Expression system characterization

Various types of validation generally required in biopharmaceutical manufacturing include process validation, facility and equipment validation, analytical method validation, software validation, cleaning validation and expression system characterization. Combined with other elements of cGMP, including lot release testing, raw material testing, vendor quality certifications, and vendor audits, the quality of product can be consistently assured. 

According to our analytical results on the solid-state redox reaction of LiNi02 based on the phenomenological expression for solid-state redox potentials of insertion electrodes [23], the reaction consists of three redox systems characterized by potentials of 4.23, 3.93, and 3.63V with re-... 

Two NKxr splice variants have been identified (Table 3). A NKxr splice variant having a very short C-terminal intracellular tail (7 instead of 96 amino acids), which has been expressed and characterized in recombinant systems (Fig. 1), was found to be expressed at higher level than the long isoform in breast cancer cells. As compared to the long receptor, the short NKxr isoform is less subjected to desensitization and internalization... 

B. subtilis is the Bacillus species used most, and also the best characterized host [36,37]. A problem with high proteolysis of secreted proteins was solved by constructing the sacB-sacY sucrose-inducible expression system [38] and developing the six protease-deficient strain WB600 or even the eight protease-deficient strain WB800 [39]. Yields can be up to 3 gL-1 media [40]. 

Schaner, M. E., et al. Functional characterization of a human purine-selective, Na+-dependent nucleoside transporter (hSPNTl) in a mammalian expression system. J. Pharmacol. Exp. Ther. 1999, 289, 1487-1491. 

Sheeley D.M., Merrill, B.M., and Taylor, L.C.E., Characterization of monoclonal antibody gly-cosylation comparison of expression systems and identification of terminal a-linked galactose, Anal Biochem., 247,102-110,1997. 

To date, most approved protein-based drugs are for therapeutic or replacement therapies. They are recombinant versions of natural proteins such as insulin and erythropoietin. Their characteristics and functions are relatively well defined and known. The next phase of biopharmaceuticals, such as antibodies and vaccines, is more complex and requires more tests and characterizations. Controls for the reliability, contamination, and fidelity of expression systems will be high on the agenda in the coming decade. 

Kimchi-Sarfaty, C., Gribar, J. J., and Gottesman, M. M. (2002) Functional characterization of coding polymorphisms in the human MDRl gene using a vaccinia virus expression system. Mol. Pharmacol. 62, 1-6. 

In a more recent study, Webster et al. (2006) report the expression and characterization of lettuce-derived measles vaccine. The MV-H protein expressed in lettuce was demonstrated to be immunogenic in mice following intraperitoneal injection in the absence of adjuvant in addition to intranasal inoculation in the presence of a mucosal adjuvant. The highest response was observed in mice primed first with MV-H DNA and then boosted with an oral formulation of freeze-dried MV-H lettuce in conjunction with a mucosal adjuvant. In addition to this, the type of immune response was found to depend largely on the manner in which MV-H is presented to the immune system. Secreted and soluble forms of MV-H were demonstrated to induce a Th2 type response, while membrane-bound MV-H protein was found to be associated with a Thl response. 

The detailed characterization of hydroxynitrile lyases from Sorghum hicolor (E.C. 4.1.2.11) and Linum usitassimum (E.C. 4.1.2.37) has been hampered for a long time due to the lack of a recombinant expression system. Therefore our studies were focused on cloning of the coding genes, recombinant expression, and characterization of these enzymes. 

The inhibition of SuSy by the divalent cations (fCi 15 aM), Zn (fC 25 aM), and Ni (fti 37 aM) was exploited for further purification of SuSyl by immobilized metal affinity chromatography (IMAC). A subsequent gel filtration yielded homogeneous SuSyl suitable for crystallization experiments [24]. The protein chemical characterization revealed a homotetrameric organization of the 93 kDa subunit. Our kinetic data for the cleavage reaction and preliminary immunoblot analysis for phosphoserine suggested that SuSyl may be phosphorylated in the yeast expression system [28]. 

Optimization of gene expression may be applied at every step of the process, from initial cloning and characterization to initiation of clinical trials. Often several rounds of optimization will be required to select the expression system that produces the highest yield with the lowest cost fermentation and purification schemes. Significant resources are allocated for optimization because the best protein expression system can lead to several hundred- to a thousandfold increased efficiency in protein produced. Under these conditions, as much as 10% of total proteins produced by the expression system are target proteins. 

Fe2S2] clusters are part of the molybdenum containing hydroxylases. Typically, apart from molybdenum and two EPR-distinct iron-sulfur centres there can be FAD as additional cofactor. In Chlostridium purinolyticum a selenium-dependent purine hydroxylase has been characterized as molybdenum hydroxylase. The EPR of the respective desulfo molybdenum (V) signal indicated that the Mo-ligands should differ from those of the well known mammalian corollary xanthine oxidase.197 For the bacterial molybdenum hydroxylase quinoline oxidoreductase from Pseudomonas putida an expression system was developed in order to be able to construct protein mutants for detailed analysis. EPR was used to control the correct insertion of the cofactors, specifically of the two [Fe2S2] clusters.198... 

There are two general approaches to cDNA expression, transient expression and stable expression. Transient expression systems are typically based on a viral vector the host cells are infected with cDNA-bearing virus and the cDNA-derived protein is produced. At some point a maximum level of cDNA-derived protein expression is obtained and the protein is harvested for use in incubations. Viral vectors often have cytopathic effects on the host cells which usually precludes analysis of xenobiotic-induced toxicity to the host cell. Stable expression systems can be based on integrating or episomal vectors. With both stable expression approaches, homogeneous, clonally derived populations of cells stably expressing the cDNA are identified and characterized. The properties, advantages and disadvantages of the two approaches are discussed below. 

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