Vibrational analysis of peptides,polypeptides and proteins

Normal mode calculations have been carried out on three low-energy structures of gramicidin S obtained from conformational energy calculations. When the results on the amide modes are compared with observed bands in the infrared and Raman spectra of crystalline gramicidin S and its N-deuterated derivative, one of the structures is clearly disfavored. Of the other two, one is slightly favored, and it corresponds to the lowest-energy structure obtained from the energy calculations. Spectra from solutions in DMSO and CH₃ OH suggest that the molecular conformation is essentially retained in these solvents.     

Characterization of recombinant human granulocyte colony stimulating factor (rHuG-CSF) by capillary zone electrophoresis, capillary isoelectric focusing electrophoresis and electrospray ionization mass spectrometry

Recombinant human granulocyte colony-stimulating factor (rHuG-CSF) is a hematopietic cytokine that stimulates and regulates the proliferation and differentiation of neutrophils. Glycosylated and non-glycosylated forms of rHuG-CSF cannot be distinguished by traditional biological assays. In addition, it is very difficult to characterize impurities of the same molecular weight in biologicals. In this study, non-glycosylated rHuG-CSF, two glycosylated rHuG-CSF isoforms and their commercial dosages were successfully separated by capillary zone electrophoresis (CZE) using 50mM Tricine containing 20mM NaCl and 2.5mM 1,4-diaminobutane (DAB) at pH 8.0, which could be employed for the qualitative discrimination assay of rHuG-CSF related products. CZE, capillary isoelectric focusing electrophoresis (CIEF), and mass spectrometry (MS) were used to effectively characterize non-glycosylated rHuG-CSF. It was found that proteins in the samples with different pIs in the CIEF profile could not be detected by CZE, while no difference was observed between these proteins and rHuG-CSF. Further analysis by electrospray ionization mass spectrometry with the resolution of 2000 showed that the components with different pIs in the non-glycosylated rHuG-CSF bulk sample are nearly equal in molecular weight. Therefore, it is necessary to combine several modern analytical techniques for quality control to get well-characterized biologicals.     

Vibrational analysis of peptides,polypeptides, and proteins

The normal modes have been calculated for three kinds of low energy γ-turn structures resulting from recent conformational energy calculations by Némethy. Frequencies have been computed for a γ-turn, a mirror-related γ-turn, and an inverse γ-turn of CH₃-CO-(L-Ala)_n-NH-CH₃, with n = 3 and n = 5, and for certain ¹⁴C and ¹⁵N derivatives of the n = 3 molecule. Correlations are evident between amide frequencies and γ-turn structures, and it is found that only amide I modes of peptide groups in the turn are relatively insensitive to the lengths of attached chains.     

On-line coupling of cyclodextrin mediated nonaqueous capillary electrophoresis to mass spectrometry for the determination of salbutamol enantiomers in urine

The usefulness of the on-line coupling of nonaqueous capillary electrophoresis (NACE) with electrospray ionization (ESI) mass spectrometry (MS) using heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-cyclodextrin (HDAS-beta-CD) was demonstrated for the enantioselective determination of low concentrations of salbutamol in human urine. After optimization of several parameters, such as sheath-liquid composition and flow rate, nebulizing gas pressure, CE counter-pressure and position of the CE capillary outlet, a limit of quantification of 18 and 20 ng/ml was obtained for salbutamol enantiomers. Moreover, the relative standard deviation values for repeatability at a concentration of 30 ng/ml were below 7% for both enantiomers. Typical regression lines obtained after application of a simple linear regression model revealed a good relationship between peak area and analyte concentration (with 0.9988 and 0.9966 as coefficients of determination). This paper proposes an easy to use and sensitive NACE-MS method to determine enantiomers of a basic chiral drug in biological fluids preceded by solid-phase extraction as sample cleanup.     

