A receptor-binding-based bioassay to determine the potency of a plasmid biopharmaceutical encoding VEGF-C

Over the last decade biological assays (bioassays) have gained much importance for quality control in biopharmaceutical development and manufacturing. Here we describe the development and validation of a bioassay to determine the biological activity (potency) of the plasmid biopharmaceutical pVGI.1 which encodes the VEGF-C (VEGF-2) protein. This assay was developed to test drug substance and drug product for release and stability testing for phase I and II clinical trials. The main focus was on fast assay development and easy handling of the assay, combined with valid results representing the specific therapeutic mechanism. The method includes the expression of the VEGF-C protein in mammalian cells and its binding to the cell surface receptor VEGFR-3. The binding activity of VEGF-C to its immobilized receptor is quantified in a colorimetric assay. IC₅₀ values of VEGF-C expressed after transfection with sample plasmid and an in-house standard plasmid are determined. The ratio of the IC₅₀ value of the test article to that of the reference standard reflects the potency of the sample. The potency assay meets the criteria generally requested by authorities for precision, linearity, accuracy, and range. Therefore the assay can be used in pharmaceutical quality control and is a suitable basis for development of related bioassays.     

Rapid and high-resolution glycoform profiling of recombinant human erythropoietin by capillary isoelectric focusing with whole column imaging detection

Human erythropoietin (hEPO) is a glycoprotein hormone produced primarily by the kidney, which stimulates red blood cell production. Recombinant human erythropoietin (rhEPO), generally produced in Chinese hamster ovary (CHO) cells, can be used as not only a therapeutic protein but also a doping agent in sports. Profiling of EPO glycoforms is a critical means for quality control in pharmaceutical industrial and anti-doping analysis of misuse in sports. However, the existing methods for the analysis of EPO are associated with either time consuming or poor resolution. In this work, a rapid and high-resolution glycoform profiling method was presented based on capillary isoelectric focusing (cIEF) with whole column imaging detection (WCID). Experimental conditions that influence the separation were investigated. Under optimized conditions, rhEPO from three different sources were resolved into distinct populations within 5 min with excellent reproducibility. As compared with existing methods, the presented method exhibited the advantages of speed and high resolution. If combined with an effective sample enrichment step and a much more sensitive WCID version, the method can be a potential alternative for the detection of rhEPO misuse in sports.     

Intact Glycoform Characterization of Erythropoietin-α and Erythropoietin-β by CZE-ESI-TOF-MS

Glycosylation of recombinant human erythropoietin (rhEPO) is a post-translational process which depends on the type of cell in which rhEPO is synthesized, but also on the cell culture conditions and the final purification steps. These glycosylation modifications alter the biological activity, solubility and lifetime of rhEPO in blood. Thus, a rapid and simple method for the elucidation of the carbohydrate microheterogeneity of rhEPO is needed in order to evaluate a certain manufacturing process or assure the quality of the final product. Based on a recently developed method [1], the accurate mass determination of the intact glycoforms from two types of commercial rhEPO (epoetin-α and epoetin-β) by capillary electrophoresis-electrospray-time of flight-mass spectrometry is presented. The sample treatment consists of a fast and simple preconcentration step of the ready-to-use drug achieved by a centrifugal filter device. Characterization of the carbohydrate composition of each single glycoform is performed, in agreement with the results in glycan and glycopeptide analysis reported by other authors. The main differences between the carbohydrate structures of both epoetins are shown: the existence of two additional basic sialic acid isoforms for epoetin-β and the higher degree of acetylation for epoetin-α. The agreement of the main glycoforms for both epoetins is shown by molecular mass agreement. The high accuracy and reproducibility of the mass measurements with a standard deviation below 1 Da is proved by repeated analysis of European Pharmacopoeia rhEPO. Summarizing, the presented method enables the fast and reliable characterization of intact rhEPO in pharmaceutical products.Presented at: CE in the Biotechnology & Pharmaceutical Industries: 7th Symposium on the Practical Applications for the Analysis of Proteins, Nucleotides and Small Molecules, Montreal, Canada, August 12–16, 2005     

