Derek H. Calam

Isolation of influenza viral proteins by size-exclusion and ion-exchange high-performance liquid chromatography: the influence of conditions on separation

Constituent proteins of influenza virus and of vaccines containing whole virus or viral antigens (surface antigen vaccines) have been separated by size-exclusion high-performance liquid chromatography. Preparations of whole virus have also been examined by high-performance anion-exchange chromatography. The separations are influenced by the conditions employed and, in particular, by the nature of detergent used to disrupt the virus and that incorporated in the eluting solvent. Individual proteins are recovered with retention of immunological activity. The method is applicable to small portions of a single human dose of vaccine.

Analysis of glycoprotein hormones and other medically important proteins by high-performance gel filtration chromatography

Abstract Glycoprotein hormones and growth hormone of human origin, and allergen extracts of plant and animal origin have been examined by high-performance liquid chromatography using size exclusion on TSK SW supports. The short times required for elution, compared with low-pressure systems, minimize changes such as aggregation and dissociation. Analysis can be performed with amounts of material too small for examination by conventional gel filtration. The elution behaviour of the glyco-proteins is intermediate between those of globular proteins and of dextrans of the same molecular weight. Native luteinizing hormone and follicle stimulating hormone are well separated from their sub-units. Aggregates and sub-units detected in samples of these hormones are not chromatographic artifacts. Differences in the chromatographic profiles of preparations of follicle stimulating hormone are associated in differences in biological and immunological properties. Commercial samples of chorionic gonadotrophin manufactured to official specification contain varying amounts of protein and differ in composition. Analysis on TSK supports confirms that the composition of growth hormone preparations depends on the procedure by which they are obtained, and the main fractions have been examined by electrophoresis. A major peak found in an extract of house dust mite Dermatophagoides pteronyssinus is attributed to the P 1 allergen. Other mite species give distinctive elution profiles on chromatography. An extract of pollen from the grass Dactylis glomerata was fractionated and the components correlated with proteins in the whole extract separable by electrophoresis. Biological and immunological studies are in progress on the fractions.

Assay of the combined formulation of ergometrine and oxytocin by high-performance liquid chromatography

Abstract The present British Pharmacopoeial monograph for the combined formulation of ergometrine and oxytocin requires a spectrophotometric assay for the ergometrine and a biological assay for the oxytocin content. Simultaneous spectrophotometric assay of the two ingredients has not previously been practicable, owing to the widely different amounts of each. Two high-performance liquid-chromatographic separations have been developed by which both ingredients can be assayed at different dilutions in both systems. One separation is on the strong cation-exchange bonded phase Partisil 5 SCX, where reproducibility of injection gave a peak-height coefficient of variation of 0.8% for ergometrine and 2.5% for oxytocin. An alternative system involves use of the ion-pair reagent, sodium tetradecyl sulphate, with a reversed-phase packing, giving a coefficient of variation for repeat injections of 1.0% (peak height) for ergometrine and 2.5% (peak area) for oxytocin.

Applications of chromatography in the standardization and control of biological products

The problems posed in the standardization of medicinal biological products and in the development of official specifications for such products, e.g., in pharmacopoeias, are outlined. Information derived from bioassay can be extended and complemented by that from physico-chemical studies. The role of chromatography, including gas--liquid, thin-layer and column methods, is assessed and illustrated with examples from the literature and from the authors studies on antibiotics and polypeptide hormones.

Studies on allergens of mammalian origin

Cat and dog saliva and extracts obtained from cat and dog hair have been examined by high-performance liquid chromatography (HPLC) to identify the likely allergenic molecules. Separation of the materials by size exclusion HPLC showed that several components in the range of apparent molecular weight from 35,000 to 200,000 had inhibitory activity in the radioallergosorbent test (RAST) when tested against pooled serum from donors hypersensitive to dogs and cats. One of these components active in our test system was identified as albumin. Despite differences in gross composition of the extracts no significant difference in the position of the active fractions was observed between cat hair and saliva, between dog hair and saliva or between hair extracts from different breeds of dog. Fractionation by anion-exchange HPLC, although promising, was complicated by problems of sensitivity.

