LC-MS Characterization and Stability Assessment Elucidate Correlation Between Charge Variant Composition and Degradation of Monoclonal Antibody Therapeutics.

Aggregation stability of monoclonal antibody (mAb) therapeutics is influenced by many critical quality attributes (CQA) such as charge and hydrophobic variants in addition to environmental factors. In this study, correlation between charge heterogeneity and stability of mAbs for bevacizumab and trastuzumab has been investigated under a variety of stresses including thermal stress at 40 °C, thermal stress at 55 °C, shaking (mechanical), and low pH. Size- and charge-based heterogeneities were monitored using analytical size exclusion chromatography (SEC) and cation exchange chromatography (CEX), respectively, while dynamic light scattering was used to assess changes in hydrodynamic size. CEX analysis revealed an increase in cumulative acidic content for all variants of both mAbs post-stress treatment attributed to increased deamidation. Higher charge heterogeneity was observed in variants eluting close to the main peak than the ones eluting further away (25-fold and 42-fold increase in acidic content for main and B1 of bevacizumab and 19-fold for main of trastuzumab, respectively, under thermal stress; 50-fold increase in acidic for main and B1 of bevacizumab and 10% rise in basic content of main of trastuzumab under pH stress). Conversely, variants eluting far away from main exhibit greater aggregation as compared to close-eluting ones. Aggregation kinetics of variants followed different order for the different stresses for both mAbs (2nd order for thermal and pH stresses and 0th order for shaking stress). Half-life of terminal charge variants of both mAbs was 2- to 8-fold less than main indicating increased degradation propensity.

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.

Structural Characterization of a Pathogenic Antibody Underlying Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT).

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but dangerous side effect of adenoviral-vectored COVID-19 vaccines. VITT had been linked to production of autoantibodies recognizing platelet factor 4 (PF4). Here, we characterize anti-PF4 antibodies obtained from a VITT patient's blood. Intact mass measurements indicate that a significant fraction of these antibodies represent a limited number of clones. MS analysis of large antibody fragments (the light chain and the Fc/2 and Fd fragments of the heavy chain) confirms the monoclonal nature of this component of the anti-PF4 antibodies repertoire and reveals the presence of a mature complex biantennary N-glycan within the Fd segment. Peptide mapping using two complementary proteases and LC-MS/MS was used to determine the amino acid sequence of the entire light chain and over 98% of the heavy chain (excluding a short N-terminal segment). The sequence analysis allows the monoclonal antibody to be assigned to the IgG2 subclass and verifies that the light chain belongs to the λ-type. Incorporation of enzymatic de-N-glycosylation into the peptide mapping routine allows the N-glycan in the Fab region of the antibody to be localized to the framework 3 region of the VH domain. This novel N-glycosylation site is the result of a single mutation within the germline sequence. Peptide mapping also provides information on lower-abundance (polyclonal) components of the anti-PF4 antibody ensemble, revealing the presence of all four subclasses (IgG1-IgG4) and both types of the light chain (λ and κ). This case study demonstrates the power of combining the intact, middle-down, and bottom-up MS approaches for meaningful characterization of ultralow quantities of pathogenic antibodies extracted directly from patients' blood.

An immunochromatographic strip sensor for rapid and sensitive detection of candesartan, olmesartan medoxomil, and irbesartan in herbal beverages.

Sartans, as a class of antihypertensive drugs, pose a threat to human health when illegally added to herbal beverages. It is crucial to detect sartans in herbal beverages. We have developed a highly sensitive monoclonal antibody against candesartan (CAN), olmesartan medoxomil (OLM), and irbesartan (IRB), with 50% inhibitory concentrations (IC50) that were obtained via indirect enzyme-linked immunosorbent assay (ic-ELISA) as 0.178 ng mL-1, 0.185 ng mL-1, and 0.262 ng mL-1 against CAN, OLM, and IRB, respectively. Based on this monoclonal antibody, we developed a rapid screening method for CAN, OLM, and IRB in herbal beverage samples using an immunochromatographic assay (ICA) strip. Test for 15 minutes after simple and rapid sample pre-treatment and the results of this method can be obtained through naked eye observation. The detection limits (LODs) of the ICA strip for CAN, OLM, and IRB in herbal beverage samples are lower than 0.15 ng mL-1, and the results of the ICA strip and ic-ELISA are consistent in spiked samples and recovery experiments. Therefore, this method can quickly, efficiently, and reliably achieve high-throughput on-site rapid detection of illegally added CAN, OLM, and IRB in herbal beverages.