Vibrational analysis of peptides,polypeptides, and proteins

The normal modes of cyclo(D-Phe-L-Pro-Gly-D-Ala-L-Pro), a cyclic pentapeptide known to contain a Λ-turn, have been calculated, and the amide I, II, III, and V modes have been compared with observed Raman and infrared bands. The observed bands are well predicted by the calculation. This gives confidence in the reliability of the calculations of the normal modes of standard Λ-turn structures.     

蛋白质及多肽的液相色谱-电喷雾离子化质谱研究进展

蛋白质及多肽的液相色谱电喷雾离子化质谱研究进展徐友宣彭师奇（北京医科大学生源药物化学中德联合实验室，北京１０００８３）多肽及蛋白质药物对临床的重要性，已引起各国药物化学界的广泛重视。在多肽或蛋白质药物的结构研究中，ＭＳ研究有重要价值。例如ＦＡＢＭ...     

A method for routine analysis of recombinant immunoglobulins (rIgGs) by capillary isoelectric focusing (cIEF)

A capillary isoelectric focusing (cIEF) method was developed for routine analysis of recombinant immunoglobulins (rlgGs). The cIEF method used a dimethyl siloxane-coated capillary and a separation matrix of 2% ampholytes in 0.4% methylcellulose (MC). The rIgGs, and internal pI marker protein standards, were mixed with carrier ampholyte in MC, focused using high voltage, and then the protein bands were mobilized past a UV detector by simultaneous application of low pressure and voltage. Qualitatively and quantitatively equivalent rIgG focusing profiles were obtained via cIEF and gel-based IEF, with individual isoform peak area percentages and calculated peak pI values being comparable for the same samples. Linear relationships were obtained for peak area response versus sample concentration, and for the pI gradient developed between the internal pI marker standards. The relative standard deviation (RSD) in rIgG peak areas was less than 2% intra-day and less than 8% inter-day (72 h). The RSD for the mobilization times of rIgG peaks was less than 1% intra-day and less than 3% inter-day (72 h). There was no observed decrease in the performance of the capillary over 150 analyses. cIEF offers several important advantages over gel IEF, e.g. direct, quantitative detection of proteins by intrinsic UV absorbance at 280 nm, rapid analyses ( < or = 30 min), capability of automation, and one-step, electronic data analysis and archival. These data demonstrate the superiority of the cIEF method for routine analysis of rIgGs.     

Microencapsulation of peptides and proteins

Microcapsules were prepared by using a double-emulsion technique. A new production method called 'induced phase separation method' was applied to encapsulate peptides and proteins. To find the optimal adjuvants a matrix was set up combining the appropriate organic solvents and the suitable surfactants. The polymer was chosen with regard to the required release period. The aqueous drug solution was intensively mixed with the organic polymer solution. An aqueous surfactant solution was slowly added to the O/W emulsion. The obtained W/O/W emulsion is stirred under partial vacuum conditions until the organic solvent was removed. After removing the solvent from the W/O/W emulsion the microcapsules were washed and lyophilized. The morphology of the microparticles (spheres, sponges, capsules, surplus polymer) was checked by microscopy, particle size distributions were measured by laser diffraction.     

Capillary electrophoretic analysis of proteins and peptides of biomedical and pharmacological interest

Capillary electrophoresis (CE) is an automated approach to electrokinetic separations that has had a deep impact in all fields of life sciences, including biomedical and biotechnological research and clinical and forensic practice. The present review highlights aspects of peptides and proteins separations, with particular emphasis on macromolecular analytes of biomedical interest. Among the various CE techniques available, a novel methodology is here illustrated consisting in separations in acidic, isoelectric buffers, which have the advantage of protonating the silica wall, thus minimizing interactions of proteinaceous material with the siliceous surface, while allowing delivery of high voltage gradients, due to their low conductivities. The review ends with applications of CE to the analysis of folding/unfolding/refolding/misfolding of proteins, a field which has deep implications in the biomedical arena, since it is connected to a host of disorders, such as prion protein diseases.     

