RESUMEN
Human serum albumin (HSA) is used in large amounts as an excipient in many biopharmaceutical formulation to prevent loss of the active ingredient through adsorption and/or degradation. Traditionally, iso-electric focusing has been used to demonstrate charge heterogeneity in HSA preparations. In an effort to develop new methods for the analysis of formulation components, a capillary zone electrophoresis method was developed for the analysis of HSA. Under initial separation conditions using untreated silica capillaries and 20 mM sodium phosphate, pH 6.0 as electrophoretic buffer, HSA migrated as a single peak. Addition of 1,4-diaminobutane allowed separation of several components which could be further resolved by varying the buffer pH. Optimal separation conditions were attained at 5 mM 1,4-diaminobutane and pH 8.5. The reproducibility of the separation conditions was verified by using capillaries from a different manufacturer. A comparative analysis of HSA preparations from different manufacturers provided evidence that the method may be used to qualitatively differentiate individual preparations. The analysis of rhEPO formulations, composed largely of HSA, showed levels of heterogeneity comparable to that of HSA preparations. Electrospray ionisation mass spectrometry (ESA-MS) was used as an independent method to confirm the heterogeneous nature of HSA.
Asunto(s)
Electroforesis Capilar , Albúmina Sérica/aislamiento & purificación , Humanos , Concentración de Iones de Hidrógeno , Sensibilidad y Especificidad , Albúmina Sérica/químicaRESUMEN
Human serum albumin (HSA) preparations and HSA-containing recombinant human erythropoietin (rhEPO) formulations were analyzed by capillary zone electrophoresis. HSA was separated into several components by the addition of 1,4-diaminobutane to 20 mM sodium phosphate, pH 6.0 as electrophoretic buffer. Resolution was improved by increasing the buffer pH to 8.5. A comparative analysis of HSA preparations from three manufacturers provided evidence that this method can be used to differentiate individual preparations. The analysis of rhEPO formulations, composed largely of HSA, showed levels of heterogeneity comparable to that of HSA preparations. Electrospray ionisation mass spectrometry was used as an independent method to confirm the heterogeneous nature of HSA.
Asunto(s)
Electroforesis de las Proteínas Sanguíneas , Electroforesis Capilar , Espectrometría de Masas , Albúmina Sérica/química , Electroforesis en Gel de Poliacrilamida , Eritropoyetina/química , Eritropoyetina/aislamiento & purificación , Humanos , Focalización Isoeléctrica , Desnaturalización Proteica , Putrescina/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Albúmina Sérica/aislamiento & purificaciónRESUMEN
A high-performance capillary electrophoresis (HPCE) method was developed for the analysis of recombinant human erythropoietin (rhEPO) in final drug preparations. All products examined were formulated with large amounts of human serum albumin (HSA) which is used as a protein excipient. Due to their similar physical characteristics in solution, rhEPO and HSA could not be resolved under HPCE conditions previously developed for the separation of bulk rhEPO. Addition of 1 mM nickel chloride to the electrophoretic buffer allowed complete separation of the two proteins as well as separation of rhEPO into several glycoform populations. The method was linear over the concentration range of 0.03-1.92 mg/ml, with limits of detection and of quantitation of 0.01 and 0.03 mg/ml, respectively. The precision of the method was evaluated from intra- and inter-day replicate injections of both rhEPO standard solution and formulation. Components of within- and between-batch variances were consistently below 5%, which constituted an acceptable level of variation. Products from two manufacturers were analyzed and showed little qualitative but appreciable quantitative lot-to-lot variations for rhEPO content when expressed in terms of units of biological activity. The method also revealed qualitative differences between the two products.
