Analysis of nonderivatized neutral and sialylated oligosaccharides by electrospray mass spectrometry.

An HPLC/MS method has been developed that allows rapid, direct analysis of underivatized sialylated as well as neutral oligosaccharides. The method involves the separation of oligosaccharides from salts and proteins using RP-HPLC with a formic acid/acetonitrile/water mobile phase system and on-line electrospray mass spectrometry analysis in the positive ion mode. Under the solution conditions employed, both neutral and acidic (sialylated) oligosaccharides are protonated and therefore detected. In contrast to MALDI-TOF MS, no loss of sialic acid is observed when operating in the positive ion mode. Furthermore, the capability of this method to provide quantitative estimates of the relative abundance of each oligosaccharide mass has been demonstrated using fetuin as a model compound.

Relating quality of center-based child care to early cognitive and language development longitudinally.

How quality of center-based child care relates to early cognitive and language development was examined longitudinally from 6 to 36 months of age in a sample of 89 African American children. Both structural and process measures of quality of child care were collected through observation of the infant classroom. Results indicated that higher quality child care was related to higher measures of cognitive development (Bayley Scales of Infant Development), language development (Sequenced Inventory of Communication Development), and communication skills (Communication and Symbolic Behavior Scales) across time, even after adjusting for selected child and family characteristics. In addition, classrooms that met professional recommendations regarding child:adult ratios tended to have children with better language skills. Classrooms that met recommendations regarding teacher education tended to have girls with better cognitive and receptive language skills. These findings, in conjunction with the growing child-care literature, provide further evidence that researchers and policymakers should strive to improve the quality of child care to enhance early development of such vulnerable children.

Development of improved high-performance liquid chromatography conditions for nonisotopic detection of isoaspartic acid to determine the extent of protein deamidation.

A rapid and accurate ion-pairing reversed-phase high-performance liquid chromatography (IP-RP-HPLC) procedure has been developed for nonisotopic detection of isoaspartic acid residues in protein or peptides resulting from deamidation of asparagine residues. The IP-RP-HPLC procedure specifically detects and quantifies S-adenosylhomocysteine (SAH). SAH is a by-product of the reaction between protein isoaspartyl methyltransferase (PIMT), S-adenosylmethionine (SAM), and isoaspartic acid residues. The HPLC conditions described in this paper have been demonstrated to offer significantly better reproducibility compared to earlier studies. The HPLC method allows determination of the extent of protein deamidation without the use of radioisotopes and therefore offers significant advantages for biopharmaceutical development laboratories.

Characterization and establishment of specifications for biopharmaceuticals.

This paper describes the role of product characterization and product specifications in the context of an overall control strategy. Due to advances in analytical characterization and biotechnology manufacturing methods, product characterization can now be used as an effective means of assessing the impact of manufacturing process changes on product quality, safety and efficacy, hence obviating the need for repeating clinical testing each time a manufacturing process change is made. The ICH specifications guidance document can serve a key role in harmonising test requirements and regulatory practices with regard to produce characterization. Consumers, regulatory agencies, and biopharmaceutical manufacturers all stand to benefit from this harmonization process.

Isolation and measurement of the endogenous cannabinoid receptor agonist, anandamide, in brain and peripheral tissues of human and rat.

Anandamide (arachidonylethanolamide) is a novel lipid neurotransmitter first isolated from porcine brain which has been shown to be a functional agonist for the cannabinoid CB1 and CB2 receptors. Anandamide has never been isolated from human brain or peripheral tissues and its role in human physiology has not been examined. Anandamide was measured by LC/MS/MS and was found in human and rat hippocampus (and human parahippocampal cortex), striatum, and cerebellum, brain areas known to express high levels of CB1 cannabinoid receptors. Significant levels of anandamide were also found in the thalamus which expresses low levels of CB1 receptors. Anandamide was also found in human and rat spleen which expresses high levels of the CB2 cannabinoid receptor. Small amounts of anandamide were also detected in human heart and rat skin. Only trace quantities were detected in pooled human serum, plasma, and CSF. The distribution of anandamide in human brain and spleen supports its potential role as an endogenous agonist in central and peripheral tissues. The low levels found in serum, plasma, and CSF suggest that it is metabolized in tissues where it is synthesized, and that its action is probably not hormonal in nature.

Protein mass spectrometry: applications to analytical biotechnology.

The advent of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS) in the last 5 years has greatly enhanced the area of protein mass spectrometry. This paper presents an overview of the applications of protein mass spectrometry in the area of analytical biotechnology, particularly as related to biopharmaceutical research and development. These applications include the determination of protein molecular mass, peptide mapping, peptide sequencing, ligand binding, determination of disulfide bonds, active site characterization of enzymes, protein self-association and protein folding/higher order structural characterization.

Biosynthesis of modified peptidoglycan precursors by vancomycin-resistant Enterococcus faecium.