Targeted CQA analytical control strategy for commercial antibody products: Replacing ion-exchange chromatography methods for charge heterogeneity with multi-attribute monitoring.

Peptide mapping with mass spectrometry (MS) is an important tool for protein characterization in the biopharmaceutical industry. Historically, peptide mapping monitors post-translational modifications (PTMs) of protein products and process intermediates during development. Multi-attribute monitoring (MAM) methods have been used previously in commercial release and stability testing panels to ensure control of selected critical quality attributes (CQAs). Our goal is to use MAM methods as part of an overall analytical testing strategy specifically focused on CQAs, while removing or replacing historical separation methods that do not effectively distinguish CQAs from non-CQAs due to co-elution. For example, in this study, we developed a strategy to replace a profile-based ion-exchange chromatography (IEC) method using a MAM method in combination with traditional purity methods to ensure control of charge variant CQAs for a commercial antibody (mAb) drug product (DP). To support this change in commercial testing strategy, the charge variant CQAs were identified and characterized during development by high-resolution LC-MS and LC-MS/MS. The charge variant CQAs included PTMs, high molecular weight species, and low molecular weight species. Thus, removal of the IEC method from the DP specification was achieved using a validated LC-MS MAM method on a QDa system to directly measure the charge variant PTM CQAs in combination with size exclusion chromatography (SE-HPLC) and capillary electrophoresis (CE-SDS) to measure the non-PTM charge variant CQAs. Bridging data between the MAM, IEC, and SE-HPLC methods were included in the commercial marketing application to justify removing IEC from the DP specification. We have also used this MAM method as a test for identity to reduce the number of QC assays. This strategy has received approvals from several health authorities.

Study of Monoclonal Antibody Aggregation at the Air-Liquid Interface under Flow by ATR-FTIR Spectroscopic Imaging.

Throughout bioprocessing, transportation, and storage, therapeutic monoclonal antibodies (mAbs) experience stress conditions that may cause protein unfolding and/or chemical modifications. Such structural changes may lead to the formation of aggregates, which reduce mAb potency and may cause harmful immunogenic responses in patients. Therefore, aggregates need to be detected and removed or ideally prevented from forming. Air-liquid interfaces, which arise during various stages of bioprocessing, are one of the stress factors causing mAb aggregation. In this study, the behavior of an immunoglobulin G (IgG) at the air-liquid interface was investigated under flow using macro attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic imaging. This chemically specific imaging technique allows observation of adsorption of IgG to the air-liquid interface and detection of associated secondary structural changes. Chemical images revealed that IgG rapidly accumulated around an injected air bubble under flow at 45 °C; however, no such increase was observed at 25 °C. Analysis of the second derivative spectra of IgG at the air-liquid interface revealed changes in the protein secondary structure associated with increased intermolecular ß-sheet content, indicative of aggregated IgG. The addition of 0.01% w/v polysorbate 80 (PS80) reduced the amount of IgG at the air-liquid interface in a static setup at 30 °C; however, this protective effect was lost at 45 °C. These results suggest that the presence of air-liquid interfaces under flow may be detrimental to mAb stability at elevated temperatures and demonstrate the power of ATR-FTIR spectroscopic imaging for studying the structural integrity of mAbs under bioprocessing conditions.

Reduction of monoclonal antibody viscosity using interpretable machine learning.

Early identification of antibody candidates with drug-like properties is essential for simplifying the development of safe and effective antibody therapeutics. For subcutaneous administration, it is important to identify candidates with low self-association to enable their formulation at high concentration while maintaining low viscosity, opalescence, and aggregation. Here, we report an interpretable machine learning model for predicting antibody (IgG1) variants with low viscosity using only the sequences of their variable (Fv) regions. Our model was trained on antibody viscosity data (>100 mg/mL mAb concentration) obtained at a common formulation pH (pH 5.2), and it identifies three key Fv features of antibodies linked to viscosity, namely their isoelectric points, hydrophobic patch sizes, and numbers of negatively charged patches. Of the three features, most predicted antibodies at risk for high viscosity, including antibodies with diverse antibody germlines in our study (79 mAbs) as well as clinical-stage IgG1s (94 mAbs), are those with low Fv isoelectric points (Fv pIs < 6.3). Our model identifies viscous antibodies with relatively high accuracy not only in our training and test sets, but also for previously reported data. Importantly, we show that the interpretable nature of the model enables the design of mutations that significantly reduce antibody viscosity, which we confirmed experimentally. We expect that this approach can be readily integrated into the drug development process to reduce the need for experimental viscosity screening and improve the identification of antibody candidates with drug-like properties.

