Biopharmaceutical Analysis Research Articles

Rapid release of N-linked glycans from glycoproteins by pressure-cycling technology.

The standard, well-established sample preparation protocol to release N-linked glycans from glycoproteins for downstream analysis requires relatively long deglycosylation times (from several hours to overnight) and relatively high endoglycosidase concentration (from 1:250 to 1:500 enzyme:substrate molar ratio). In this paper, we significantly improve this standard protocol by the use of pressure-cycling technology (PCT) to increase the speed and decrease the relative amount of PNGase F during the release of N-linked glycans from denatured glycoproteins. With the application of pressure cycling from atmospheric to as high as 30 kpsi, >95% release of the asparagine-linked glycans from bovine ribonuclease B, human transferrin, and polyclonal human immunoglobulin was rapidly achieved in a few minutes using as low as 1:2500 enzyme:substrate molar ratio. The deglycosylation rate was first examined by SDS-PAGE at the protein level. The released glycans were then quantitated by capillary electrophoresis with laser induced fluorescence detection (CE-LIF). This new sample preparation protocol readily supports large-scale glycan analysis of biopharmaceuticals with rapid deglycosylation times.

Phytochemical and Biopharmaceutical Analysis of Willow Bark

Phytochemical and Biopharmaceutical Analysis of Willow Bark

A Series of Collaborations Between Various Pharmaceutical Companies and Regulatory Authorities Concerning the Analysis of Biomolecules Using Capillary Electrophoresis

An international project team (including members from US, Canada and UK) has been formed from a number of interested biopharmaceutical companies and regulatory authorities to conduct a cross-organisation collaboration exercise. The results from this exercise demonstrate the robustness of CE-SDS across eight different organisations that used instruments of the same equipment model, the same reagents, and the same methodology. Data generated from the analysis of a series of molecular weight markers showed very good precision with regards to relative migration time (RMT) both within and between organisations. The apparent molecular weight of bovine serum albumin (BSA) was measured with good precision to within approximately 2% RSD across the participants. A representative IgG sample showed similar results with regards to relative migration time of its 3 main components, IgG Light Chain, IgG Non-glycosylated Heavy Chain, and IgG Heavy Chain. Fractional peak area for each peak also showed good agreement, with less than 9% RSD for all peaks. This exercise will facilitate both increased regulatory and industrial opinion of CE for biopharmaceutical analysis.

Study of multiple solid-phase microextraction combined off-line with high performance liquid chromatography: Application in the analysis of pharmaceuticals in urine

Solid-phase microextraction (SPME) was applied to the extraction of five common pharmaceuticals: quinine, naproxen, haloperidol, ciprofloxacin and paclitaxel. SPME combined off-line with HPLC analysis was studied independently for each analyte. Polydimethylsiloxane and polyacrylate fibres were used for the direct immersion SPME from isotonic analyte solutions and spiked human urine samples. Analyte desorption occurred in HPLC autosampler inserts containing 200 μL of methanol; next 80 μL of the resulting solution were injected on the HPLC. Extraction optimisation involved parameters such as addition of salts, pH value and saturation time. The pharmaceuticals were assayed independently by multiple extraction from isotonic aqueous solutions and urine samples. A protocol of multiple SPME provided higher yields for the four of the five examined analytes. The results showed the potential of using multiple SPME (but also single SPME) combined off-line to HPLC in bio-pharmaceutical analysis.

Analysis of Pichia pastoris Components in Recombinant Human Serum Albumin by Immunological Assays and by HPLC with Pulsed Amperometric Detection

We have developed a recombinant human serum albumin (rHSA) from Pichia pastoris which expresses high levels of heterologous proteins. rHSA is used clinically in high concentration (approximately 250 mg/ml in a 50 mL vial). We had to consider not only proteins from host cells as impurities but also mannan, which exhibits harmful effects on humans. However, the analysis of mannan in biopharmaceuticals produced from yeast has not been reported. Contaminating mannans in the final product were one important index to assess the clinical safety of rHSA. We have developed a highly sensitive enzyme immunoassay (EIA), utilizing an avidin-biotin system, for the detection of either the protein or mannan polysaccharide components from P. pastoris components (PPC) in rHSA. In addition, we used anion exchange chromatography with pulsed amperometric detection (AE-PAD) for monosaccharide analysis of glycoconjugates for the detection of mannan from PPC in rHSA. The detection limits of the EIA for PPC (PPC EIA) and the AE-PAD were 1 ng of protein/250 mg of rHSA and 180 ng of mannose/mg of rHSA, respectively. The mannan content in partially purified rHSA as determined by the AE-PAD was about same as the PPC content as determined by the PPC EIA. We showed that the PPC EIA and the AE-PAD are useful methods for the purity analysis of biopharmaceuticals produced from yeast.