Site-specific qualitative and quantitative analysis of the N- and O-glycoforms in recombinant human erythropoietin

Recombinant human erythropoietin (rhEPO) has been extensively used as a pharmaceutical product for treating anemia. Glycosylation of rhEPO affects the biological activity, immunogenicity, pharmacokinetics, and in-vivo clearance rate of rhEPO. Characterization of the glycosylation status of rhEPO is of great importance for quality control. In this study, we established a fast and comprehensive approach for reliable characterization and relative quantitation of rhEPO glycosylation, which combines multiple-enzyme digestion, hydrophilic-interaction chromatography (HILIC) enrichment of glycopeptides, and tandem mass spectrometry (MS) analysis. The N-linked and O-linked intact glycopeptides were analyzed with high-resolution and high-accuracy (HR–AM) mass spectrometry using an Orbitrap. In total, 74 intact glycopeptides from four glycosylation sites at N₂₄, N₃₈, N₈₃, and O₁₂₆ were identified, with the simultaneous determination of peptide sequences and glycoform compositions. The extracted ion chromatograms based on the HR–AM data enabled relative quantification of glycoforms. Our results could be extended to quality control of rhEPO or could help establish detection approaches for glycosylation of other proteins. Graphical Abstract

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Fractionation of glycoforms of recombinant human erythropoietin by preparative capillary isotachophoresis

This feasibility study deals with the use of preparative capillary isotachophoresis (CITP), operating in a discontinuous fractionation mode, to the separations and isolations of glycoforms of recombinant human erythropoietin (rhEPO). The preparative CITP separations were monitored by capillary zone electrophoresis (CZE) with a hydrodynamically closed separation unit. Such a CZE system, suppressing fluctuations of the migration data linked with fluctuations of EOF and hydrodynamic flow, made possible to evaluate and compare the preparative CITP separations performed within a longer time frame. Preparative CITP, carried out in the separation unit with coupled columns of enhanced sample loadability, separating 100 microg of rhEPO in a run lasting ca. 30 min, gave the production rate higher than 55 ng/s for the rhEPO glycoforms. The preparative separations included valve isolations of the glycoforms from the ITP stack into four or six fractions. Such numbers of the fractions corresponded to typical numbers of the major glycoform peaks as resolved in CZE of rhEPO. With respect to close effective mobilities of the glycoforms and a multicomponent nature of rhEPO, the fractions contained mixtures of glycoforms with the dominant glycoforms enriched 10-100-fold, relative to the original rhEPO sample.     

用N,N-二甲基六烷基溴化铵涂层的毛细管对重组促红细胞生长素及其酶解产物进行毛细管电泳和毛细管电泳-电喷雾质谱分析

采用毛细管电泳技术研究了重组促红细胞生长素(rhEPO)的分离问题。用N,N-二甲基六烷基溴化铵（6,6-ionene）涂层的毛细管测定了rhEPO中唾液酸的微多相性,同时采用毛细管电泳-质谱(CE-MS)联用技术在22 min内鉴定了rhEPO 20段胰酶消化肽中的11段。该方法简单快捷,重现性好,可用于蛋白质一级结构的测定。     

UPLC-MS/MS Method for the Identification of Recombinant Human Erythropoietin Analogues in Horse Plasma and Urine

Recombinant human erythropoietin (rhEPO) analogues are known to have been used in horse sports for their assumed performance enhancing properties. Recently, several authors have published liquid chromatographic-tandem mass spectrometric (LC-MS/MS) methods for confirming the presence of rhEPO analogues in horse plasma. In the current study, an improved LC-MS/MS confirmatory procedure for rhEPO, darbepoetin (DPO) and continuous erythropoietin receptor activator (CERA) in horse plasma was developed and validated. The method was also adapted for and applied to urine samples for the first time. Similar to previously published plasma assays, the methods utilise size exclusion and immunoaffinity extraction prior to tryptic cleavage, enzymatic deglycosylation and LC-MS/MS analysis of the resulting signature tryptic peptides (rhEPO/CERA T5, rhEPO/CERA/DPO T6 and DPO T9). However, the novel application of UPLC chromatography significantly improves the run time of the method compared to nano- or micro-LC and its robustness compared to nano-LC. Furthermore, recombinant canine EPO was found to serve as an effective internal standard, thus allowing confidence in interpretation of the success/failure of every step in the procedure. Limits of detection for confirming the presence of rhEPO, CERA and DPO in plasma were 0.1, 0.25 and 0.05 ng mL^?1, respectively, which were equal to or lower than limits achieved using previously published LC-MS/MS based methods. Limits of detection for confirming the presence of rhEPO, CERA and DPO in urine were 0.05, 0.15 and 0.025 ng mL^?1 and the analysis of urine samples collected from horses administered rhEPO (Eprex?) or DPO (Aranesp?) demonstrated the use of this matrix as a suitable alternative in situations where plasma is not available.     