Evaluation of the impact of antibody fragments on aggregation of intact molecules via size exclusion chromatography coupled with native mass spectrometry.

Aggregates are recognized as one of the most critical product-related impurities in monoclonal antibody (mAb)-based therapeutics due to their negative impact on the stability and safety of the drugs. So far, investigational efforts have primarily focused on understanding the causes and effects of mAb self-aggregation, including both internal and external factors. In this study, we focused on understanding mAb stability in the presence of its monovalent fragment, formed through hinge cleavage and loss of one Fab unit (referred to as "Fab/c"), a commonly observed impurity during manufacturing and stability. The Fab/c fragments were generated using a limited IgdE digestion that specifically cleaves above the IgG1 mAb hinge region, followed by hydrophobic interaction chromatographic (HIC) enrichment. Two IgG1 mAbs containing different levels of Fab/c fragments were incubated under thermally accelerated conditions. A method based on size exclusion chromatography coupled with native mass spectrometry (SEC-UV-native MS) was developed and used to characterize the stability samples and identified the formation of heterogeneous dimers, including intact dimer, mAb-Fab/c dimer, Fab/c-Fab/c dimer, and mAb-Fab dimer. Quantitative analyses on the aggregation kinetics suggested that the impact of Fab/c fragment on the aggregation rate of individual dimer differs between a glycosylated mAb (mAb1) and a non-glycosylated mAb (mAb2). An additional study of deglycosylated mAb1 under 25°C accelerated stability conditions suggests no significant impact of the N-glycan on mAb1 total aggregation rate. This study also highlighted the power of SEC-UV-native MS method in the characterization of mAb samples with regard to separating, identifying, and quantifying mAb aggregates and fragments.

Predicting deamidation and isomerization sites in therapeutic antibodies using structure-based in silico approaches.

Asparagine (Asn) deamidation and aspartic acid (Asp) isomerization are common degradation pathways that affect the stability of therapeutic antibodies. These modifications can pose a significant challenge in the development of biopharmaceuticals. As such, the early engineering and selection of chemically stable monoclonal antibodies (mAbs) can substantially mitigate the risk of subsequent failure. In this study, we introduce a novel in silico approach for predicting deamidation and isomerization sites in therapeutic antibodies by analyzing the structural environment surrounding asparagine and aspartate residues. The resulting quantitative structure-activity relationship (QSAR) model was trained using previously published forced degradation data from 57 clinical-stage mAbs. The predictive accuracy of the model was evaluated for four different states of the protein structure: (1) static homology models, (2) enhancing low-frequency vibrational modes during short molecular dynamics (MD) runs, (3) a combination of (2) with a protonation state reassignment, and (4) conventional full-atomistic MD simulations. The most effective QSAR model considered the accessible surface area (ASA) of the residue, the pKa value of the backbone amide, and the root mean square deviations of both the alpha carbon and the side chain. The accuracy was further enhanced by incorporating the QSAR model into a decision tree, which also includes empirical information about the sequential successor and the position in the protein. The resulting model has been implemented as a plugin named "Forecasting Reactivity of Isomerization and Deamidation in Antibodies" in MOE software, completed with a user-friendly graphical interface to facilitate its use.

Identification and quantification of chain-pairing variants or mispaired species of asymmetric monovalent bispecific IgG1 monoclonal antibody format using reverse-phase polyphenyl chromatography coupled electrospray ionization mass spectrometry.

