Establishment and validation of a microfluidic capillary gel electrophoresis platform method for purity analysis of therapeutic monoclonal antibodies

Capillary and microfluidic chip electrophoresis technologies are heavily utilized for development, characterization, release, and stability testing of biopharmaceuticals. Within the biopharmaceutical industry, CE‐SDS and M‐CGE are commonly used for purity determination by separation and quantitation of size‐based variants. M‐CGE is used primarily as an R&D tool for product and process development, while cGMP release and stability testing applications are commonly reserved for CE‐SDS. This paper describes the establishment of an M‐CGE platform method to be used for R&D and cGMP applications, including release and stability testing, for monoclonal antibodies. The M‐CGE platform method enables testing for product development support and cGMP release and stability using the same method, and utilization of one CE technology for the entire lifecycle of a biopharmaceutical product. Critical method parameters were identified, and the analytical design space of those critical parameters was defined using design of experiments (DOE) studies. Once defined through DOE studies, the method design space was validated according to ICH Q2 (R1) guidelines. Additional molecules of the same validated class were verified for use in the method by experimental confirmation of accuracy, specificity, and stability indicating capabilities. The platform method model facilitates rapid utilization of the method in development and GMP testing environments, and eliminates the need for individual validations for assets of the same class entering early stage development.     

Analysis of monoclonal antibody by a novel CE‐UV/MALDI‐MS interface

mAbs are highly complex proteins that present a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product‐ and time‐consuming. CE‐MS couplings, especially to MALDI, appear really attractive methods for the characterization of biological samples. In this work, we report the last instrumental development and performance of the first totally automated off‐line CE‐UV/MALDI‐MS/MS. This interface is based on the removal of the original UV cell of the CE apparatus, modification of the spotting device geometry, and creation of an integrated delivery matrix system. The performance of the method was evaluated with separation of five intact proteins and a tryptic digest mixture of nine proteins. Intact protein application shows the acquisition of electropherograms with high resolution and high repeatability. In the peptide mapping approach, a total number of 154 unique identified peptides were characterized using MS/MS spectra corresponding to average sequence coverage of 64.1%. Comparison with NanoLC/MALDI‐MS/MS showed complementarity at the peptide level with an increase of 42% when using CE/MALDI‐MS coupling. Finally, this work represents the first analysis of intact mAb charge variants by CZE using an MS detection. Moreover, using a peptide mapping approach CE‐UV/MALDI‐MS/MS fragmentation allowed 100% sequence coverage of the light chain and 92% of the heavy chain, and the separation of four major glycosylated peptides and their structural characterization.     

Quick analysis of baicalin in Scutellariae Radix by enzyme-linked immunosorbent assay using a monoclonal antibody

To establish an immunoassay for baicalin (BA), a hybridoma cell line (9D6) secreting a monoclonal antibody (MAb) against BA was prepared by cell fusion with splenocytes derived from a mouse immunized with BA-bovine serum albumin (BSA) conjugate and a myeloma cell line, SP2/0-Ag14. MAb 9D6 shows specific reactivity against BA and its aglycone, baicalein, but not against other natural products. We developed an enzyme-linked immunosorbent assay (ELISA) using MAb 9D6 in a competitive manner, ranging from 200 ng/mL to 2 μg/mL. After validating the developed ELISA on the basis of intra- and inter-assays and a recovery experiment, it was found that the ELISA was not only simple, but also sufficiently reliable and accurate for quality control of Scutellariae Radix. It allowed determination of BA in complex and mixed materials, such as Kampo medicines.     