兴奋剂重组人促红细胞生成素及其类似物检测方法的研究进展

重组人促红细胞生成素(rhEPO)是一种激素类兴奋剂,近年来被滥用在一些耐力性比赛项目中。由于重组与内源性EPO的氨基酸序列相同,区别很小,并且在尿样或血样中的浓度低,代谢快,给检测带来了很大的难度。本文从直接方法和间接方法两个方面综述了近几年来兴奋剂rhEPO及其类似物检测的研究进展,并结合本小组的工作展望了rhEPO检测的发展方向。     

Simultaneous liquid chromatographic determination of ezetimibe and simvastatin in pharmaceutical products

A reversed-phase liquid chromatographic (LC) method was developed and validated for the simultaneous determination of ezetimibe and simvastatin in pharmaceutical dosage forms. The LC method was carried out on a Synergi fusion C18 column (150 mm x 4.6 mm id) maintained at 45 degrees C. The mobile phase consisted of phosphate buffer 0.03 M, pH 4.5-acetonitrile (35 + 65, v/v) run at a flow rate of 0.6 mL/min, and detection was made using a photodiode array detector at 234 nm. The chromatographic separation was obtained within 15.0 min, and calibration graphs were linear in the concentration range of 0.5-200 microg/mL. Validation parameters such as specificity, linearity, precision, accuracy, and robustness were evaluated, giving results within the acceptable range for both compounds. Moreover, the proposed method was successfully applied for the routine quality control analysis of pharmaceutical products.     

Quantification of recombinant human erythropoietin by amino acid analysis using isotope dilution liquid chromatography–tandem mass spectrometry

Herein, we describe an accurate method for protein quantification based on conventional acid hydrolysis and an isotope dilution-ultra performance liquid chromatography–tandem mass spectrometry method. The analyte protein, recombinant human erythropoietin (rhEPO), was effectively hydrolyzed by incubation with 8 mol/L hydrochloric acid at 130 °C for 48 h, in which at least 1 μmol/kg of rhEPO was treated to avoid possible degradation of released amino acids during hydrolysis. Prior to hydrolysis, sample solution was subjected to ultrafiltration to eliminate potential interfering substances. In a reversed-phase column, the analytes (phenylalanine, proline, and valine) were separated within 3 min using gradient elution comprising 20 % (v/v) acetonitrile and 10 mmol/L ammonium acetate, both containing 0.3 % (v/v) trifluoroacetic acid. The optimized hydrolysis and analytical conditions in our study were strictly validated in terms of accuracy and precision, and were suitable for the accurate quantification of rhEPO. Certified rhEPO was analyzed using a conventional biochemical assay kit as an additional working calibrant for the quantification of EPO and improved the accuracy. The optimized protocol is suitable for the accurate quantification of rhEPO and satisfactorily serves as a reference analytical procedure for the certification of rhEPO and similar proteins.

