In-Membrane Enrichment and Peptic Digestion to Facilitate Analysis of Monoclonal Antibody Glycosylation.

The number of therapeutic monoclonal antibodies (mAbs) is growing rapidly due to their widespread use for treating various diseases and health conditions. Assessing the glycosylation profile of mAbs during production is essential to ensuring their safety and efficacy. This research aims to rapidly isolate and digest mAbs for liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of glycans and monitoring of glycosylation patterns, potentially during manufacturing. Immobilization of an Fc region-specific ligand, oFc20, in a porous membrane enables the enrichment of mAbs from cell culture supernatant and efficient elution with an acidic solution. Subsequent digestion of the mAb eluate occurred in a pepsin-modified membrane within 5 min. The procedure does not require alkylation and desalting, greatly shortening the sample preparation time. Subsequent LC-MS/MS analysis identified 11 major mAb N-glycan proteoforms and assessed the relative peak areas of the glycosylated peptides. This approach is suitable for the glycosylation profiling of various human IgG mAbs, including biosimilars and different IgG subclasses. The total time required for this workflow is less than 2 h, whereas the conventional enzymatic release and labeling of glycans can take much longer. Thus, the integrated membranes are suitable for facilitating the analysis of mAb glycosylation patterns.

Size exclusion chromatography of biopharmaceutical products: From current practices for proteins to emerging trends for viral vectors, nucleic acids and lipid nanoparticles.

The 21st century has been particularly productive for the biopharmaceutical industry, with the introduction of several classes of innovative therapeutics, such as monoclonal antibodies and related compounds, gene therapy products, and RNA-based modalities. All these new molecules are susceptible to aggregation and fragmentation, which necessitates a size variant analysis for their comprehensive characterization. Size exclusion chromatography (SEC) is one of the reference techniques that can be applied. The analytical techniques for mAbs are now well established and some of them are now emerging for the newer modalities. In this context, the objective of this review article is: i) to provide a short historical background on SEC, ii) to suggest some clear guidelines on the selection of packing material and mobile phase for successful method development in modern SEC; and iii) to highlight recent advances in SEC, such as the use of narrow-bore and micro-bore columns, ultra-wide pore columns, and low-adsorption column hardware. Some important innovations, such as recycling SEC, the coupling of SEC with mass spectrometry, and the use of alternative detectors such as charge detection mass spectrometry and mass photometry are also described. In addition, this review discusses the use of SEC in multidimensional setups and shows some of the most recent advances at the preparative scale. In the third part of the article, the possibility of SEC for the characterization of new modalities is also reviewed. The final objective of this review is to provide a clear summary of opportunities and limitations of SEC for the analysis of different biopharmaceutical products.

Charge heterogeneity of therapeutic monoclonal antibodies by different cIEF systems: views on the current situation.

Therapeutic mAbs show a specific "charge fingerprint" that may affect safety and efficacy, and, as such, it is often identified as a critical quality attribute (CQA). Capillary iso-electric focusing (cIEF), commonly used for the evaluation of such CQA, provides an analytical tool to investigate mAb purity and identity across the product lifecycle. Here, we discuss the results of an analysis of a panel of antibody products by conventional and whole-column imaging cIEF systems performed as part of European Pharmacopoeia activities related to development of "horizontal standards" for the quality control of monoclonal antibodies (mAbs). The study aimed at designing and verifying an independent and transversal cIEF procedure for the reliable analysis of mAbs charge variants. Despite the use of comparable experimental conditions, discrepancies in the charge profile and measured isoelectric points emerged between the two cIEF systems. These data suggest that the results are method-dependent rather than absolute, an aspect known to experts in the field and pharmaceutical industry, but not suitably documented in the literature. Critical implications from analytical and regulatory perspectives, are herein thoughtfully discussed, with a special focus on the context of market surveillance and identification of falsified medicines.

Physical origin of the peak tailing of monoclonal antibodies in size-exclusion chromatography using bio-compatible systems and columns.