Investigations of the allergens of cocksfoot grass (Dactylis glomerata) pollen.

The pollen of cocksfoot grass (Dactylis glomerata) is an important cause of allergic reactions in man. Preliminary studies, which established that constituents of an extract of this pollen could be separated, and then recovered efficiently, by size-exclusion chromatography on TSK G3000 SW, have been extended. A comparative examination has been made by this procedure of cocksfoot pollen extracts from different sources and of several batches of extract from one source. The recovery and distribution of biological activity has been assessed by the radioallergosorbent test, and the results have been used for the selection of fractions for further investigation by chromatography and electrophoresis. Two constituents active in the radioallergosorbent test have been purified from the extracts.

Production and certification of an enzyme reference material for pancreatic α-amylase (CRM 476)

Abstract We describe the preparation of a lyophilized material containing purified human pancreatic α-amylase and the certification of its catalytic concentration. The enzyme was purified from human pancreas by ammonium sulphate precipitation and chromatography successively on DEAE-Sephacel, CM-Sepharose and Sephadex G-75. The purified enzyme had a specific activity of 52.9 kU/g protein and was >99% pure on polyacrylamide gel electrophoresis. Only trace amounts of lipase and lactate dehydrogenase were detected in the purified fraction. The purified pancreatic a-amylase had a molar mass of 57 500 g/mol and an isoelectric point at 7.1. The material was prepared by diluting the purified α-amylase in a matrix containing PIPES buffer 25 mmol/l, pH 7.0, sodium chloride 50 mmol/l, calcium chloride 1.5 mmol/l, EDTA 0.5 mmol/1 and human serum albumin 30 g/l, dispensing in ampoules and freeze-drying. The ampoules were homogeneous and the yearly loss of activity on the basis of accelerated degradation studies was less than 0.01% at −20° C. The certified value for α-amylase catalytic concentration in the reconstituted reference material is 555 U/l ± 11 U/l when measured by the specified method at 37°C. The material can be used to verify the comparability of results from different laboratories, for intra-laboratory quality control or for calibration of α-amylase catalytic concentration measurements.

Determination of vitamin D3 in cod-liver oil by high-performance liquid chromatography

The present British Pharmacopoeia monograph for cod-liver oil requires a bioassay for the vitamin D3 content which is both time-consuming and complex. Alternative assays employing chromatographic procedures have been described but all these involve prior saponification of the oil. A selective extraction for vitamin D3 without the need for saponification is reported in this paper. The extraction utilizes only chromatographic assay using argentation on reversed-phase silica, with vitamin D2 as the internal standard. Reproducibility of injection gave a coefficient of variation of 0.6%, and repeatability of extraction for six samples gave a coefficient of variation of 6.8%.

High-performance liquid chromatographic investigations on some enzymes of papaya latex

Papaya latex and commercial chymopapain have been examined by cation-exchange chromatography on a TSK SP 5PW column. Multiple components are observed and the resolution is superior to that obtained by low-pressure ion exchange. Most components display amidase activity. Fractions obtained from chymopapain by preliminary chromatography on SP-Sephadex have also been examined by the same procedure and by N-terminal and amino acid analysis. The results are consistent with the existence of chymopapain in multiple forms, the proportions of which alter. The chromatographic profile of chymopapain is influenced by the presence of cysteine in the sample.

Chromatographic analysis of allergens

SummaryExtracts of insect, mite, fungal and mammalian origin, known to provoke an allergic response in man, have been examined and fractionated by high-performance size exclusion chromatography. The mild conditions employed for separation have permitted direct immunological assessment of the fractions obtained, and recognition of the biologically important components. These are being isolated for further study. This chromatographic procedure has proved to be of value for assessing different extracts from the same source material as potential reference preparations. It can also be employed as a sensitive method for identification of extracts derived from a variety of sources.