Carboxymethyl chitosan-coated capillary and its application in CE of proteins

A hydrophilic basic polysaccharide, carboxymethyl chitosan (CMC) as a capillary coating is presented with a simple preparation procedure. The CMC-coated capillary showed a long lifetime of more than 100 runs, and had good tolerance to some organic solvents, 0.1 M HCl, 0.1 M NaOH, and 5 M urea. The run-to-run, day-to-day, and capillary-to-capillary RSDs for the CMC-coated capillary were all below 2.0% for the determination of EOF. Moreover, the coatings with different concentrations and molecular weights of CMC were also investigated. The CMC-coated capillary was successfully applied to separate basic proteins and recombinant human erythropoietin (rhEPO). Furthermore, several experimental parameters, such as the concentration and pH of the running buffer, temperature, and applied voltage, were optimized for the separation of rhEPO glycoforms. Comparison of an uncoated capillary with chitosan- and CMC-coated capillaries for the separation of rhEPO glycoforms was also discussed. The results demonstrated that rhEPO glycoforms can be well separated by a CMC-coated capillary within 8 min with good reproducibility and resolution. Finally, the volatile BGE HAc-NH4Ac was utilized to separate rhEPO for its further application with CE-MS, achieving a satisfactory result.     

Analysis of recombinant human erythropoietin and novel erythropoiesis stimulating protein digests by immunoaffinity capillary electrophoresis–mass spectrometry

In this work, we demonstrate that detection of a specific peptide marker by immunoaffinity capillary electrophoresis–mass spectrometry (IA-CE–MS) could be used to confirm the presence of recombinant human erythropoietin (rhEPO) in solution. Besides the carbohydrate content, the amino acid sequence of novel erythropoiesis stimulating protein (NESP) differs from human erythropoietin (hEPO) at five positions (Ala30Asn, His32Thr, Pro87Val, Trp88Asn, and Pro90Thr). After digesting both glycoproteins in solution by trypsin and PNGase F, two specific proteotypic peptides, EPO (77–97) and NESP (77–97) which differ in three amino acids, were selected as rhEPO and NESP markers, respectively. Both digests and their mixtures were analyzed by IA-CE–MS. The IA stationary phase was prepared from a custom made polyclonal anti-EPO (81–95) antibody immobilized on a solid support of CNBr-Sepharose 4B and was packed in a microcartridge near the inlet of the separation capillary. As the antibody was directed to a synthetic peptide EPO (81–95), only the proteotypic peptide EPO (77–97) was retained. The retained peptide was eluted, separated by electrophoresis and detected by MS. The method was specific to confirm the presence of rhEPO in solution. Although the limits of detection for the peptide marker were similar to those obtained with CE–MS (a few mg/L), these results show the potential of this novel approach to detect in the future rhEPO and its analogues selectively and unambiguously at the levels expected in biological fluids.     

High-performance anion-exchange chromatography combined with intrinsic fluorescence detection to determine erythropoietin in pharmaceutical products

A high-performance anion-exchange chromatography (HPAEC) method was developed for determination of recombinant human erythropoietin (EPO) in pharmaceutical products. A fluorescence detector was added to the HPLC system as intrinsic fluorescence detection compared favourably to UV detection regarding sensitivity and selectivity. The HPLC method has been successfully applied to analyse erythropoietin products even in the presence of albumin as excipient. The intrinsic fluorescence chromatograms of both proteins revealed various peaks attributed to either micro-heterogeneous erythropoietin or albumin variants. The intrinsic fluorescence signal was linear over the range 10-200 microg/ml erythropoietin corresponding to pharmaceutically relevant concentrations. The HPLC method appeared to be a suitable method for differentation between recombinant human erythropoietin epoetin-alpha and -beta as they revealed different intrinsic fluorescence elution profiles. In conclusion, this study contributes to the development of a straightforward physicochemical method for specific quantification of recombinant human erythropoietin in pharmaceutical preparations.     

Ionene-dynamically coated capillary for analysis of urinary and recombinant human erythropoietin by capillary electrophoresis and online electrospray ionization mass spectrometry