Ultra high performance liquid chromatography method development for separation of omeprazole and related substances on core‐shell columns using a Quality by Design approach

An updated and improved method for analysis of omeprazole/esomeprazole and related substances on core‐shell columns was developed using Fusion LC Method Development?. The method was optimized with respect to column type, column temperature, mobile phase pH level, and gradient time. Four different core‐shell columns were examined to develop a method suitable for both high performance‐ and ultra‐high performance liquid chromatography using a Quality by Design approach. The final method offers two alternative columns: Poroshell EC C18 (3.0 × 100 mm, 2.7 µm) or Poroshell HPH (3.0 × 100 mm, 2.7 µm) with the same gradient elution condition and mobile phase composition. Total run time is 18 min with 12 min of gradient elution. Phosphate buffer (15 mM, pH 7.8) is selected as the aqueous mobile phase and acetonitrile as the organic mobile phase. Column temperature is set at 40°C and ultraviolet detection at 302 nm. Furthermore, by studying parameters in a systematic way, an understanding of the effect of the input parameters enhances the method robustness and should allow for regulatory flexibility in terms of post‐approval changes. Compared to the current United States Pharmacopeia method, the updated method is faster, more efficient and performs well above acceptance criteria.     

Isolation and characterization of therapeutic antibody charge variants using cation exchange displacement chromatography

In this report, we have demonstrated the isolation and enrichment of charge variants of a monoclonal antibody IgG1 using cation exchange displacement chromatography. We successfully achieved the separation of acidic, main and basic charge variants with high recovery (>70%) and purity (>90%) by using a commercially available stationary phase in conjunction with a commercially available displacer. In addition, we have isolated and enriched a trace methionine-oxidized variant of the monoclonal antibody allowing a secondary means of identification of this variant while providing sufficient enrichment for further analysis, stability tests and potency determination. Further characterization of the displacement trains by SEC indicate the possibility of enrichment of high and low molecular weight species. Glycan analysis of the displacement fractions indicates minimal variation in glycan distribution patterns among a wide spectrum of charge variants. These results provide a case study demonstrating the utility of cation exchange displacement chromatography as a viable approach to isolate and enrich antibody charge variants for enhanced molecular characterization.     

Production of a monoclonal antibody and development of enzyme-linked immunosorbent assay for alkyl ethoxylates

A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) has been developed for the determination of alkyl ethoxylates (AEs) that are the most widely used nonionic surfactants in the world. Three types of hapten, hemi-succinated AEs (C12EO7suc, C16EO23suc, and C18EO10suc), were synthesized and conjugated to bovine serum albumin (BSA) for mouse immunization. The mice immunized with the C12EO7suc-BSA that showed the high immune responses were used for cell fusion. The obtained monoclonal antibody (TFG2-76) was specific to AEs, which had alkyl (C) and ethoxy (EO) chain lengths of C10-12 and EO5-15, respectively. Two types of solid support, namely, a polystyrene tube and a 96-well microplate, were used for antibody immobilization. The working ranges of the tube-type and plate-type ELISAs were 2-100 and 20-1000 μg/L with IC₅₀ values of 12 and 71 μg/L AE (C12EO7), respectively. Moreover, the lowest quantification limit of plate-type ELISA could be lowered to 5 μg/L by decreasing the coated antibody concentration. Cross-reactivities with non-AE surfactants were determined, and the assay proved highly selective for AEs. The application of plate-type ELISA to determine spiked AEs in distilled water, tap water and river water provided good recoveries without matrix effects.     

Quality by design-assisted development of a capillary electrophoresis method for the enantiomeric purity determination of tenofovir

A CE method was developed and validated for the assessment of the chiral purity of the drug tenofovir applying a quality by design approach. Following selection of a quaternary ammonium β-CD as chiral selector, a fractional factorial resolution V+ design was employed for identification of the critical process parameters, while a central composite design served for method optimization. The final method used a 40/50.2 cm, 50 μm id fused-silica capillary, a BGE composed of a 100 mM sodium phosphate buffer, pH 6.4, containing 45 mg/mL quaternary ammonium β-CD, an applied voltage of 18 kV, and a capillary temperature of 22°C. Robustness was assessed by a Plackett–Burman design. The method was validated according to guideline Q2(R1) of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use and enabled the determination of the (S)-enantiomer of tenofovir at the 0.1% level.     