The analysis of mixtures containing monoclonal antibody (mAb) (approximately 150 kDa molecular weight) and sub-unit impurities (approximately 100 kDa) is challenging, even when adopting the latest ultra-high-pressure liquid chromatography (UHPLC) columns (4.6 mm [Formula: see text] 150 mm coated hardware, 1.7 [Formula: see text]m 250 BEH[Formula: see text] Surface-modified Particles) and systems (ACQUITY[Formula: see text] UPLC[Formula: see text] I-class Bio Plus). The main issue still encountered is a persistent tail of the mAb peak. Here, the physical origin(s) of such peak tailing in size-exclusion chromatography (SEC) are investigated from both fundamental and practical approaches. Up to five relevant physical origins are analyzed: sample heterogeneity (glycoforms), UHPLC system dispersion, strong residual binding of the mAb to the SEC particles (via hydrophobic and/or electrostatic interactions) and to the stainless steel column/system hardware, slow escape kinetics of the mAb from the SEC particles, and flow heterogeneity caused by the non-ideal slurry packing of SEC columns. Experiments (testing sample heterogeneity, system dispersion, and strong residual interactions) and calculations (predicting the transient absorption/escape kinetics in a single SEC particle and the two-dimensional peak concentration profiles) altogether unambiguously demonstrate that the observed mAb peak tailing is caused primarily by the long-range velocity biases across the SEC column combined with the slow transverse dispersion of mAbs. Therefore, improvement in the resolution between mAb and sub-unit fragment impurities can only be achieved by increasing the column length, e.g., by applying recycling chromatography at acceptable pressures.

Automated Bioanalytical Workflow for Ligand Binding-Based Pharmacokinetic Assay Development.

The growth of therapeutic monoclonal antibodies (mAbs) continues to accelerate due to their success as treatments for many diseases. As new therapeutics are developed, it is increasingly important to have robust bioanalytical methods to measure the pharmacokinetics (PK) of circulating therapeutic mAbs in serum. Ligand-binding assays such as enzyme-linked immunosorbent assays (ELISAs) with anti-idiotypic antibodies (anti-IDs) targeting the variable regions of the therapeutic antibody are sensitive and specific bioanalytical methods to measure levels of therapeutic antibodies in a biological matrix. However, soluble circulating drug mAb targets can interfere with the anti-IDs binding to the therapeutic mAb, thereby resulting in an underestimation of total drug concentration. Therefore, in addition to a high binding affinity for the mAb, the selection of anti-IDs and the assay format that are not impacted by soluble antigens and have low matrix interference is essential for developing a robust PK assay. Standardized automated approaches to screen and select optimal reagents and assay formats are critical to increase efficiency, quality, and PK assay robustness. However, there does not exist an integrated screening and analysis platform to develop robust PK assays across multiple formats. We have developed an automated workflow and scoring platform with multiple bioanalytical assay parameters that allow for ranking of candidate anti-IDs. A primary automated indirect electrochemiluminescence (ECL) was utilized to shortlist the anti-IDs that were selected for labeling and screening in pairs. A secondary screen using an ECL sandwich assay with labeled-anti-ID pairings was used to test multiple PK assay formats to identify the best anti-ID pairing/PK assay format. We developed an automated assay using fixed plate maps combined with a human-guided graphical user interface-based scoring system and compared it to a data-dependent scoring system using Gaussian mixture models for automated scoring and selection. Our approach allowed for screening of anti-IDs and identification of the most robust PK assay format with significantly reduced time and resources compared with traditional approaches. We believe that such standardized, automated, and integrated platforms that accelerate the development of PK assays will become increasingly important for supporting future human clinical trials.

Size exclusion chromatography as green support for forced degradation study of adalimumab.

