Principles of Downstream Processing

Recombinant proteins synthesized by bacterial, animal, plant or fungal cells are either stored in or secreted from these cells. For easy downstream processing, the secretion of proteins from cells is preferred since the proteins can be harvested fairly easily from the simpler extracellular environment. It is common for animal and fungal cells to secrete recombinant proteins. However, in simple prokeryotic bacterial cells and in plants recombinant proteins are generally stored inside the cell in some sort of storage space, a vesicle, granule or vacuole for example. This results in the relatively more difficult isolation and purification of protein from the more complicated cytoplasm of the cell.

If isolating and purifying recombinant proteins from bacterial cells, the cell must be treated in various ways to release the recombinant proteins from their storage sites in the cytoplasm. Once released the protein can be separated from the rest of the cellular contents by centrifugation or by filtration. Human proteins made in bacterial cells may not be folded or may be improperly folded so a refolding step or steps might be necessary before final isolation and purification. Proteins made in transgenic plants would present similar technical problems.

Proteins made in animal and fungal cells are, by-in-large, secreted so they are easier to separate from the surrounding medium. The first step in downstream processing of such secreted proteins would be to separate the medium containing the proteins from the cells that secreted them. In downstream processing using bioreactors and suspension cells this would usually be effected by centrifugation or filtration. In downstream processing of recombinant proteins secreted by transgenic animals the fluid containing human proteins is often ascites fluid or milk. If the protein is in ascites fluid secreted by a hybridoma in a mouse, a syringe could draw off the 10ml or so of fluid rich in human protein; if the protein is in the milk of a goat or sheep, a simple mechanical milking could remove the fluid containing the human protein.

Liquid chromatography and membrane filtration processes are then used to removed the desired human protein from other proteins and contaminants in the medium and to concentrate the desired human protein.

In liquid chromatography a solid support is used to trap a desired protein: the desired protein in its fluid medium is loaded onto a column, is trapped by the solid support, the column is washed, and the desired protein is bumped off the column using an eluting buffer with a predetermined pH and ionic strength. Today, the types of chromatography most often used to separate a desired human protein from other materials are ion exchange and affinity chromatography. Another type of liquid chromatography that is gaining more interest is gel filtration (also known as molecular sieving or size exclusion) chromatography. Ion exchange chromatography, based on electrostatic interactions between the solid support and the molecular species to be separated, is used extensively because it is fairly inexpensive. Affinity chromatography is very expensive. However, it is the most selective medium because it is based on lock and key interactions between the solid support and the molecular species to be separated.

Preparative chromatography is carried out during research and development. During preparative chromatography, methods are worked out to ensure that the protein of interest will be separated at the lowest cost with the highest yield and purity. Bench scale liquid chromatography systems and fraction collectors, along with protein standards, are employed to isolate the protein of interest from the medium and determine the best separation conditions. Protein electrophoresis is used as a companion to liquid chromatography and the fractions are electrophoresed with standards to confirm the location of a particular protein in a particular fraction, to determine the purity of the protein of interest and to determine the protein's molecular weight. The isoelectric point (pI) of the protein can also be determined using a protein gel box and a technique known as isoelectric focusing (IEF). The isoelectric porint is the point at which the protein carries equal numbers of negative and positive charges. Knowing the pI can be helpful in separating a protein by liquid chromatography. A "rule of thumb" is that the protein can be most easily separated at a pH a little above or below its isoelectric point.

Listed in the table below are the molecular weights and isoelectric points of the two human proteins (tPA and HSA) we will be characterizing, isolating and purifying by chromatography and electrophoresis. Also listed are the molecular weights and isoelectric points of the proteins found in fetal bovine serum (FBS) which is a component of the CHO cell medium but which becomes a contaminant of the medium during isolation and purification. These proteins include bovine serum albumin (BSA), transferrin, and immunoglobin G (IgG).

Preparative chromatography and electrophoresis would be accompanied by bench-scale methods development, followed by large scale column chromatography using the same or a slightly modified process that would be validated. Among other things, the expected yield would be calculated for the protein of interest for each batch of medium of a certain size received from fermentation or upstream processing. A measure of the efficiency of column packing or (HETP) would also be made each time the column is packed to help to ensure reproducibility of the chromatographic process.

There could be many contaminants in the medium which must be removed during the chromatographic process or before or after chromatography used to isolate the protein of interest. Contaminants of animal cells might include: prions, viruses, microorganisms such as Mycoplasma species, and DNA. There do not seem to be many contaminants of yeast cells and other fungi. Contaminants of bacteria include lipid A of the outer membrane of Escherichia coli and other gram negative microorganisms. Lipid A can cause toxic shock. Procedures must be in place to both remove the offending substances and organisms and to assay for their presence or removal. The limulus amoebocyte lysis (LAL) assay is used as an assay to test for the presence of lipid A.

Although, in this course we make tissue plasminogen activator (tPA) in both bacteria and Chinese Hamster Ovary (CHO) cells, we will concentrate on the isolation and purification of human proteins from the culture medium of CHO cells and fungal cells (Pichia pastoris yeast cells). Since the CHO cell medium contains fetal bovine serum (FBS), students can get a look at the difficulties inherent in separating a single human protein from other contaminating proteins.

REFERENCES:

WALSH, GARY AND DENIS HEADON 1994 Chapter 3: Downstream processing. In Protein Biotechnology, John Wiley and Sons, New York, NY.

BIOTOL 1992 Chapter 5: Separation metods; Chapter 4: Centrifugation; Chapter 3: Methods of cell disruption. In Techniques Used in Bioproduct Analysis, Butterworth-Heinemann, Stoneham, MA.

Burton, Steven J. 1996 Affinity chromatography: production and regulatory considerations. Biotechnology Laboratory, April: 64-66.

Orr, Tom 1996 Chromatography companies draw on old and new purification technologies. Genetic Engineering News, February 15: 6.

Datar, Rajiv V., Terence Cartwright and Carl-Gustaf Rosen 1993 Review-Process economics of animal cell culture and bacterial fermenations: a case study analysis of tissue plasminogen activator. Bio/Technology 11: 349-357.

Weiss, Mark D. 1995 Innovations increase the throughput of chromatographic purifications. Genetic Engineering News, February 15: 8.

Sonia Wallman, NHCTC. 1997