Polymers for controlled parenteral delivery of peptides and proteins

This review summarizes the current state of parenteral delivery of proteins and peptides. Although an attempt has been made to provide a comprehensive review, the more recent developments, such as delivery of LHRH analogues, vaccine delivery systems and regulated systems, have been emphasized. Non-regulated delivery systems have been divided into non-degradable and degradable and each has been subdivided into hydrophilic and hydrophobic systems. Regulated systems have been divided into externally regulated and self-regulated.     

Isoaspartate in peptides and proteins: formation, significance, and analysis

Formation of isoaspartyl peptide bonds (isoAsp) is one of the most common forms of non-enzymatic degradation of peptides and proteins under mild conditions. IsoAsp arises when certain Asn-Xaa and Asp-Xaa sites undergo a spontaneous intramolecular rearrangement to form a succinimide which subsequently hydrolyzes to generate a mixture of isoAsp-Xaa and Asp-Xaa linkages in a ratio of approximately 2:1. This pathway is responsible for the much greater susceptibility of asparagine, compared with glutamine, to deamidation at neutral and alkaline pH. Rearrangement occurs most readily at Asn-Gly, Asn-Ser, and Asp-Gly sequences where the local polypeptide chain flexibility is high. Formation of isoAsp can decrease the biological activity of a protein pharmaceutical, alter its susceptibility to proteolytic degradation, and elicit autoimmunity. The enzyme protein L-isoaspartyl methyltransferase can be used to measure isoAsp sites in the low pmol range with or without the use of radioisotopes.     

Controlled delivery of peptides and proteins

The final aim/target of Pharmaceutical Sciences is to design successful dosage forms for effective therapy, considering individual patient needs and compliance. Development of new drug entities, particularly using peptides and proteins, is growing in importance and attracting increased interest, as they are specifically effective at a comparably low dose. These very potent and specific peptides and proteins can now be produced in large quantities due to increased knowledge and advancements in biotechnological and pharmaceutical applications. A number of peptide and protein products are now available on the market, and numerous studies investigating them have been published in the literature. Although many peptide/protein like products are generally designed for parenteral administration, some other noninvasive routes have also been used. For example, desmopressin is delivered nasally and deoxyribonuclease by inhalation. Although peptides and proteins are generally orally inactive, cyclosporine is an exception. In order to design and develop long-acting, more effective peptide/protein drugs, the controlled release mechanisms and effective parameters need to be understood and clarified. Therefore, we review herein various peptide/protein delivery systems, including biodegradable and nondegradable microspheres, microcapsules, nanocapsules, injectable implants, diffusion-controlled hydrogels and other hydrophilic systems, microemulsions and multiple emulsions, and the use of iontophoresis or electroporation, and discuss the results of recent researches.     

Studies on 1-alkylamine adduct formation in electrospray ionization mass spectrometry for quantitative analysis

We investigated the formation of 1-alkylamine adduct ions using 1,2-, 1,3-, and 1,4-cyclohexanediol (CHD) as model compounds and the relationships of the peak intensity between the protonated molecules ([M+H]+), sodium adduct ions ([M+Na]+), and 1-alkylamine adduct ions ([M+A+H]+) of 16 model compounds using electrospray ionization mass spectrometry. When 1-octylamine was added to 1,2-, 1,3-, and 1,4-CHD solutions, the peak intensity of the 1-octylamine adduct ions ([M+Oct+H]+) was higher than those of [M+H]+ and [M+Na]+. The highest peak intensity of [M+Oct+H]+ was observed in 1,2-CHD, followed by 1,3-CHD and 1,4-CHD and this order was the same as that of [M+Na]+ for CHDs in the solution without 1-octylamine. These results suggest that the mechanism of formation of [M+Oct+H]+ is similar to that of [M+Na]+ and that adduct formation seems to occur between 1-octylamine and two oxygen atoms in CHD in a similar manner to [M+Na]+. Based on these results, 16 model compounds including CHDs were investigated with respect to the relationship between [M+Na]+ and [M+A+H]+. A positive correlation was observed between the peak intensities of [M+Na]+ and [M+A+H]+, supporting that the formation mechanism of [M+A+H]+ is potentially similar to the [M+Na]+ formation mechanism. These data indicate that a sensitivity enhanced quantitative analysis using [M+A+H]+ could be a feasible approach for compounds generating [M+Na]+.     