Size exclusion chromatography (SEC) has become a powerful tool for analysing size variants of biologic drugs in their native form. Modern SEC can be defined by the use of chromatographic columns packed with sub-3 µm particles, allowing an increase in method throughput compared to that of conventional SEC. We performed the forced degradation study of adalimumab, the first genetically engineered fully humanised immunoglobulin G1 monoclonal antibody, and evaluated tha possibilities of an advanced SEC column packed with sub-3 µm particles for elucidation of the degradation pathway. Our results revealed the main adalimumab degradation products to be antibody fragments. Acidic and basic conditions had the most intensive effect on the degradation of the adalimumab while the drug exhibits relative stability under thermal and photolytic stress conditions. The AGREE and AGREEprep calculators were used for the evaluation of the environmental performance of the forced degradation procedure. The results of the green score evaluation are presented as round pictograms with a circle in the centre that shows the overall score of 0.81 and 0.61, respectively. Both pictograms are highlighted in green, indicating the eco-friendly conditions.

Multi-Attribute Method (MAM) Analytical Workflow for Biotherapeutic Protein Characterization from Process Development to QC.

The multi-attribute method (MAM) has emerged significantly in recent years to support biotherapeutic protein characterization from process development to the QC environment. MAM is a liquid chromatography mass spectrometry (LC-MS) based peptide mapping approach, which combines the benefits from liquid chromatography coupled to high resolution accurate mass mass spectrometry (LC-HRAM MS), enabling direct assessment of protein sequence and product quality attributes with site specificity. These product quality attributes may impact efficacy, safety, stability, and process robustness. MAM is intended to replace conventional analytical approaches as it offers a more streamlined strategy for parallel monitoring of multiple attributes in a single analysis with high sensitivity and confidence, and ultimately supports more robust Quality by Design (QbD) approaches and faster decision cycles for biotherapeutic development. MAM consists of three main stages. The first stage is sample digestion, which typically entails proteolytic digestion of the protein. The second stage is reversed-phase chromatographic separation of the generated peptides and detection by HRAM MS in two phases. During MAM Phase I (discovery phase), data-dependent acquisition (DDA) MS/MS is performed to enable confident identification of peaks and development of a peptide workbook. During MAM Phase II (monitoring phase), full MS acquisition is only carried out for the monitoring of predefined product quality attributes (PQAs). The third stage is data processing, which entails analysis and reporting for each of the two phases including evaluation of sequence coverage, assessment of PQAs and peptide workbook creation during phase I, and targeted monitoring of predefined product attributes and new peak detection (NPD) during phase II. The latter is a comparative analysis that uses a base peak alignment algorithm to determine any non-monitored differences between the LC-MS chromatograms of a test sample and a reference standard. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: In-solution sample digestion Alternate Protocol: Automated sample digestion Basic Protocol 2: Reversed-phase chromatographic separation and detection by HRAM-MS (RPLC-HRAM MS) Basic Protocol 3: Data processing and reporting.

Continuous monitoring of a monoclonal antibody by size exclusion chromatography reveals a correlation between system suitability parameters and column aging.

Size exclusion chromatography (SEC) is a foundational analytical method to assess product purity of biological molecules. To ensure accurate and reproducible data that meet regulatory agency standards, it is critical to monitor the chromatographic column with efficient and continuous approaches. In this study, 19 SEC columns (Waters Acquity BEH200) were evaluated using an in-house monoclonal antibody made at Regeneron. System suitability parameters (SSPs) were used to monitor the performance of the SEC assay, including USP resolution, USP plate count, USP tailing factor, asymmetry factor, elution time, peak width, and peak height. A general linear model was built and revealed that elution time, peak width, asymmetry factor, and tailing factor increased with injection number, while peak height, resolution, and plate count decreased. After 1000 injections, tailing factor and peak width increased by more than 10%, while resolution and plate count decreased by more than 10% from their respective starting values.

A Magnetic-Bead-Based Immunoassay with a Newly Developed Monoclonal Antibody for Rapid and Highly Sensitive Detection of Forchlorfenuron.