Improved reversed-phase high performance liquid chromatography columns for biopharmaceutical analysis

Reversed-phase high performance liquid chromatography continues to grow in importance for the analysis of peptides and proteins in biomolecular and pharmaceutical research. The mobile-phase conditions for separation of proteins and peptides are essentially fixed. Most separations of these solutes are conducted with shallow gradients using aqueous buffers modified with acetonitrile. Therefore, changing selectivity in peptide and protein separations is often best accomplished by a change in bonded-phase chemistry of the column (e.g. by using CN- or C3-bonded phases rather than C8 or C18). In general, however, short-chain bonded phases are more unstable and irreproducible than bonded phases having longer chains, due to increased susceptibility to hydrolysis as hydrophobicity of the bonded phase decreases. These problems are minimized using bonded phases that are protected through use of sterically bulky side-groups (StableBond Technology). This paper describes a series of comparisons between traditional polymeric bonded-phase silica columns and sterically protected, highly purified, silica stationary phases. These studies compare lot-to-lot reproducibility, stability of the bonded phase, selectivity effects between bonded phases, and operational advantages that can be obtained by high temperature operation using a series of stable bonded phases.

Advances in biopharmaceutical analysis in the People's Republic of China: 1991–1993

The methodological studies on biopharmaceutical analysis of drugs and their metabolites by liquid and gas chromatography with various detectors have been reviewed. Research articles were selected from well known journals published in the People's Republic of China between January 1991 and March 1993. The applications of these methods in bioavailability, pharmacokinetics, therapeutic drug monitoring and metabolic studies have also been discussed.

Precision, accuracy, and data acceptance criteria in biopharmaceutical analysis.

Accuracy and precision are the most important criteria in the assessment of an analytical method, and monitoring quality control during sample analysis is essential to ensure the validity of reported results. Various approaches to testing accuracy, precision, and quality control were applied to 10 analytes from seven chromatographic bioanalytical methods. These methods include fixed interval bias and significance testing for accuracy; fixed interval percentage relative standard deviation (%RSD) and analysis of variance (ANOVA) approaches for precision; +/- 20% fixed range, 99% confidence interval, multiple rules, and range chart for individuals approaches for quality control acceptance criteria. Quality control approaches were also applied to the entire run and to a bracketed approach whereby results are considered valid only if bracketed by acceptable quality control. Accuracy and precision were assessed for six runs of each analyte at three concentrations established to represent the calibration range of the analytical method. Quality control acceptance criteria were evaluated using all data sets from each of the analytical methods collected during the course of running various numbers of real samples. The data suggest that the fixed interval bias criteria for accuracy was a more liberal method of accuracy assessment because three of the seven methods would have been rejected according to the significance testing criteria whereas all were acceptable by the fixed internal bias criteria. Precision can be effectively assessed for between- and within-run data by criteria set on unconfounded %RSD values or by separation of the sources of variation using an ANOVA approach applied to confounded data.(ABSTRACT TRUNCATED AT 250 WORDS)

Dosages biopharmaceutiques et automatisation

Pharmacokinetic and drug metabolism studies request the development and implementation of efficient, fast, validated analytical methods. Through the application of flexible and cartesian robots to the sample preparation, considerable progress is being made in automating both the development and the routine use of analytical methods in biopharmaceutical analysis.