Liposomal delivery of proteins and peptides

Introduction: A number of delivery issues exist for biotech molecules including peptides, proteins and gene-based medicines that now make up over 60% of the drug pipeline. The problems comprise pharmaceutical ad biopharmaceutical issues. One of the common approaches to overcome these issues is the use of a carrier and liposomes as carriers have been investigated extensively over the last decade.

Areas covered: The review has been discussed in terms of formulation and preclinical development studies and in vivo studies encompassing different delivery routes including parenteral, oral, buccal, pulmonary, intranasal, ocular and transdermal involving liposomes as carriers. Important research findings have been tabulated under each side heading and an expert opinion has been summarised for each delivery route.

Expert opinion: The conclusion and expert opinion – conclusion sections discuss in detail troubleshooting aspects related to the use of liposomes as carriers for delivery of biopharmaceutical moieties and scrutinises the aspects behind the absence of a protein/peptide-containing liposome in market.     

Applications of multi-angle laser light-scattering detection in the analysis of peptides and proteins

The proliferation of new peptides and proteins requiring characterisation is a direct result of recent advances in genomics and proteomics, but protein aggregation is particular problem in the biotechnology industry, where aggregation is encountered throughout the lifetime of a therapeutic protein, including during refolding, purification, sterilization, shipping, and storage process. To ensure that it meets quality standards, the size, molecular weight and/or molecular weight distribution, and aggregate state must be accurately determined. Traditional analytical methods for determining molecular weight include size-exclusion chromatography (SEC), gel electrophoresis, analytical ultracentrifugation and time-of-flight mass spectrometry. These technologies are time-consuming (some take days), provide data based on relative standards, or cannot characterise very high molecular weight aggregates. Laser light-scattering (LS) detection coupled with SEC system have been used for over a decade to determine the size and molecular weight of bio-molecules such as proteins, peptides, polysaccharides, oligonucleotides, and antibodies, the method of choice being for molar mass determinations and the study of self-association and heterogeneous interaction under native, equilibrium conditions in solution. The purpose of the current review is to describe and discuss the capability of the SEC/LS system to determine absolute molecular weight of proteins and their complexes and the association state of the conjugate, either with itself or with protein receptor/ligands. For this, the "two or three detector" methods, each with its advantages and limitations, can be used to calculate the molecular weight of a simple protein or glycoprotein, and the stoichiometry of their complexes. Also, some alternative techniques for determining the molecular weight are discussed in this review. Applications of all these methodologies are described.     

Orally active-targeted drug delivery systems for proteins and peptides

Introduction: In the past decade, extensive efforts have been devoted to designing ‘active targeted’ drug delivery systems (ATDDS) to improve oral absorption of proteins and peptides. Such ATDDS enhance cellular internalization and permeability of proteins and peptides via molecular recognition processes such as ligand–receptor or antigen?antibody interaction, and thus enhance drug absorption.

Areas covered: This review focuses on recent advances with orally ATDDS, including ligand–protein conjugates, recombinant ligand–protein fusion proteins and ligand-modified carriers. In addition to traditional intestinal active transport systems of substrates and their corresponding receptors, transporters and carriers, new targets such as intercellular adhesion molecule-1 and β-integrin are also discussed.

Expert opinion: ATDDS can improve oral absorption of proteins and peptides. However, currently, no clinical studies on ATDDS for proteins and peptides are underway, perhaps due to the complexity and limited knowledge of transport mechanisms. Therefore, more research is warranted to optimize ATDDS efficiency.     