Quality by Design approach in the development of a solvent-modified micellar electrokinetic chromatography method: Finding the design space for the determination of amitriptyline and its impurities

A solvent-modified micellar electrokinetic chromatography method was set up for the simultaneous determination of the tricyclic antidepressant amitriptyline (AMI) and its main impurities. The method was developed following Quality by Design (QbD) principles according to ICH Guideline Q8(R2). QbD approach made it possible to find the design space (DS), where quality was assured. After a scouting phase, aimed at selecting a suitable capillary electrophoresis pseudostationary phase, risk assessment tools were employed to define the critical process parameters (CPPs) to be considered in a screening phase (applied voltage, concentration and pH of the background electrolyte, concentration of the surfactant sodium dodecyl sulphate, of the cosurfactant n-butanol and of the organic modifiers acetonitrile and urea). The effects of the seven selected CPPs on critical quality attributes (CQAs), namely resolution values between critical peak pairs and analysis time, were investigated throughout the knowledge space by means of a symmetric screening matrix. Response surface study was then carried out on four selected CPPs by applying a Doehlert Design. Monte-Carlo simulations were performed in order to estimate the probability of meeting the desired specifications on CQAs, and thus to define the DS by means of a risk of failure map. Additional points at the edges of the DS were tested in order to verify the requirements for CQAs to be fulfilled. A control strategy was implemented by defining system suitability tests. The developed method was validated following ICH Guideline Q2(R1), including robustness assessment by Plackett–Burman design, and was applied to the analysis of real samples of amitriptyline coated tablets.     

Innovative high-performance liquid chromatography method development for the screening of 19 antimalarial drugs based on a generic approach, using design of experiments, independent component analysis and design space

An innovative methodology based on design of experiments (DoE), independent component analysis (ICA) and design space (DS) was developed in previous works and was tested out with a mixture of 19 antimalarial drugs. This global LC method development methodology (i.e. DoE-ICA-DS) was used to optimize the separation of 19 antimalarial drugs to obtain a screening method. DoE-ICA-DS methodology is fully compliant with the current trend of quality by design. DoE was used to define the set of experiments to model the retention times at the beginning, the apex and the end of each peak. Furthermore, ICA was used to numerically separate coeluting peaks and estimate their unbiased retention times. Gradient time, temperature and pH were selected as the factors of a full factorial design. These retention times were modelled by stepwise multiple linear regressions. A recently introduced critical quality attribute, namely the separation criterion (S), was also used to assess the quality of separations rather than using the resolution. Furthermore, the resulting mathematical models were also studied from a chromatographic point of view to understand and investigate the chromatographic behaviour of each compound. Good adequacies were found between the mathematical models and the expected chromatographic behaviours predicted by chromatographic theory. Finally, focusing at quality risk management, the DS was computed as the multidimensional subspace where the probability for the separation criterion to lie in acceptance limits was higher than a defined quality level. The DS was computed propagating the prediction error from the modelled responses to the quality criterion using Monte Carlo simulations. DoE-ICA-DS allowed encountering optimal operating conditions to obtain a robust screening method for the 19 considered antimalarial drugs in the framework of the fight against counterfeit medicines. Moreover and only on the basis of the same data set, a dedicated method for the determination of three antimalarial compounds in a pharmaceutical formulation was optimized to demonstrate both the efficiency and flexibility of the methodology proposed in the present study.     

Harnessing the power of electrophoresis and chromatography: Offline coupling of reverse phase liquid chromatography-capillary zone electrophoresis-tandem mass spectrometry for analysis of host cell proteins in monoclonal antibody producing CHO cell line

