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Chapter: Pharmaceutical Biotechnology: Fundamentals and Applications : Production and Downstream Processing of Biotech Compounds

Issues to Consider in Production And purification of Proteins

N- and C-Terminal Heterogeneity, Chemical Modification/Conformational Changes, Glycosylation, Proteolytic Processing , Protein Inclusion Body Formation.

ISSUES TO CONSIDER IN PRODUCTION ANDPURIFICATION OF PROTEINS

 

 N- and C-Terminal Heterogeneity


A major problem connected with the production of biotech products is the problem associated with the amino (NH2)-terminus of the protein e.g., in E. coli systems, where protein synthesis always starts with f-methyl-methionine. Obviously, it has been of great interest to develop methods (Christensen et al., 1990) that generate proteins with an NH2-terminus as found in the authentic protein. When the proteins are not produced in the correct way, the final product may contain several methionyl variants of the protein in question or even contain proteins lacking one or more residues from the amino terminus. This is called the amino terminal heterogeneity. This heterogeneity can also occur with recombinant proteins (e.g.,a-interferon) susceptible to proteases that are eithersecreted by the host or introduced by serum-containing media. These proteases can clip off amino acids from the C-terminal and/or N-terminal of the desired product (amino- and/or carboxy-terminal heterogeneity) (Garnick et al., 1988).

Amino- and/or carboxy-terminal heterogeneity is not desirable since it may cause difficulties in purification and characterization of the proteins. In case of the presence of an additional methionine at the N-terminal end of the protein, its secondary and tertiary structure can be altered. This could affect the biological activity and stability and may make it immunogenic. Moreover, N-terminal methionine and/or internal methionine are sensitive to oxidation (Sharma, 1990).


Chemical Modification/Conformational Changes


Although mammalian cells are able to produce proteins structurally equal to endogenous proteins, some caution is advisable. Transcripts containing the full-length coding sequence could result in conforma-tional isomers of the protein because of unexpected secondary structures that affect translational fidelity (Sharma, 1990). Another factor to be taken into account is the possible existence of equilibria between the desired form and other forms such as dimers. The correct folding of proteins after biosynthesis is important, because it determines the specific activity of the protein (Berthold and Walter, 1994). Therefore, it is important to determine if all molecules of a given recombinant protein secreted by a mammalian ex-pression system are folded in their native conforma-tion. In some cases it may be relatively easy to detect misfolded structures, but in other cases it may be extremely difficult. Sometimes selection and purifica-tion of the native protein may require the develop-ment of novel preparative and analytical technologies for process development and quality assurance.

Apart from conformational changes, proteins can undergo chemical alterations, such as proteolysis, deamidation, hydroxyl and sulfhydryl oxidations during the purification process. These alterations can result in (partial) denaturation of the protein. Vice versa, denaturation of the protein may cause chemical modifications as well (e.g., as a result of exposure of sensitive groups) (Ptitsyn, 1987).


Glycosylation


Many therapeutic proteins produced by recombinant DNA technology are glycoproteins (Sharma, 1990). The presence and nature of oligosaccharide side chains in proteins affect a number of important characteristics, like the proteins’ serum half-life, solubility, stability and sometimes even the pharma-cological function (Cumming, 1991). Darbepoietin, a

second generation, genetically modified erythropoie-tin, has a carbohydrate content of 80% compared to 40% for the native molecule, which increases the in vivo half-life after intravenous administration from 8 hours for erythropoietin to 25 hours for darbepoietin (Sinclair and Elliott, 2005).

As a result, the therapeutic profile may be “glycosylation” dependent. As mentioned previously, protein glycosylation is not determined by the DNA sequence. It is an enzymatic modification of the protein after translation, and can depend on the environment in the cell. Although mammalian cells are very well able to glycosylate proteins, it is hard to fully control glycosylation. Carbohydrate heterogene-ity is detected by variations in the size of the chain, type of oligosaccharide, and sequence of the carbohy-drates. This has been demonstrated for a number of recombinant products including interleukin-4, chor-ionic gonadotropin, erythropoietin and tissue plasmi-nogen activator. Carbohydrate structure and composition in recombinant proteins may differ from their native counterparts, because the enzymes re-quired for synthesis and processing vary among different expression systems (e.g., glycoproteins in insect cells are frequently smaller than the same glycoproteins expressed in mammalian cells) or even from one mammalian system to another.