Calibration and validation of linearity in chromatographic biopharmaceutical analysis

Calibration in chromatographic biopharmaceutical analysis is a major determinate of method performance and many methods have been proposed to evaluate an appropriate calibration model, to determine the linear range and to evaluate the goodness of fit. Ten chromatographic bioanalytical methods have been evaluated in this work by observation of concentration-response curves, linearity plots, calculation of concentration residuals, correlation coefficients and lack of fit analysis. These methods were applied to univariant linear regression, weighted regression, polynomial regression and power fit models in order to determine the most appropriate way to establish and evaluate calibration functions. It was found that weighted linear regression provided the most appropriate calibration function for eight of the 10 methods studied, whereas unweighted regression and the power fit model proved appropriate for one each of the other two methods. The choice of calibration function was best accomplished through observation of calculated concentration residuals. Linearity and sensitivity plots were of little value for assessment of linearity through the selected calibration range if conventional (+/- 5%) tolerance limits are employed. Validation of the calibration model can be accomplished by demonstrating the concentration residuals and the slope of the log concentration-log response plots are within reasonable tolerance limits or by lack of fit analysis. Correlation coefficients were demonstrated to be of little value for this purpose and the quadratic approach to linearity validation was in disagreement with other methods in four of the 10 methods evaluated.

A basic program to determine outliers in biopharmaceutical analysis using the Nair criterion

In bioanalysis, as in other areas of analysis, difficulties can arise in judging whether to accept or reject individual values from a set of replicate analytical results. There appear to be no definitive procedures described in the literature which are applicable to bioanalytical results; operations systems of individual laboratories range from the purely subjective to sophisticated statistical techniques. In the authors’ laboratory, the analysis of a sample can yield a set of triplicate results, two of which are close and the other an apparent outlier. If this is judged to be so, then the outlier is rejected and the mean of the two remaining values is quoted as the analytical result. If it is judged otherwise, the mean of the three results is reported. However, a problem arises when two operators judge the same data. A finite number of instances will occur where there is disagreement. To overcome this problem, a number of statistical tests were examined. Of these, the Nair criterion was found to be the test that mnat nftc.n RQVP a vem,lt CS~.T..C.;~R whc lll”UC “Ib”II 5u.u u lrLIUl& uglrrlllg “Ill1 Cllr UULll”l.J ““ll JUUg~lllrllC. II”“II”cII) .3lllticI Ill&D is a time-consuming exercise, ways of reducing the time spent were evaluated. The application described in the present communication has been written into a Basic program, use of which enables inexperienced investigators to make consistently objective data-handling decisions without the need to consult superiors or colleagues.

Applications of column-switching technique in biopharmaceutical analysis: I. High-performance liquid chromatographic determination of amitriptyline and its metabolites in human plasma

A new high-performance liquid chromatographic method for the determination of amitriptyline and its metabolites, nortriptyline, 10-hydroxynortriptyline and 10-hydroxyamitriptyline, in plasma is described which uses direct injection and a column-switching valve. The method is based on the enrichment of drugs on a reversed-phase concentration column, packed with Corasil RP. The enriched drugs were then separated, using back-flush mode on a bonded-phase CN column using an isocratic acetonitrile-acetate buffer (60:40, v/v) mobile phase. The validation of the method showed excellent sensitivity, precision and reproducibility. The limit of detection, using a 250-μl direct injection of plasma, was between 5 and 10 ng/ml for each of the four drugs. The mean coefficient of variation for intra- and inter-assay was better than 5%. The method showed obvious advantages over conventional extraction procedures in terms of speed and ease of sample handling. The method has been successfully applied to the samples from patients receiving oral doses of amitriptyline.

Applications of column-switching techniques in biopharmaceutical analysis: II. High-performance liquid chromatographic determination of tripelennamine in bovine plasma and milk

Applications of column-switching techniques in biopharmaceutical analysis: II. High-performance liquid chromatographic determination of tripelennamine in bovine plasma and milk

Some aspects of pharmaceutical analysis using high performance liquid chromatography

The history and general principles of high performance liquid chromatography are reviewed. Liquid chromatographic theory and the various modes of chromatography are discussed, including adsorption, partition, reversed phase and ion-exchange modes. Equipment used in pharmaceutical analysis is described including pumping systems, mobile phases, sample introduction systems, columns and column fittings, detectors and data handling systems. Applications are reviewed, with particular reference to antibiotic analysis, optical isomer resolution, biopharmaceutical analysis and pharmaceutical production. Future developments are considered, including column technology, detector development, automation and the analysis of biopolymers.

Determination of drugs and metabolites in biological fluids

The determination of drugs and metabolites in biological fluids (biopharmaceutical analysis) is becoming increasingly important in the pharmaceutical and biomedical sciences. Successful analyses require sensitivities at ppb-level or less, high selectivity, and minimal interferences from artifacts. Modern approaches to biopharmaceutical analysis rely principally on chromatographic and immunochemical methods.