Abstract Purpose Peptides are an important class of therapeutics. Their quality is evaluated using a series of analytical tests, many of which depend on well-characterized reference standards to determine identity, purity, and strength. Objective Discuss approaches to producing peptide reference standards, including vialing, lyophilization, analytical testing and stability studies. Methods Case studies are used to illustrate analytical approaches to characterize reference standards, including methods for value assignment, content uniformity, and identity testing. Methods described include NMR, mass spectrometry, and chromatography techniques for identity testing and HPLC and GC methods for assessing peptide content and impurities. Results This report describes the analytical strategy used to establish peptide reference standard and illustrates how results from multiple labs are integrated to assign a value to the final lyophilized vial. A two-step process for value assignment is described, which uses a mass balance approach to assign a quantitative value to a bulk peptide material. The bulk material is then used as a standard to assign a final value to the vialed material. Testing to confirm peptide identity and to ensure consistency of the vialed material is also described. Considerations for addressing variability, identifying outliers, and implementing stability studies are also presented. Conclusion The methods and case studies described provide a benchmark for best practices in establishing the preparation, analytical testing, handling, and storage of peptide reference standards for the pharmaceutical industry. Some peptide features, such as chiral or isobaric amino acids, may require additional techniques to ensure a full characterization of the peptide reference standard.
The methionine analog norleucine was produced during the synthesis of bovine somatotropin by Escherichia coli strain W 3 110G containing the recombinant plasmid pBGH1.Norleucine was generated by the leucine biosynthetic pathway from pyruvate or a-ketobutyrate in place of a-ketoisovalerate as the initial substrate.The intracellular level of norleucine was high enough to permit the analog to compete successfully with methionine for incorporation into protein.Two ways were found to prevent either the formation of norleucine or its incorporation into protein.The endogenous synthesis of norleucine was eliminated by deleting the leucine operon.The addition of sufficient methionine or 2-hydroxy-4-methylthiobutanoic acid, a precursor of methionine, to the culture medium prevented any norleucine from being incorporated into protein.
Inulin, a polyfructan, is found as the reserve carbohydrate in the roots and tubers of various plants. The (beta)-fructofuranosidase (inulase) from the yeast Kluyveromyces fragilis is of interest because of its industrial potential in fructose syrup and alcohol production from inulin containing plants. The enzyme was purified and characterized with respect to its physical and kinetic parameters. The relationship between the enzyme and the yeast cell wall was also investigated. It was found that the inulase of Kluyveromyces fragilis could be immobilized in the yeast cells by glutaraldehyde treatment. Although the exact nature of the immobilization is not fully understood the kinetic parameters of the immobilized enzyme are similar to those of the soluble enzyme. The cells are resistant to physical and enzymatic destruction and warrant further investigation as to their use in an enzymatic reactor for the production of high fructose syrup from inulin.
A 75-kilobase plasmid from Bacillus thuringiensis var. kurstaki (HD-244) was associated with the k-73 type insecticidal crystal protein production by mating into B. cereus and subsequent curing of excess plasmids. This plasmid was partially digested with endonuclease R · Sau 3A and the fragments were cloned into Escherichia coli (HB101) on vector pBR322. Candidate clones were screened for plasmid vectors which contained the expected insert size (at least 3 kilobases) and then with an enzyme-linked immunosorbent assay, using antisera prepared against electrophoretically purified, solubilized insecticidal crystal protein of 130,000 daltons. Several positive clones were isolated and were analyzed for expression, toxicity, and genetic content by restriction enzyme analysis. Electrophoretic transfer blots of proteins from a candidate E. coli clone, analyzed by enzyme-linked immunosorbent assay, demonstrated a predominant cross-reacting protein of about 140,000 daltons. Ouchterlony analysis also showed a single precipitin band. Extensive bioassays with Manduca sexta larvae revealed that the E. coli clones make toxin with a specific activity (50% lethal dose per microgram of cross-reacting protein) equivalent to that of the parental B. thuringiensis strain or a B. cereus trancipient carrying the toxin-encoding, 75-kilobase plasmid.
A β-glucosidase (EC 3.2.1.21) from the fungus Aspergillus terreus was purified to homogeneity as indicated by disc acrylamide gel electrophoresis. Optimal activity was observed at pH 4.8 and 50°C. The β-glucosidase had K m values of 0.78 and 0.40 mM for p -nitrophenyl-β- d -glucopyranoside and cellobiose, respectively. Glucose was a competitive inhibitor, with a K i of 3.5 mM when p -nitrophenyl-β- d -glucopyranoside was used as the substrate. The specific activity of the enzyme was found to be 210 IU and 215 U per mg of protein on p -nitrophenyl-β- d -glucopyranoside and cellobiose substrates, respectively. Cations, proteases, and enzyme inhibitors had little or no effect on the enzyme activity. The β-glucosidase was found to be a glycoprotein containing 65% carbohydrate by weight. It had a Stokes radius of 5.9 nm and an approximate molecular weight of 275,000. The affinity and specific activity that the isolated β-glucosidase exhibited for cellobiose compared favorably with the values obtained for β-glucosidases from other organisms being studied for use in industrial cellulose saccharification.
Inulin, a polyfruction, is found as the reserve carbohydrate in the roots and tubers of various plants (i.e. Jerusalem artichoke, chicory, and dahlia tubers). The beta-fructofuranosidase (inulase) from the yeast Kluyveromyces fragilis is of interest because of its industrial potential in fructose syrup and alcohol production from inulin containing plants. We have found that the inulase of K. fragilis can be immobilized in the yeast cells by glutaraldehyde treatment. These cells are resistant to physical and enzymatic destruction. Although the exact nature of the immobilization is not fully understood, the kinetic parameters of the immobilized enzyme are similar to those of the soluble enzyme. No reduction of enzyme activity was observed after glutaraldehyde treatment and glutaraldehyde concentration did not affect enzyme activity. A 96% hydrolysis of dahlia inulin was achieved in 10.5 h with a 9.5% (w/v) fixed enzyme suspension. A Jerusalem artichoke extract containing 16.8%polyfructan was completely hydrolyzed in 3.5 h with a 0.24% (w/v)fixed enzyme suspension. This is a time frame feasible for industrial consideration.
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