Developing a knob-into-hole asymmetric bispecific IgG1 monoclonal antibody (mAb) poses manufacturing challenges due to the expression of chain pairing variants, also called mispaired species, in the desired product. The incorrect pairing of light and heavy chains could result in heterogeneous mispaired species of homodimers, heterodimers, light chain swapping, and low molecular weight species (LMWS). Standard chromatography, capillary electrophoretic, or spectroscopic methods poorly resolve these from the main variants. Here, we report a highly sensitive reverse-phase polyphenyl ultra-high-performance liquid chromatography (RP-UHPLC) method to accurately measure mispaired species of Duet mAb format, an asymmetric IgG1 bispecific mAb, for both process development and quality control analytical tests. Coupled with electrospray ionization mass spectrometry (ESI-MS), it enabled direct online characterization of mispaired species. This single direct assay detected diverse mispaired IgG-like species and LMWS. The method resolved eight disulfide bonds dissociated LMWS and three mispaired LMWS. It also resolved three different types of IgG-like mispaired species, including two homodimers and one heterodimer. The characterization and quantification simultaneously enabled the cell line selection that produces a lesser heterogeneity and lower levels of mispaired species with the desired correctly paired product. The biological activity assessment of samples with increased levels of these species quantified by the method exhibited a linear decline in potency with increasing levels of mispaired species in the desired product. We also demonstrated the utility of the technique for testing in-process intermediate materials to determine and assess downstream purification process capability in removing diverse mispaired IgG-like species and LMWS to a certain level during the downstream purification process. Our investigation demonstrates that adopting this method was vital in developing asymmetric bispecific mAb from the initial stage of cell line development to manufacturing process development. Therefore, this tool could be used in the control strategy to monitor and control mispaired species during manufacturing, thus improving the quality control of the final product.

How spray drying processing and solution composition can affect the mAbs stability in reconstituted solutions for subcutaneous injections. Part II: Exploring each protein stabilizer effect.

Despite extensive research in spray drying of biopharmaceuticals, identifying the optimal formulation composition and process conditions to minimize the various stresses a biopharmaceutical undergoes during this drying process. The current study extends previous research on investigating how spray drying processing and solution composition can affect the stability of monoclonal antibodies (mAbs) in reconstituted solutions for subcutaneous injections. The decoupling process stresses on a model mAb (mAb-A) compared to the effect of coupled spray-drying stresses revealed that excipients and protein concentration had a more pronounced effect on stabilizing mAb-A against shear and thermal/dehydration stresses than spray drying operating conditions. These results prompted the continuation of the study, with the aim to investigate in greater depth the effect of mAb-A concentration in the formulation designated to spray-drying and then the effect of type and the concentration of individual excipients (sugars, amino acids and surfactants). The outcomes of this investigation suggest that a general increase in the concentration of excipients, particularly surfactants, correlates with a reduction in aggregation and turbidity observed in the reconstituted spray-dried mAb-A powders. These results, contribute to the identification of a suitable composition for a spray-dried mAb-A powder that ensures robust stability of the protein in reconstituted solutions intended for subcutaneous injection. This valuable insight has important implications for advancing the development of pharmaceutical formulations with enhanced stability and efficacy.

The search for novel proline analogs for viscosity reduction and stabilization of highly concentrated monoclonal antibody solutions.

Administration of monoclonal antibodies (mAbs) is currently focused on subcutaneous injection associated with increased patient adherence and reduced treatment cost, leading to sustainable healthcare. The main bottleneck is low volume that can be injected, requiring highly concentrated mAb solutions. The latter results in increased solution viscosity with pronounced mAb aggregation propensity because of intensive protein-protein interactions. Small molecule excipients have been proposed to restrict the protein-protein interactions, contributing to reduced viscosity. The aim of the study was to discover novel compounds that reduce the viscosity of highly concentrated mAb solution. First, the chemical space of proline analogs was explored and 35 compounds were determined. Viscosity measurements revealed that 18 proline analogs reduced the mAb solution viscosity similar to or more than proline. The compounds forming both electrostatic and hydrophobic interactions with mAb reduced the viscosity of the formulation more efficiently without detrimentally effecting mAb physical stability. A correlation between the level of interaction and viscosity-reducing effect was confirmed with molecular dynamic simulations. Structure rigidity of the compounds and aromaticity contributed to their viscosity-reducing effect, dependent on molecule size. The study results highlight the novel proline analogs as an effective approach in viscosity reduction in development of biopharmaceuticals for subcutaneous administration.

Host cell protein networks as a novel co-elution mechanism during protein A chromatography.