In this article, a series of ionene polymers were synthesized and used to coat fused-silica capillaries for the separation of recombinant and urinary human erythropoietin (rhEPO and uEPO) standards by CE. The influence of the charge density of coatings on the separation of rhEPO and uEPO glycoforms was investigated. Then, we further studied the method for fast separation and detection of rhEPO and uEPO standards by CE-ESI-MS. The influence of several CE and MS operating parameters, such as the concentration of CE running buffer, applied external pressure, and the composition and flow rate of sheath liquid on CE-ESI-MS was studied. The results demonstrated that when the capillary was permanently coated with 6,6-ionene and the pH value of acetic acid-ammonium acetate running buffer was 4.80 and 5.50, respectively, a significantly reproducible separation was achieved for rhEPO and uEPO glycoforms. In the online CE-ESI-MS experiments, we not only achieved the online MS signal of uEPO, but also obtained baseline separation of three major rhEPO glycoforms successfully and reproducibly on the 6,6-ionene-coated capillaries. Furthermore, the standard mixture of rhEPO and uEPO was separated, and two incompletely resolved peaks that were identified to be rhEPO and uEPO by the unique MS "fingerprint" were obtained. Additionally, the molecular weight of rhEPO and uEPO were verified and compared to the results by MALDI-TOF-MS. It can be concluded that, in contrast to other indirect methods, the online CE-ESI-MS technique with the combination of the advantages of both CE and MS shows great potential for the separation and detection of rhEPO doping directly in competitive sports.     

Validation of liquid chromatography and liquid chromatography/tandem mass spectrometry methods for the determination of etoricoxib in pharmaceutical formulations

Reversed-phase liquid chromatography (LC) and LC/tandem mass spectrometry (LC/MS/MS) methods were developed and validated for the determination of etoricoxib in pharmaceutical dosage forms. The LC method was performed by reversed-phase chromatography on a Synergi fusion C18 column (150 x 4.6 mm id) maintained at ambient temperature. The mobile phase consisted of 0.01 M phosphoric acid, pH 3.0-acetonitrile (62 + 38, v/v) at a flow rate of 1.0 mL/min, and photodiode array detection at 234 nm was used. The chromatographic separation was obtained within 7.0 min, and calibration curves were linear in the concentration range of 0.02-150 microg/mL. The LC/MS/MS method was performed on a Luna C18 column (50 x 3.0 mm id). The mobile phase consisted of acetonitrile-water (95 + 5)-0.1% acetic acid (90 + 10, v/v). Detection was performed by positive electrospray ionization in the multiple reaction monitoring mode, monitoring the transitions 359.3 > 280.0 and 332.0 > 95.0 for etoricoxib and piroxicam (internal standard), respectively. The chromatographic separation was obtained within 2.0 min, and calibration curves were linear in the concentration range of 1-5000 ng/mL. Validation parameters, such as specificity, linearity, precision, accuracy, and robustness, were evaluated, which gave results within the acceptable range for both methods. Moreover, the proposed methods were successfully applied for routine quality control analysis of pharmaceutical products and showed significant correlation (r = 0.9999) of the results.     

Quantitative analysis of erythropoietin in human plasma by tandem mass spectrometry

The extended use of protein drugs in therapeutics has created the need for their quantification in human plasma. A methodology using the therapeutic protein itself as internal standard for quantitative analysis by multiple reaction monitoring (MRM) has been designed and applied to epoetin beta, a recombinant human erythropoietin (rhEPO). After depletion of major proteins, plasma samples were desalted and enriched in rhEPO by reversed phase liquid chromatography prior to tryptic cleavage. Differential isotopic labeling of peptides was performed by derivatization with 2-methoxy-4,5-dehydro-imidazole. A light version (four hydrogen atoms) of this reagent was used for plasma peptides. Tryptic peptides obtained from pure rhEPO were derivatized with a heavy version (four deuterium atoms) of the same reagent and used as internal standards. Two rhEPO tryptic peptides with three MRM transitions per peptide were selected for quantification. This strategy provided a quantification limit close to 50 amol of epoetin beta per microliter of plasma (equivalent to 1.7 ng/mL), i.e., well below the expected therapeutic concentrations in plasma (around 100–500 amol/μL).     