Host cell proteins (HCPs) are widely regarded as a critical quality attribute for a biotherapeutic product. Bottom up MS is the present gold standard for HCP analysis but suffers from incomplete protein identification due to complex nature of the HCP mixture and limited separation efficiency of the preceding LC-based systems. In this paper, we present for the first time an application involving use of LC-CE-MS/MS platform for analysis of HCPs. It has been demonstrated that the proposed platform has been able to successfully identify 397 HCPs from the supernatants of recombinant Chinese hamster ovary cells, twice and thrice the number of proteins identified by the state-of-the-art LC-MS/MS (189 HCPs) and CE-MS/MS (128 HCPs) analyses, respectively. Of these, 225 HCPs were unique to the LC-CE-MS/MS approach and were not identified by either LC-MS/MS or CE-MS/MS. It is observed that the LC-CE-MS/MS platform combines the benefits of LC-MS/MS and CE-MS/MS techniques and identifies peptides in a wider range of size, pI, and hydrophobicity. Additionally, LC-CE-MS/MS also identified more HCPs associated with cellular components, molecular functions, biological processes, peptidases, and secretory proteins. The proposed approach would thus be a useful addition in HCP analysis and secretome studies of mAb-producing Chinese hamster ovary cells.     

Quality by design: A systematic and rapid liquid chromatography and mass spectrometry method for eprosartan mesylate and its related impurities using a superficially porous particle column

The present work describes the systematic development of a robust, precise, and rapid reversed‐phase liquid chromatography method for the simultaneous determination of eprosartan mesylate and its six impurities using quality‐by‐design principles. The method was developed in two phases, screening and optimization. During the screening phase, the most suitable stationary phase, organic modifier, and pH were identified. The optimization was performed for secondary influential parameters—column temperature, gradient time, and flow rate using eight experiments—to examine multifactorial effects of parameters on the critical resolution and generated design space representing the robust region. A verification experiment was performed within the working design space and the model was found to be accurate. This study also describes other operating features of the column packed with superficially porous particles that allow very fast separations at pressures available in most liquid chromatography instruments. Successful chromatographic separation was achieved in less than 7 min using a fused‐core C₁₈ (100 mm × 2.1 mm, 2.6 μm) column with linear gradient elution of 10 mM ammonium formate (pH 3.0) and acetonitrile as the mobile phase. The method was validated for specificity, linearity, accuracy, precision, and robustness in compliance with the International Conference on Harmonization Q2 (R1) guidelines. The impurities were identified by liquid chromatography with mass spectrometry.     

Development and Optimization of a New UPLC-UV/MS Method through DoE and MLR for Detecting Substandard Drug Products to Treat Tuberculosis

Drug products used for treating tuberculosis are one of the most widely reported medicines to be classified as falsified or substandard in low- and middle-income countries, representing a major hazard to health. The aim of this study was, firstly, to develop an ultra-performance liquid chromatography (UPLC) method which is able to analyze fixed combination tablets with up to four active pharmaceutical ingredients, including isoniazid, pyrazinamide, rifampicin, and ethambutol. Secondly, we aimed to optimize it through the design of experiments and multi-linear regression based on a central composite design and to validate it according to the guidelines of the International Conference on Harmonization. The application of this tools enabled the identification of the influential factors (flow rate, formic acid, and temperature) and their effects on the studied responses (retention factor and percentage for each drug) as part of the quality by design approach. The method proved to be to be linear in the range from 5.0 to 15 µg/mL for isoniazid, pyrazinamide, and rifampicin, being precise (<1%) and accurate (97–101%). In addition, the method validated for ethambutol proved to be linear from 1.4 to 4.2 µg/mL, as well as precise (0.54%) and accurate (97.3%). The method was stability indicated for all the active pharmaceutical ingredients studied and was able to detect two substandard formulations sampled on the African market.     

Quantitative analysis of boldine alkaloid in natural extracts by cyclic voltammetry at a liquid-liquid interface and validation of the method by comparison with high performance liquid chromatography