Host cell proteins (HCPs) are process-related impurities of therapeutic proteins produced in for example, Chinese hamster ovary (CHO) cells. Protein A affinity chromatography is the initial capture step to purify monoclonal antibodies or Fc-based proteins and is most effective for HCP removal. Previously proposed mechanisms that contribute to co-purification of HCPs with the therapeutic protein are either HCP-drug association or leaching from chromatin heteroaggregates. In this study, we analyzed protein A eluates of 23 Fc-based proteins by LC-MS/MS to determine their HCP content. The analysis revealed a high degree of heterogeneity in the number of HCPs identified in the different protein A eluates. Among all identified HCPs, the majority co-eluted with less than three Fc-based proteins indicating a drug-specific co-purification for most HCPs. Only ten HCPs co-purified with over 50% of the 23 Fc-based proteins. A correlation analysis of HCPs identified across multiple protein A eluates revealed their co-elution as HCP groups. Functional annotation and protein interaction analysis confirmed that some HCP groups are associated with protein-protein interaction networks. Here, we propose an additional mechanism for HCP co-elution involving protein-protein interactions within functional networks. Our findings may help to guide cell line development and to refine downstream purification strategies.

Updates of the current strategies of labeling for N-glycan analysis.

This mini review summarizes the current methods used for screening N-glycosylation of glycoproteins, with a specific focus on therapeutic proteins and on techniques involving the release of N-glycans. With the continuous development of biopharmaceuticals, particularly monoclonal antibodies (mAbs), which are N-glycosylated proteins, monitoring has gained importance in recent decades. Glycosylation of therapeutic glycoproteins is considered a critical quality attribute because it can impact the efficacy and safety of these therapeutic drugs. The protocols and instrumentation have evolved with the advancement of technologies. Nowadays, methods are becoming increasingly robust, rapid, and sensitive. For the release of N-glycans, the most commonly used method is enzymatic release using PNGase F. The latter is discussed in light of the advent of rapid release that is now possible. The strategy for separating N-glycans using either liquid chromatography (LC) with hydrophilic interaction liquid chromatography (HILIC) chemistry or capillary electrophoresis will be discussed. The selection of the labeling agent is a crucial step in sample preparation for the analysis of released N-glycans. This review also discusses labeling agents that are compatible with and dependent on the separation and detection techniques employed. The emergence of multiplex labeling agents is also summarized. The latter enables the analysis of multiple samples in a single run, but it requires MS analysis.

High throughput multidimensional liquid chromatography approach for online protein removal and characterization of polysorbates and poloxamer in monoclonal antibody formulations.

The majority of commercially available monoclonal antibody (mAb) formulations are stabilized with one of three non-ionic surfactants: polysorbate 20 (PS20), polysorbate 80 (PS80), or poloxamer 188 (P188). All three surfactants are susceptible to degradation, which can result in functionality loss and subsequent protein aggregation or free fatty acid particle formation. Consequently, quantitative, and qualitative analysis of surfactants is an integral part of formulation development, stability, and batch release testing. Due to the heterogeneous nature of both polysorbates and poloxamer, online isolation of all the compounds from the protein and other excipients that may disturb the subsequent liquid chromatography with charged aerosol detection (LC-CAD) analysis poses a challenge. Herein, we present an analytical method employing LC-CAD, utilizing a combination of anion and cation exchange columns to completely remove proteins online before infusing the isolated surfactant onto a reversed-phase column. The method allows high throughput analysis of polysorbates within 8 minutes and poloxamer 188 within 12 minutes, providing a separation of the surfactant species of polysorbates (unesterified species, lower esters, and higher esters) and poloxamer 188 (early eluters and main species). Accuracy and precision assessed according to the International Council for harmonisation (ICH) guideline were 96 - 109 % and ≤1 % relative standard deviation respectively for all three surfactants in samples containing up to 110 mg/mL mAb. Subsequently, the method was effectively applied to quantify polysorbate 20 and polysorbate 80 in nine commercial drug products with mAb concentration of up to 180 mg/mL.

Mixed-mode size-exclusion silica resin for polishing human antibodies in flow-through mode.

The polishing step in the downstream processing of therapeutic antibodies removes residual impurities from Protein A eluates. Among the various classes of impurities, antibody fragments are especially challenging to remove due to the broad biomolecular diversity generated by a multitude of fragmentation patterns. The current approach to fragment removal relies on ion exchange or mixed-mode adsorbents operated in bind-and-gradient-elution mode. However, fragments that bear strong similarity to the intact product or whose biophysical features deviate from the ensemble average can elude these adsorbents, and the lack of a chromatographic technology enabling robust antibody polishing is recognized as a major gap in downstream bioprocessing. Responding to this challenge, this study introduces size-exclusion mixed-mode (SEMM) silica resins as a novel chromatographic adsorbent for the capture of antibody fragments irrespective of their biomolecular features. The pore diameter of the silica beads features a narrow distribution and is selected to exclude monomeric antibodies, while allowing their fragments to access the pores where they are captured by the mixed-mode ligands. The static and dynamic binding capacity of the adsorbent ranged respectively between 30-45 and 25-33 gs of antibody fragments per liter of resin. Selected SEMM-silica resins also demonstrated the ability to capture antibody aggregates, which adsorb on the outer layer of the beads. Optimization of the SEMM-silica design and operation conditions - namely, pore size (10 nm) and ligand composition (quaternary amine and alkyl chain) as well as the linear velocity (100 cm/h), ionic strength (5.7 mS/cm), and pH (7) of the mobile phase - afforded a significant reduction of both fragments and aggregates, resulting into a final antibody yield up to 80% and monomeric purity above 97%.