Forchlorfenuron (CPPU) is a widely used plant growth regulator in agriculture, and CPPU residue in food can cause harm to human health. Thus, it is necessary to develop a rapid and sensitive detection method for CPPU monitoring. In this study, a new monoclonal antibody (mAb) against CPPU with high affinity was prepared by a hybridoma technique, and a magnetic bead (MB)-based analytical method was established for the determination of CPPU by a one-step procedure. Under optimized conditions, the detection limit of the MB-based immunoassay was as low as 0.0004 ng/mL, which was five times more sensitive than the traditional indirect competitive ELISA (icELISA). In addition, the detection procedure took less than 35 min, a significant improvement over the 135 min required for icELISA. The selectivity test of the MB-based assay also showed negligible cross-reactivity with five analogues. Furthermore, the accuracy of the developed assay was assessed by the analysis of spiked samples, and the results agreed well with those obtained by HPLC. The excellent analytical performance of the proposed assay suggests its great potential for routine screening of CPPU, and it provides a basis for promoting the application of more immunosensors in the quantitative detection of low concentrations of small organic molecules in food.

Imaged capillary isoelectric focusing associated with multivariate analysis: A powerful tool for quality control of therapeutic monoclonal antibodies.

Monoclonal antibodies are increasingly used in cancer therapy. To guarantee the quality of these mAbs from compounding to patient administration, characterization methods are required (e.g. identity). In a clinical setting, these methods must be fast and straightforward. For this reason, we investigated the potential of image capillary isoelectric focusing (icIEF) combined with Principal Component Analysis (PCA) and Partial least squares-discriminant analysis (PLS-DA). icIEF profiles obtained from monoclonals antibodies (mAbs) analysis have been pre-processed and the data submitted to principal component analysis (PCA). This pre-processing method has been designed to avoid the impact of concentration and formulation. Analysis of four commercialized mAbs (Infliximab, Nivolumab, Pertuzumab, and Adalimumab) by icIEF-PCA led to the formation of four clusters corresponding to each mAb. Partial least squares-discriminant analysis (PLS-DA) applied to these data allowed us to build models to predict which monoclonal antibody is analyzed. The validation of this model was obtained from k-fold cross-validation and prediction tests. The selectivity and the specificity of the model performance parameters were assessed by the excellent classification obtained. In conclusion, we established that the combination of icIEF and chemometric approaches is a reliable approach for unambiguously identifying compounded therapeutic monoclonal antibodies (mAbs) before patient administration.

Development of a highly sensitive imaged cIEF immunoassay for studying AAV capsid protein charge heterogeneity.

Post-translational modifications (PTMs) of adeno-associated virus (AAV) capsid proteins tune and regulate the AAV infective life cycle, which can impact the safety and efficacy of AAV gene therapy products. Many of these PTMs induce changes in protein charge heterogeneity, including deamidation, oxidation, glycation, and glycosylation. To characterize the charge heterogeneity of a protein, imaged capillary isoelectric focusing (icIEF) has become the gold standard method. We have previously reported an icIEF method with native fluorescence detection for denatured AAV capsid protein charge heterogeneity analysis. Although well suited for final products, the method does not have sufficient sensitivity for upstream, low-concentration AAV samples, and lacks the specificity for capsid protein detection in complex samples like cell culture supernatants and cell lysates. In contrast, the combination of icIEF, protein capture, and immunodetection affords significantly higher sensitivity and specificity, addressing the challenges of the icIEF method. By leveraging different primary antibodies, the icIEF immunoassay provides additional selectivity and affords a detailed characterization of individual AAV capsid proteins. In this study, we describe an icIEF immunoassay method for AAV analysis that is 90 times more sensitive than native fluorescence icIEF. This icIEF immunoassay provides AAV stability monitoring, where changes in individual capsid protein charge heterogeneity can be observed in response to heat stress. When applied to different AAV serotypes, this method also provides serotype identity with reproducible quantification of VP protein peak areas and apparent isoelectric point (pI). Overall, the described icIEF immunoassay is a sensitive, reproducible, quantitative, specific, and selective tool that can be used across the AAV biomanufacturing process, especially in upstream process development where complex sample types are often encountered.

Identification of monoclonal antibody drug substances using non-destructive Raman spectroscopy.