In the presence of bacitracin, vancomycin-resistant Enterococcus faecium (vanA phenotype) accumulate UDP-N-acetylmuramyl(UDP-Mur-NAc)-tetrapeptide and a UDP-MurNAc-depsipentapeptide containing lactate substituted for the carboxy-terminal-D-alanine residue. In an in vitro peptidoglycan polymerization assay, the modified precursors function and confer resistance to vancomycin.

The effects of formulation variables on the stability of freeze-dried human growth hormone.

Formulation often has a dramatic effect on degradation of proteins during the freeze-drying process as well as impacting on the "shelf-life" stability of the freeze-dried product. This research presents the results of a formulation optimization study of the "in-process" and shelf-life stability of freeze-dried human growth hormone (hGH). Chemical decomposition via methionine oxidation and deamidation of asparagine residues as well as irreversible aggregation were characterized by HPLC assay methodology. In-process degradation and stability of low moisture freeze-dried solids were studied at 25 and 40 degrees C in a nominal nitrogen headspace (approximately 0.5% O2). Formulation variables included pH, level of salts, and the nature of the lyoprotectant. Studies of the effect of shear on aggregation in solutions indicated that shear comparable to that experienced during filtration does not induce aggregation. Irreversible changes in hGH during the freeze-drying process were minimal, but chemical decomposition via methionine oxidation and asparagine deamidation and aggregation did occur on storage of the freeze-dried solid. Decomposition via methionine oxidation was significant. A combination of mannitol and glycine, where the glycine remains amorphous, provided the greatest protection against decomposition and aggregation. It is postulated that an excipient system that remains at least partially amorphous is necessary for stabilization. However, the observation that dextran 40 formulations showed poor stability toward aggregation demonstrates that an amorphous excipient system is not a sufficient condition for stability. Stability of the solid was optimal when produced from solutions in the pH range, 7-7.5, with severe aggregation being observed at high pH. The level of sodium phosphate buffer affected stability of the solid, although this relationship was complex. Freeze-drying in the presence of NaCl produced severe aggregation and precipitation during the freeze-drying process as well as acceleration of oxidation and/or deamidation.

Cross-reactivity of monomeric and dimeric biosynthetic human growth hormone in commercial immunoassays.

Commercial kits give different measurements for concentrations of growth hormone (GH, somatotropin) in serum. Most notably, a two-site monoclonal-antibody-based immunoradiometric assay (IRMA) from Hybritech routinely yields lower values than do conventional RIAs in which polyclonal antibodies are used. We used purified dimeric biosynthetic human GH as a model compound to investigate the specificity of five commercial immunoassays for size variants of GH. In all five assays, biosynthetic monomeric GH was significantly more potent than pituitary-derived standard GH supplied with the kits. Dimeric GH was significantly less potent than monomer in four of the five assays, and cross-reactivities varied more than fivefold, from 15% to 84%. Using three commercial kits selected for their specificity for dimeric GH, we measured GH in serum samples from 18 normal adults. The mean GH concentrations in serum--0.7 (Hybritech, IRMA), 1.8 (Diagnostic Products, RIA), and 3.1 (Cambridge, RIA) micrograms/L--differed significantly, but in the same rank order as that obtained in the experiments on dimer cross-reactivity.

Capillary zone electrophoresis of peptide fragments from trypsin digestion of biosynthetic human growth hormone.

Capillary zone electrophoresis (CZE) was applied to the separation of the 19 peptide fragments produced by enzymatic digestion of human growth hormone (hGH). The fragments of hGH produced by trypsin digestion under non-reducing conditions were identified in the electropherogram. Almost all of the fragments were resolved by CZE in less than 15 min. There is a marked difference in selectivity between reversed-phase high-performance liquid chromatography (RP-HPLC) and CZE. CZE is demonstrated to be a powerful complement to RP-HPLC for routine identification of hGH using trypsin digests.

Application of free-solution capillary electrophoresis to the analytical scale separation of proteins and peptides.

The application of free solution capillary electrophoresis (FSCE) to the separation of protein and peptide mixtures is presented. Both qualitative and quantitative aspects of FSCE separations are considered. In addition, a brief introduction describing the separation principle behind FSCE separations and a discussion of electrophoretic mobility are included. The applications were chosen in order to highlight the selectivity of FSCE separations and to demonstrate applications of potential practical interest to the bioanalytical chemist. Comparison of FSCE relative to traditional analytical separation alternatives is stressed throughout. The examples are presented in three broad categories: protein separations, peptide separations, and the application of both to the analysis of recombinant protein products. In the first section, FSCE separations of peptide mixtures are presented which demonstrate the suitability of FSCE for the analysis of the purity of peptide samples, the homogeneity of peptide samples prior to sequencing, the identity of peptides by using electrophoretic mobility values, and the reduction of an intrachain disulfide bridge. In the second section, protein separations are presented that show the resolution of glycoproteins having the same primary structure and the separation of immune complexes from free unreacted antibody and antigen. In the final section, highly purified and well-characterized samples of biosynthetic human insulin (BHI), biosynthetic human growth hormone (hGH), and their derivatives were used to evaluate FSCE as a complement and/or alternative to conventional analytical separation techniques for the determination of purity and identity of biosynthetic human proteins. In addition, the quantitative aspects of FSCE analysis such as linearity of response, precision, and limit of detection were examined.

