/or intramolecular disulfide bonds, the number and types of subunits in the protein molecule, and the presence of prosthetic groups. 

Interactions between protein and polyelectrolytes have been used to fractionate protein solutions (1-3), recover whey proteins (1, 3-6) and isolate serum glycoproteins (7) and recA protein (8). If the potential for protein recovery and purification from aqueous solution by precipitation with polyelectrolytes is to be fully exploited, several factors must be evaluated. The efficacy of the protein precipitation as well as the characteristics of the resulting precipitates depend on several variables. 

Cross-flow filtration (CFF) also known as tangential flow filtration is not of recent origin. It began with the development of reverse osmosis (RO) more than three decades ago. Industrial RO processes include desalting of sea water and brackish water, and recovery and purification of some fermentation products. The cross-flow membrane filtration technique was next applied to the concentration and fractionation of macromolecules commonly recognized as ultrafiltration (UF) in the late 1960 s. Major UF applications include electrocoat paint recovery, enzyme and protein recovery and pyrogen removal. 

As a result of experiments originally conducted on Apollo 16 (34), commercial electrophoretic efforts have been undertaken on space shuttle flights (45,46). The apparatus involved employs free flow electrophoresis of a protein mixture for the purification and recovery of a very high valued product. The process itself is proprietary. However, data and models are available from earlier flights and other experiments (34,47,48). 

The purification of heparinase has been followed by SDS gel electrophoresis. The crude sonicate gave more than 20 major bands the HA purified enzyme, 3 major bands and the IEF purified enzyme, 2 major bands. A summary of the specific activities, protein recoveries, and enzyme purity obtained using our purification procedures is listed in Table I. 

Leser EW, Asenjo JA (1994) Protein recovery, separation and purification. Selection of optimal techniques using an expert system. Mem Inst Oswaldo Cruz RJ 89(1) 99-109 Liao YH, Brown MB, Martin GP (2004) Investigation of the stabilisation of freeze-dried lysozyme and the physical properties of the formulations. Eur J Pharm Biophaim 58(l) 15-24... 

---