Proteolytic Processing


Proteases play an important role in processing, maturation, modification, or isolation of recombinant proteins (Sharma and Hopkins, 1981). Proteases from mammalian cells are involved in secreting proteins into the cultivation medium. If secretion of the recombinant protein occurs co-translationally, then the intracellular proteolytic system of the mammalian cell should not be harmful to the recombinant protein. Proteases are released if cells die or break (e.g., during cell break at cell harvest) and undergo lysis. It is therefore important to control growth and harvest conditions in order to minimize this effect. Another source of proteolytic attack is found in the compo-nents of the medium in which the cells are grown. For example, serum contains a number of proteases and protease zymogens that may affect the secreted recombinant protein. If present in small amounts, and if the nature of the proteolytic attack on the desired protein is identified, appropriate protease inhibitors to control proteolysis could be used. It is best to document the integrity of the recombinant protein after each purification step.

Proteins become much more susceptible to proteases at elevated temperatures. Purification stra-tegies should be designed to carry out all the steps at 4 C (Sharma, 1990) if proteolytic degradation occurs.

Alternatively, Ca2þ complexing agents (e.g., citrate) can be added as many proteases depend on Ca2þ for their activity. From a manufacturing perspective, however, providing cooling to large scale chromato-graphic processes, although not impossible, is a complicating factor in the manufacturing process.


Protein Inclusion Body Formation

In bacteria soluble proteins can form dense, finely granular inclusions within the cytoplasm. These “inclusion bodies” often occur in bacterial cells that overproduce proteins by plasmid expression. The protein inclusions appear in electron micrographs as large, dense bodies often spanning the entire diameter of the cell. Protein inclusions are probably formed by a build-up of amorphous protein aggregates held together by covalent and non-covalent bonds. The inability to measure inclusion body proteins directly may lead to the inaccurate assessment of recovery and yield and may cause problems if protein solubility is essential for efficient, large-scale purification (Berthold and Walter, 1994). Several schemes for recovery of proteins from inclusion bodies have been described (Krueger et al., 1989). The recovery of proteins from inclusion bodies requires cell breakage and inclusion body recovery. Dissolution of inclusion proteins is the next step in the purification scheme and typically takes place in extremely dilute solutions which tend to have the effect of increasing the volumes of the unit operations during the manufac-turing phases. This can make process control more difficult if for example low temperatures are required during these steps. Generally, inclusion proteins dissolve in denaturing agents such as sodium dodecylsulfate (SDS), urea, or guanidine hydrochlor-ide. Because bacterial systems generally are incapable of forming disulfide bonds, a protein containing these bonds has to be re-folded under oxidizing conditions to restore these bonds and to generate the biologically active protein. This so-called renaturation step is increasingly difficult if more S–S bridges are present in the molecule and the yield of renatured product could be as low as only a few percent. Once the protein is solubilized, conventional chromatographic separations can be used for further purification of the protein.

Aggregate formation at first sight may seem undesirable, but there may also be advantages as long as the protein of interest will unfold and refold properly. Inclusion body proteins can easily be recovered to yield proteins with >50% purity, a substantial improvement over the purity of soluble proteins (sometimes below 1% of the total cell protein). Furthermore, the aggregated forms of the proteins are more resistant to proteolysis (Krueger et al., 1989), because most molecules of an aggregated form are not accessible to proteolytic enzymes. Thus the high yield and relatively cheap production using a bacterial system can offset a low yield renaturation process. For a non-glycosylated, simple molecule this is still the production system of choice.

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Pharmaceutical Biotechnology: Fundamentals and Applications : Production and Downstream Processing of Biotech Compounds : Issues to Consider in Production And purification of Proteins |


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