Asunto(s)
Eritropoyetina/análisis , Western Blotting , Calibración , Fenómenos Químicos , Química Física , Cromatografía en Gel , Electroforesis Capilar , Humanos , Proteínas Recombinantes , Reproducibilidad de los Resultados , Albúmina Sérica/químicaRESUMEN
Toxin generated by activation of the Bacillus thuringiensis CryIA(c) crystal protein (protoxin) with bovine trypsin was separated into two components by anion-exchange chromatography. One component (T2) was DNA-associated toxin, and the other was the DNA-free toxin (T1). Only one major protoxin component was observed, and it was found to be associated with DNA. The DNA from the T2 toxin varied in size from 100 to 300 base pairs, whereas the crystal and the solubilized protoxin contained 20-kilobase DNA as the major DNA component. DNase treatment converted the T2 toxin to the DNA-free T1 toxin. In contrast, the DNA in the crystal and the solubilized protoxin was resistant to DNase digestion and was not dissociated from the protein by 1.5 M NaCl. The protoxin and DNA appeared to elute as a complex with a molecular mass of > 2 x 10(6) Da on gel-filtration chromatography. No toxin was generated from the protoxin with trypsin after extensive digestion of the protoxin with DNase or dissociation of the DNA by succinylation of the lysine residues. It is proposed that DNA binds to the COOH-terminal half of the crystal protein and is essential for maintaining the conformational integrity required for crystal formation and generation of toxin.
Asunto(s)
Bacillus thuringiensis/química , Proteínas Bacterianas/química , Toxinas Bacterianas/química , ADN Bacteriano/química , Endotoxinas , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/aislamiento & purificación , Toxinas Bacterianas/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , ADN Bacteriano/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Proteínas Hemolisinas , Microscopía Fluorescente , Peso Molecular , Espectrofotometría Ultravioleta , TripsinaRESUMEN
Bacillus thuringiensis produces a 130-140 kDa insecticidal protein in the form of a bipyramidal crystal. The protein in the crystals from the subspecies kurstaki HD-1 and entomocidus was found to contain 16-18 cysteine residues per molecule, present primarily in the disulphide form as cystine. Evidence that all the cysteine residues form symmetrical interchain disulphide linkages in the protein crystal was obtained from the following results: (i) the disulphide diagonal procedure [Brown & Hartley (1966) Biochem. J. 101, 214-228] gave only unpaired cysteic acid peptides in diagonal maps; (ii) the disulphide bridges were shown to be labile in dilute alkali and the crystal protein could be released quantitatively with 1 mM-2-mercaptoethanol; (iii) the thiol groups of the released crystal protein were shown by competitive labelling [Kaplan, Stevenson & Hartley (1971) Biochem. J. 124, 289-299] to have the same chemical properties as exposed groups on the surface of the protein; (iv) the thiol groups in the released crystal protein reacted quantitatively with iodoacetate or iodoacetamide. The finding that all the disulphide linkages in the protein crystal are interchain and symmetrical accounts for its alkali-lability and for the high degree of conservation in the primary structure of the cystine-containing regions of the protein from various subspecies.
Asunto(s)
Bacillus thuringiensis , Proteínas Bacterianas , Toxinas Bacterianas , Cisteína/análisis , Endotoxinas , Aminoácidos/análisis , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/aislamiento & purificación , Disulfuros/análisis , Proteínas Hemolisinas , Peso Molecular , Elastasa Pancreática , Pepsina A , Mapeo PeptídicoRESUMEN
A procedure for the selective isolation of the C-terminal peptides from enzymatic digests of proteins is described. The methodology is based on the diagonal electrophoretic procedure described by R. G. Duggleby and H. Kaplan (1975) Anal. Biochem. 65, 346-354). The carboxyl groups in the protein are amidated with [14C]-methylamine followed by enzymatic digestion. Since only the C-terminal peptides lack a free carboxyl group, these peptides will lie on a diagonal line of a two-dimensional electrophoretogram run at pH 2.1 and 4.4. The diagonal line is delineated by autoradiography using [14C]taurine (net charge = 0 at pH 2.1 and 4.4) and [14C]choline (net charge = +1 at pH 2.1 and 4.4). Radioactive C-terminal peptides lie between these markers and can be directly excised for analysis. This procedure permits the detection and selective isolation of C-terminal peptides with minimal losses. The procedure was applied to the test proteins alpha-chymotrypsin and ribonuclease A. It was used to determine the C-terminus of the Bacillus thuringiensis toxin generated by tryptic cleavage of the protoxin.