Development of a capillary zone electrophoretic method to determine six antidepressants in their pharmaceutical preparations. Experimental design for evaluating the ruggedness of method

A capillary zone electrophoresis method is proposed for the separation of six of the antidepressants most used for the treatment of mental illness (clomipramine, paroxetine, fluoxetine, citalopram, fluvoxamine, and trazodone). Optimum conditions for their separation were investigated. A background electrolyte solution consisting of 50 mM phosphate buffer adjusted to pH 2.0, hydrodynamic injection, and 25 kV as separation voltage were used. Relative standard deviations (RSD) were <0.38% and <2.93% for migration time and corrected peak area (n = 24), respectively. Detection limits obtained for the six antidepressants ranged from 0.03 to 0.11 mg L(-1). Stability of the solutions, linear concentration range, accuracy, and precision were examined during validation of the method. A ruggedness test of this method was performed using the fractional factorial model of Plackett-Burman, requiring in our case design of a matrix of 15 experiments, in which the influence of seven factors at three different levels was tested on different electrophoretic results: efficiency; resolution; and corrected peak area. Statistical evaluation of electrophoretic results was achieved by Youden and Steiner's method. The described method is rapid, sensitive, and rugged and it was tested for the pharmaceutical formulation analysis, giving recoveries between 95.6 and 99.1% with respect to the nominal content.     

Analysing traces of autoinducer-2 requires standardization of the <Emphasis Type="Italic">Vibrio harveyi</Emphasis> bioassay

Autoinducer-2 (furanosyl borate diester) is a biologically active compound whose role as a universal bacterial signalling molecule is currently under intense investigation. Because of its instability and the low concentrations of it found in biological samples, its detection relies at present on a bioassay that measures the difference in the timing of the luminescence of the Vibrio harveyi BB170 sensor strain with and without externally added AI-2. Here we systematically investigated which parameters affected the fold induction values of luminescence obtained in the bioassay and developed a modified protocol. Our experiments showed that growth and luminescence of V. harveyi BB170 are strongly influenced by trace elements. In particular, addition of Fe³⁺ within a certain concentration range to the growth medium of the preinoculum culture improved the reproducibility and reduced the variance of the bioassay. In contrast, trace elements and vitamins introduced directly into the bioassay caused inhibitory effects. The initial density and luminescence of the sensor strain are very important and the values required for these parameters were defined. Borate interferes with the detection of AI-2 by giving false positive results. The response of V. harveyi BB170 to chemically synthesized AI-2 in the bioassay is nonlinear except over a very small concentration range; it is maximum over three orders of magnitude and shows inhibition above 35 μM. Based on the modified protocol, we were able to detect AI-2 in the absence of inhibitors with maximum fold induction values for the positive control (chemically synthesized AI-2) of >120 with a standard deviation of ~30% in a reliable and reproducible way.     

毛细管电泳及其质谱联用技术分析重组人促红细胞生成素

用高效毛细管电泳测定了重组人促红细胞生成素（ｒｈＥＰＯ）的微多相性、肽图，同时采用毛细管电泳质谱联用技术鉴定了部分非糖基化胰酶消解片段和Ｏ－１２６糖基化肽的结构。方法简便、快速，适用于ｒｈＥＰＯ半成品的质量控制。     

Development and validation of an agar diffusion assay for determination of ceftazidime in pharmaceutical preparations

Ceftazidime (CFZ) is a broad spectrum parenteral beta-lactam antibiotic of the cephalosporin family. This paper reports the development and validation of an agar diffusion microbiological assay using the cylinder-plate method for determination of CFZ in powder for injection. The validation carried out yielded good results in terms of linearity, precision, accuracy, selectivity, and robustness. The assay is based on the inhibitory effect of CFZ upon the strain of Pseudomonas aeruginosa ATCC 27853 used as the test microorganism. The results of the assays were treated statistically by analysis of variance and were found to be linear (correlation coefficient = 0.999998) in the selected range of 8.0-32.0 microg/mL; precise [repeatability: relative standard deviation (RSD) = 1.11%; intermediate precision: between-day RSD = 1.37% and between-analyst RSD = 1.41%]; and accurate. The selectivity of the bioassay was evaluated by analysis of degraded samples at 50 degrees C, and the results were compared with a pharmacopeial liquid chromatographic method at the time 0, 24, and 48 h. The results demonstrated the validity of the proposed bioassay, which allows reliable quantitation of CFZ in pharmaceutical samples and can be used as a useful alternative methodology for CFZ analysis in routine quality control.