Liquid chromatographic control of the identity, purity and "potency" of biomolecules used as drugs.

The use of high-performance liquid chromatography (HPLC) in the control of rDNA-derived human insulin and human growth hormone is described. Powerful identity tests based upon reversed-phase HPLC separation of enzymatic digests have been developed. Size exclusion and reversed-phase assays are used to control higher molecular weight materials and monomeric derivatives, respectively, for both proteins. Finally, HPLC is used to control the relevant protein content, which in concert with other information controls the biopotency of the protein preparations.

Isolation and characterization of a sulfoxide and a desamido derivative of biosynthetic human growth hormone.

Two derivatives of biosynthetic human growth hormone, a sulfoxide and a mixture of two monodesamido isomers, have been isolated and characterized. The sulfoxide derivative arises from an oxidation of Met-14. The major site of deamidation is at Asn-149 with a minor site at Asn-152. In addition, a fraction has been isolated from a sample of human growth hormone that was maintained at 40 degrees C for 2 weeks. This fraction, the isolated impurities fraction, contains the sulfoxide and the desamido forms, thereby demonstrating that these derivatives are the primary degradation products of biosynthetic human growth hormone. The sulfoxide, the desamido, and the isolated impurities fraction exhibit full biological activity.

High-performance size-exclusion chromatographic determination of the potency of biosynthetic human growth hormone products.

A new high-performance size-exclusion chromatography method has been developed for the determination of potency of human growth hormone products. This method has been extensively validated and shown to correlate well with the hypophysectomized rat bioassay which has been used traditionally. The method is much more precise than the traditional bioassay and thus provides more reliable means of producing consistent biosynthetic human growth hormone batches.

Chemical, physical, and biological characterization of a dimeric form of biosynthetic human growth hormone.

A dimer of biosynthetic human growth hormone (HGH) has been isolated and characterized. This entity, which is the predominant dimeric species in biosynthetic HGH, is chemically identical to monomeric HGH and exists in a noncovalent dimeric form which is dissociated to monomeric HGH on polyacrylamide electrophoresis gels or in aqueous solutions containing 30% acetonitrile. This substance, found in all production lots of pituitary HGH, biosynthetic HGH, and biosynthetic methionyl HGH examined, is much less biopotent than monomeric HGH and can be distinguished from monomeric HGH by a monoclonal antibody. These data demonstrate that polyacrylamide gel electrophoresis is not a valid method for measuring this dimer and that size-exclusion chromatography under aqueous conditions is required.

A reversed-phase high-performance liquid chromatographic method for characterization of biosynthetic human growth hormone.

An isocratic reversed-phase high-performance liquid chromatographic method for the determination of human growth hormone (HGH) purity is described. This method offers superior resolution of HGH-related substances (e.g., sulfoxide and desamido derivatives) from unmodified HGH when compared to a number of alternative chromatographic and electrophoretic techniques.

In vitro activity of clindamycin against strains of Chlamydia trachomatis, Mycoplasma hominis, and Ureaplasma urealyticum isolated from pregnant women.

Chlamydia trachomatis, Mycoplasma hominis, and Ureaplasma urealyticum are genital agents that are being increasingly implicated in infectious pregnancy complications and abnormal pregnancy outcomes. We measured the in vitro activity of clindamycin against strains of these three agents which were isolated from pregnant women. For 30 strains of C. trachomatis, the median minimal inhibitory concentration was 1.0 microgram/ml (range, 0.25 to 2.0 micrograms/ml). For 27 strains of M. hominis, the median minimal inhibitory concentration was 0.12 microgram/ml (range, 0.06 to 0.25 microgram/ml) and the median minimal bactericidal concentration was 0.5 microgram/ml (range, 0.06 to 2.0 micrograms/ml). For 27 strains of U. urealyticum, the mean minimal inhibitory concentration was 4 micrograms/ml (range, 1.0 to 32.0 micrograms/ml) and the mean minimal bactericidal concentration was 32.0 micrograms/ml (range, 4.0 to 128 micrograms/ml). Thus in vitro clindamycin would appear to be highly active against pregnancy-associated strains of M. hominis, less active against strains of C. trachomatis, and least active against strains of U. urealyticum. Since M. hominis has been strongly linked to postabortal fever and to postpartum fever and endometritis, our results indicate that clindamycin should be evaluated in treatment trials in pregnancy aimed at prevention of M. hominis-induced morbidity as well as in treatment of the complications themselves.