Alternative delivery systems for peptides and proteins as drugs

The emergence of recombinant DNA technology has resulted in the large-scale production of a myriad of genetically engineered proteins and peptides, making many of them available for the first time for potential use as therapeutic entities. In addition, increased knowledge in the area of peptide/polypeptide hormones has resulted in an expansion of research efforts utilizing peptide synthetic chemistry, aimed toward developing novel therapeutic peptide drugs. Proteins and peptides cannot readily be administered by the conventional oral route, and thus alternative delivery methods to circumvent the necessity of frequent injections are being explored. This article reviews the current state-of-the-art technology of such methodologies, encompassing localized administration, administration to various body cavities (i.e., nasal, rectal, etc.), as well as controlled-release injectable or implantable systems. These different approaches result in quite different pharmacokinetics that may in part dictate which approach is best suited for a particular protein or peptide.     

Taylor dispersion analysis compared to dynamic light scattering for the size analysis of therapeutic peptides and proteins and their aggregates

Purpose  

To evaluate Taylor dispersion analysis (TDA) as a novel method for determination of hydrodynamic radius of therapeutic peptides and proteins in non-stressed and stressed formulations and to compare it with dynamic light scattering (DLS).     

Comprehensive quantitative and qualitative liquid chromatography-radioisotope-mass spectrometry analysis for safety testing of tolbutamide metabolites without standard samples

Liquid chromatography-radioisotope-mass spectrometry (LC-RI-MS) analysis was used to determine the structures of 12 (four previously unknown) (14) C-tolbutamide (TB) metabolites in rat biological samples (plasma, urine, bile, feces, and microsomes). The four novel metabolites are ω-carboxy TB, hydroxyl TB (HTB)-O-glucuronide, TB-ortho or meta-glutathion, and tolylsulphoaminocarbo-glutathion. In rat plasma, after oral administration of (14) C-TB at therapeutic dose (1 mg/kg) and microdose (1.67 μg/kg), the total RI and six metabolites [HTB, carboxy TB (CTB), M1: desbutyl TB, M2: ω-hydroxyl TB, M3: α-hydroxyl TB, and M4: ω-1-hydroxyl TB] were quantified by LC-RI-MS. The plasma concentrations were calculated using their response factors (MS-RI intensity ratio) without standard samples, and the area under the curve (AUC) of plasma concentration per time for evaluation of Safety Testing of Drug Metabolites (MIST) was calculated using the ratio of TB metabolites AUC/total RI AUC. The ratios were as follows: TB 94.5% and HTB 2.5% for the microdose (1.67 μg/kg) and TB 95.6%, HTB 0.96%, CTB 0.065%, M1 0.62%, M2 0.0035%, M3 0.077%, and M4 0.015% for the therapeutic dose (1 mg/kg). These values were less than 10% of the MIST criteria.     

Simultaneous chromatographic fingerprinting and quantitative analysis of flavonoids in Pollen Typhae by high-performance capillary electrophoresis

To evaluate the quality of Pollen Typhae as used in traditional Chinese medicine, a high-performance capillary electrophoresis (HPCE) method has been developed, validated and applied to chromatographic fingerprinting and quantitation of its eight main bioactive flavonoids (naringenin, isorhamnetin 3-O-(2^G-α-l-rhamnosyl)-rutinoside, rhamnetin 3-O-neohesperidoside, isorhamnetin, quercetin 3-O-(2^G-α-l-rhamnosyl)-rutinoside, quercetin 3-O-neohesperidoside, kaempferol and quercetin). Fingerprinting was based on the selection of nine characteristic chromatographic peaks. In quantitative analysis, the recovery of all eight compounds was in the range 98.5–102.2% with good linearity (r²>0.9919) over a relatively wide concentration range. The assay was successfully applied to the analysis of the eight bioactive flavonoids in 14 different samples. The results indicate that the assay is reproducible and precise and can be used for convenient quality assessment of Pollen Typhae.