The quantitative determination of boldine alkaloid in boldo leaf extracts by employing cyclic voltammetry, at a liquid/liquid interface as well as the validation of this methodology against the reference method, high performance liquid chromatography (HPLC), are reported in the present paper. The voltammetric analysis was performed successfully and economically using two kinds of liquid/liquid interfaces: water/1,2-dicholoroethane and water/PVC (polyvinyl chloride)-gelled 1,2-dichloroethane. Linear calibration curves in the concentration range of 1.04 × 10⁻⁵ mol L⁻¹ to 5.19 × 10⁻⁴ mol L⁻¹ were obtained with a detection limit equal to (6.1 ± 0.7) × 10⁻⁵ mol L⁻¹ and the quantitative determination of this alkaloid, in complex matrixes such as boldo leaf extracts, by the electrochemical technique proposed was found to be equal to the values obtained using the standard HPLC method. The validation analysis of this methodology against HPLC demonstrated that accuracy, linearity, limit of detection (LOD), limit of quantification (LOQ), specificity and precision are acceptable. The electroanalytical technique proposed is economical and selective, involves simple equipment and can be applied for the quantitative determination of boldine alkaloid in complex matrixes such as leaf extracts without special drug separation. Moreover, cyclic voltammetry (CV) experiments applied at the liquid/liquid interface under different experimental conditions allowed us to study the transfer mechanism of boldine, and determine a value of pK_a^w = 6.90 for protonated boldine, from the variation of voltammetric peak current with pH.     

Quality by design-guided development of a capillary electrophoresis method for the simultaneous chiral purity determination and impurity profiling of tamsulosin

Analytical Quality by Design principles using the design of experiments were applied for the development of a capillary electrophoresis method for the determination of enantiomeric purity and chemically related impurities of tamsulosin. From initial scouting experiments, a dual cyclodextrin (CD) system composed of sulfated β-CD and carboxymethyl-α-CD was selected as the chiral selector. A fractional factorial resolution V+ design was used for the identification of the critical process parameters, while a face-centered central composite design and Monte Carlo simulations were employed for final optimization and defining the design space of the method. The experimental conditions of the working point were: 30 mM sodium phosphate buffer, pH 3.0, containing 40 mg/mL sulfated β-CD and 7 mg/mL carboxymethyl-α-CD, capillary temperature 18°C, applied voltage -23 kV. Following the assessment of robustness by applying a Plackett-Burman design, the method was validated according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use guideline Q2(R1). The method allowed the quantification of the chiral impurity and three other related impurities at the 0.1 % level with acceptable accuracy and precision.     

Analytical quality by design in the development of a cyclodextrin‐modified capillary electrophoresis method for the assay of metformin and its related substances

Quality by Design (QbD) is a new paradigm of quality to be applied to pharmaceutical products and processes, recently encouraged by International Conference on Harmonisation guidelines. In this paper QbD approach was applied to the development of a CE method for the simultaneous assay of metformin hydrochloride (MET) and its main impurities. QbD strategy was focused on electrophoretic process understanding, and the analytical method was thoroughly evaluated by applying risk assessment and chemometric tools. Method scouting allowed CD‐CZE based on the addition of carboxymethyl‐β‐CD to Britton‐Robinson acidic buffer to be chosen as operative mode. Seven critical process parameters (CPPs) were selected, related to capillary, injection, BGE and instrumental settings. The effect of the different levels of the CPPs on critical quality attributes (CQAs), e.g. critical resolution values and analysis time, was evaluated in a screening study. Response surface methodology led to draw contour plots and sweet spot plots. The definition of design space was accomplished by applying Monte‐Carlo simulations, thus identifying by risk of failure maps a multivariate zone where the CQAs fulfilled the requirements with a selected probability. Finally, a control strategy was designed and the method was applied to a real sample of MET tablets.     

Quantitative analysis of multicomponents by a single marker and quality evaluation of Venenum Bufonis from different geographical origins

Bufadienolides are the main bioactive components of Venenum Bufonis (VB) and have been widely used to treat different types of human cancers for decades. The bufadienolide content in VB varies significantly in materials from different geographical origins. In this work, a new strategy for the quality assessment of VB was developed through quantitative analysis of multi‐components by single marker (QAMS). Cinobufagin was selected as the internal reference substance; seven bufadienolides were separated and simultaneously determined based on relative correction factors. The correlation coefficient value (r ≥ 0.9936) between QAMS and the normal external standard method proved the consistency of the two methods. According to the outcomes of 30 batches of VB samples, the contents of the seven bufadienolides were used for further chemometric analysis. All of the samples of VB from various geographical origins were divided into three categories based on hierarchical cluster analysis and radar plot, which indicated the crucial influence of geographical origins on VB. This study showed that QAMS combined with chemometristry could be used to comprehensively evaluate and effectively control the quality of VB from different geographical origins.     