Monoclonal antibodies provide highly specific and effective therapies for the treatment of chronic diseases. These protein-based therapeutics, or drug substances, are transported in single used plastic packaging to fill finish sites. According to good manufacturing practice guidelines, each drug substance needs to be identified before manufacturing of the drug product. However, considering their complex structure, it is challenging to correctly identify therapeutic proteins in an efficient manner. Common analytical techniques for therapeutic protein identification are SDS-gel electrophoresis, enzyme linked immunosorbent assays, high performance liquid chromatography and mass spectrometry-based assays. Although effective in correctly identifying the protein therapeutic, most of these techniques need extensive sample preparation and removal of samples from their containers. This step not only risks contamination but the sample taken for the identification is destroyed and cannot be re-used. Moreover, these techniques are often time consuming, sometimes taking several days to process. Here, we address these challenges by developing a rapid and non-destructive identification technique for monoclonal antibody-based drug substances. Raman spectroscopy in combination with chemometrics were used to identify three monoclonal antibody drug substances. This study explored the impact of laser exposure, time out of refrigerator and multiple freeze thaw cycles on the stability of monoclonal antibodies. and demonstrated the potential of using Raman spectroscopy for the identification of protein-based drug substances in the biopharmaceutical industry.

An interlaboratory capillary zone electrophoresis-UV study of various monoclonal antibodies, instruments, and ε-aminocaproic acid lots.

Capillary zone electrophoresis ultraviolet (CZE-UV) has become increasingly popular for the charge heterogeneity determination of mAbs and vaccines. The ε-aminocaproic acid (eACA) CZE-UV method has been used as a rapid platform method. However, in the last years, several issues have been observed, for example, loss in electrophoretic resolution or baseline drifts. Evaluating the role of eACA on the reported issues, various laboratories were requested to provide their routinely used eACA CZE-UV methods, and background electrolyte compositions. Although every laboratory claimed to use the He et al. eACA CZE-UV method, most methods actually deviate from He's. Subsequently, a detailed interlaboratory study was designed wherein two commercially available mAbs (Waters' Mass Check Standard mAb [pI 7] and NISTmAb [pI 9]) were provided to each laboratory, along with two detailed eACA CZE-UV protocols for a short-end, high-speed, and a long-end, high-resolution method. Ten laboratories participated each using their own instruments, and commodities, showing excellence method performance (relative standard deviations [RSDs] of percent time-corrected main peak areas from 0.2% to 1.9%, and RSDs of migration times from 0.7% to 1.8% [n = 50 per laboratory], analysis times in some cases as short as 2.5 min). This study clarified that eACA is not the main reason for the abovementioned variations.

Ultrasensitive Quantification Method for Understanding Biologically Relevant Concentrations of Host Cell Proteins in Therapeutics.

Certain host cell proteins (HCPs) in biotherapeutic drugs may be detrimental to drug product quality even when they are present at the subppm level. Therefore, an analytical method that can reliably quantify trace amounts of HCPs is desirable. This study demonstrates a novel strategy to quantify HCPs present at subppm levels with ProteoMiner enrichment coupled with limited digestion followed by targeted analysis with nano-liquid chromatography-parallel reaction monitoring. The method can achieve LLOQ values as low as 0.06 ppm, with an accuracy of 85%-111% of the theoretical value, and inter-run and intrarun precision within 12% and 25%, respectively. The approach was applied to the quantification of five high-risk HCPs in drug products. The results indicated that 2.5 ppm lysosomal acid lipase, 0.14 ppm liver carboxylesterase, 1.8 ppm palmitoyl-protein thioesterase 1, and 1 ppm cathepsin D affected the stability of drug products, whereas drug products could safely contain 1.5 ppm lipoprotein lipase, 0.1 ppm lysosomal acid lipase, or 0.3 ppm cathepsin D. In combination with lipase activity analysis, the accurate quantification of lipases/esterases in drug products enables better understanding and comparison of the enzymatic activity of polysorbate degradation from endogenous proteins.

Evaluation and Screening of Biopharmaceuticals using Multi-Angle Dynamic Light Scattering.

Biopharmaceuticals are large, complex and labile therapeutic molecules prone to instability due to various factors during manufacturing. To ensure their safety, quality and efficacy, a wide range of critical quality attributes (CQAs) such as product concentration, aggregation, particle size, purity and turbidity have to be met. Size exclusion chromatography (SEC) is the gold standard to measure protein aggregation and degradation. However, other techniques such as dynamic light scattering (DLS) are employed in tandem to measure the particle size distribution (PSD) and polydispersity of biopharmaceutical formulations. In this study, the application of multi-angle dynamic light scattering (MADLS) was evaluated for the determination of particle size, particle concentration and aggregation in 3 different protein modalities, namely bovine serum albumin (BSA) and two biopharmaceuticals including a monoclonal antibody (mAb) and an enzyme. The obtained calibration curve (R2 > 0.95) for the particle number concentration of the 3 proteins and the observed correlation between MADLS and SEC (R2 = 0.9938) for the analysis of aggregation in the enzyme can be employed as a 3-in-1 approach to assessing particle size, concentration and aggregation for the screening and development of products while also reducing the number of samples and experiments required for analysis prior to other orthogonal tests.

Competitive ELISA based on pH-responsive persistent luminescence nanoparticles for autofluorescence-free biosensor determination of ochratoxin A in cereals.

An accurate and sensitive competitive enzyme-linked immunosorbent assay (ELISA) based on persistent luminescence nanoparticles Zn2GeO4:Mn2+, Eu3+ (ZGME) was developed for detecting ochratoxin A (OTA), a powerfully toxic mycotoxin usually found in grains. As a signal output element of autofluorescence-free biosensors, ZGME can be integrated into ELISA with glucose oxidase (GOx)-binding OTA molecules due to its excellent pH-responsive persistent luminescence. In the absence of OTA, the OTA-GOx conjugate was captured by the anti-OTA monoclonal antibody (anti-OTA mAb) pre-coated on the 96-well plate. The results indicate a decrease in the pH value of the solution, which triggered the quenching of ZGME luminescence due to GOx-dependent gluconic acid production. The presence of OTA inhibited the binding of OTA-GOx on the plate, thus decreasing the production of gluconic acid and increasing the persistent luminous intensity of ZGME. Under the optimized concentrations of anti-OTA mAb and OTA-GOx, quantitative determination of OTA was achieved by plotting the increase or decrease in persistent luminescence intensity of ZGME at 535 nm. In this study, the linear range was from 0.1 µg L-1 to 63 µg L-1, and the limit of detection (LOD) was as low as 0.045 µg L-1. In five food samples (corn grit, brown rice, soybean, rice, and wheat), the results exhibited good stability and repeatability, with a recovery range from 81.3% to 94.4% and a relative standard deviation (RSD) of less than 4.2%. Hence, the established method provides a sensitive, accurate, and autofluorescence-free approach for the determination of OTA in different grain samples.

Integrating ultra-high-performance liquid chromatography tandem mass spectrometry and imaged capillary isoelectric focusing for in-depth characterization of complex fusion proteins.

RATIONALE: Fc-fusion proteins represent a successful class of biopharmaceutical products, which combine the tailored pharmacological properties of biological ligands with the multiple functions of the fragment crystallizable domain of immunoglobulins. There is great diversity in terms of possible biological ligands creating highly diverse structures, therefore the analytical characterization of fusion proteins is far more complex than that of monoclonal antibodies and requires the use and development of additional product-specific methods over conventional generic/platform methods. METHODS: Employing etanercept analogues as studied fusion proteins, the Orbitrap mass analyzer with ultra-high performance liquid chromatography (UHPLC-MS) and imaged capillary isoelectric focusing (icIEF) were utilized for the in-depth fusion protein characterization. RESULTS: The amino acid sequence coverage, peptide mapping, and post-translational modifications of etanercept analogues were analyzed by UHPLC-MS. The post-translational modification results were complemented by imaged capillary isoelectric focusing to produce quality research on etanercept analogues. CONCLUSIONS: The developed workflow integrating UHPLC-MS and icIEF provided an innovative strategy for characterizing complex fusion proteins in the process of quality control and manufacturing.