Quantitative analysis of a quaternary ammonium drug: ipratropium bromide by LC/ESI-MS(n) in horse plasma and urine

A quantitative method, using LC/ESI-MS(n) with a quadrupole linear ion trap mass analyzer, has been developed for the analysis of ipratropium cation in horse plasma and urine. The method applies solid-phase extraction with WCX cartridges for plasma and MM2 cartridges for urine, prior to analysis by LC/ESI-MS(n). The efficiency of extraction combined with the sensitivity and the selectivity of MS(n) allows for the quantification of ipratropium cation at picogram per milliliter levels. The analytical capabilities of the method have been successfully checked by the quantitative analysis of ipratropium cation in post-administration samples collected from horses treated by nebulization.     

Determination of newly synthesized lipoic acid–niacin dimer in rat plasma by UPLC/electrospray ionization tandem mass spectrometry: assay development,validation and application to a pharmacokinetic study

A simple, sensitive and specific ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC‐MS/MS) method was developed to determine the newly synthesized compound lipoic acid–niacin dimer (N2L) in plasma. Plasma samples were precipitated by methanol using tetrahydropalmatine as internal standard. Chromatographic separation was achieved on an Acquity BEH C₁₈ (2.1 × 50 mm i.d., 1.7 µm) column; the mobile phase contains methanol and buffer solution (water with 0.5% formic acid and 10 mmol/L ammonium acetate). Multiple reaction monitoring (m/z 353.9 → 148.6 for N2L and m/z 356.0 → 192.0 for internal standard) was performed for detection and quantification. The method was validated to be rapid, specific, accurate and precise over the concentration range of 1–750 ng/mL; N2L was not stable on the bench‐top or during freeze–freeze‐thaw cycles in plasma, but was stable in the stock solution and after preparation in the autosampler for 24 h. The utility of the assay was confirmed by pharmacokinetic study of N2L in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of (4‐(1,3‐dioxo‐1,3‐dihydro‐2H‐isoindol‐ 2‐yl)‐N′‐[(4‐ethoxyphenyl) methylidene] benzohydrazide,a novel anti‐inflammatory agent,in biological fluids by UPLC‐MS/MS: Assay development,validation and in vitro metabolic stability study

A thalidomide analog, (4‐(1,3‐dioxo‐1,3‐dihydro‐2H‐isoindol‐2‐yl)‐N ′‐[(4‐ethoxyphenyl) methylidene] benzohydrazide), has been identified as a promising broad‐spectrum anti‐inflammatory agent in previous study. In this study, a sensitive and selective UPLC‐MS/MS assay was developed and validated for its determination in rat plasma samples. The chromatographic separation was performed on an Aquity BEH C₁₈ column using mobile phase comprising of acetonitrile and 10 mm ammonium acetate in the ratio of 85: 15, at flow rate of 0.3 mL/min. The detection and quantification were performed in positive multiple reaction monitoring mode by parent to daughter ion transition of 414.06 ˃ 148.05 for analyte and 411.18 ˃ 191.07 for internal standard (risperidone), respectively using electrospray ionization source. The sample extraction process consisted of liquid–liquid extraction method using diethyl ether as the extracting solvent. The assay was validated by following FDA guidelines and all parameters were found to be within acceptable limits. The linearity was between 10.1 and 2500 ng/mL and the lower limit of quantification was 10.1 ng/mL. The reported results indicate that the assay could meet the requirement for analysis of this compound in amounts expected to the present in actual samples. Further, in vitro metabolic stability study was performed in rat liver microsomes by using the validated assay.