A multiparameter optimization in middle-down analysis of monoclonal antibodies by LC-MS/MS.

In antibody-based drug research, a complete characterization of antibody proteoforms covering both the amino acid sequence and all posttranslational modifications remains a major concern. The usual mass spectrometry-based approach to achieve this goal is bottom-up proteomics, which relies on the digestion of antibodies but does not allow the diversity of proteoforms to be assessed. Middle-down and top-down approaches have recently emerged as attractive alternatives but are not yet mastered and thus used in routine by many analytical chemistry laboratories. The work described here aims at providing guidelines to achieve the best sequence coverage for the fragmentation of intact light and heavy chains generated from a simple reduction of intact antibodies using Orbitrap mass spectrometry. Three parameters were found crucial to this aim: the use of an electron-based activation technique, the multiplex selection of precursor ions of different charge states, and the combination of replicates.

High-throughput capillary electrophoresis analysis of biopharmaceuticals utilizing sequential injections.

The complexity of biotherapeutic products implies an ever-increasing list of product quality attributes that need to be monitored and characterized. In addition, the growing interest in implementing process analytical technology in biopharmaceutical production has further increased the testing burden, together with the need for rapid testing that can facilitate real-time or near-real-time decision-making. Capillary electrophoresis (CE) has made a place in biopharmaceutical analysis but is regarded as a low-throughput method, with the instrument dead time constituting more than 80% of the total time of analysis. In this study, the dead time of CE was utilized to analyse 3 mAb samples in a single-CE run. This approach resulted in an up to 77% reduction in the total analysis time and increased the productivity by up to 300%, compared to traditional single CE-ultraviolet runs, without compromising resolution or relative peak areas. Additionally, good method reproducibility was observed. The compatibility of the method has been demonstrated with protein A eluate and cation exchange chromatography fractions. We, thus, propose that sequential injections can be applied for fast and robust CE analysis of biopharmaceuticals.

Fractionation and online mass spectrometry based on imaged capillary isoelectric focusing (icIEF) for characterizing charge heterogeneity of therapeutic antibody.

Imaged capillary isoelectric focusing (icIEF) technology has been proved to be robust for the characterization of protein charge heterogeneity due to its high-resolution pI discrimination and high-throughput. Although high performance liquid chromatography (HPLC) tandem mass spectrometry (MS) and offline fraction collection technologies including isoelectric focusing (IEF), ion exchange chromatography (IEX) and free flow electrophoresis (FFE) have been widely utilized for protein charge variant characterization, there are a few applications of MS coupling with icIEF as a front-separation technique and related fractionation technologies for protein charge heterogeneity. However, the application of icIEF-MS has been much less frequent due to difficulties in MS interface, compatible ampholyte and coated capillary cartridge designation, ultimately impeding the breadth of icIEF applications in protein charge heterogeneity. In this study, a therapeutic monoclonal antibody (mAb-M-AT) was used for its charge variant characterization on an integrated icIEF platform with functions including analytical profiling, MS online coupling and fraction collection for charge heterogeneities. The main protein component and its four charge variants were identified using direct icIEF-MS coupling. Additionally, the two major acidic and basic charge variants were collected using preparative fractionation after the protein focused in the separation capillary. The identity of the fractions was confirmed by LC-MS at intact protein level and the results were consistent with those using icIEF-MS online coupling. The multiple operation modes of the icIEF platform described above can be rapidly and flexibly switched just by changing customized capillary separation cartridges without drastically altering instrument configuration. The whole workflow of icIEF-based profiling, fractionation and MS online coupling for protein heterogeneity is straightforward, reliable, and accurate, thus providing comprehensive solutions for in-depth protein heterogeneity characterization.