A validated LC-MS/MS method for the quantitation of daratumumab in rat serum using rapid tryptic digestion without IgG purification and reduction.

Daratumumab, a humanized monoclonal antibody utilized in treating immunoglobulin light-chain amyloidosis and relapsed/refractory multiple myeloma, was quantified in rat serum through a simple, economical and effective liquid chromatography tandem-mass spectrometry (LC-MS/MS) method. A surrogate peptide, LLIYDASNR, derived from trypsin hydrolysis, was quantitatively analyzed with LLIYDASN [13C6, 15N4] RAT as an internal standard. This corrected variations from sample pretreatment and mass spectrometry response, involving denaturation and trypsin hydrolysis in a two-step process lasting approximately 1 hour. Methodological validation demonstrated a linear range of 1 µg/mL to 1000 µg/mL in rat serum. Precision, accuracy, matrix effect, sensitivity, stability, selectivity, carryover, and interference met acceptance criteria. The validated LC-MS/MS approach was successfully applied to a pharmacokinetic study of daratumumab in rats at an intravenous dose of 15 mg/kg.

Identification of Surfactant Impact on a Monoclonal Antibody Characterization via HPLC-Separation Based and Biophysical Methods.

PURPOSE OR OBJECTIVE: Surfactants, including polysorbates and poloxamers, play a crucial role in the formulation of therapeutic proteins by acting as solubilizing and stabilizing agents. They help prevent protein aggregation and adsorption, thereby enhancing the stability of drug substance and products., However, it is important to note that utilizing high concentrations of surfactants in protein formulations can present significant analytical challenges, which can ultimately affect the product characterization. METHODS: In our study, we specifically investigated the impact of elevated surfactant concentrations on the characterization of monoclonal antibodies. We employed various analytical techniques including size-exclusion chromatography (SEC), capillary electrophoresis (CE-SDS), a cell based functional assay, and biophysical characterization. RESULTS: The findings of our study indicate that higher levels of Polysorbate 80 (PS-80) have adverse effects on the measured purity, biological activity, and biophysical characterization of biologic samples. Specifically, the elevated levels of PS-80 cause analytical interferences, which can significantly impact the accuracy and reliability of analytical studies. CONCLUSIONS: Our study results highlight a significant risk in analytical investigations, especially in studies involving the isolation and characterization of impurities. It is important to be cautious of surfactant concentrations, as they can become more concentrated during common sample manipulations like buffer exchange. Indeed, the research presented in this work emphasizes the necessity to evaluate the impact on analytical assays when there are substantial alternations in the matrix composition. By doing so, valuable insights can be gained regarding potential challenges associated with assay development and characterization of biologics with complex formulations.

Generation of novel anti-apoE monoclonal antibodies that selectively recognize apoE isoforms.

Apolipoprotein E (apoE) is a regulator of lipid metabolism, cholesterol transport, and the clearance and aggregation of amyloid ß in the brain. The three human apoE isoforms apoE2, apoE3, and apoE4 only differ in one or two residues. Nevertheless, the functions highly depend on the isoform types and lipidated states. Here, we generated novel anti-apoE monoclonal antibodies (mAbs) and obtained an apoE4-selective mAb whose epitope is within residues 110-117. ELISA and bio-layer interferometry measurements demonstrated that the dissociation constants of mAbs are within the nanomolar range. Using the generated antibodies, we successfully constructed sandwich ELISA systems, which can detect all apoE isoforms or selectively detect apoE4. These results suggest the usability of the generated anti-apoE mAbs for selective detection of apoE isoforms.

Multi-dimensional technology - Recent advances and applications for biotherapeutic characterization.

This review provides an overview of the latest advancements and applications in multi-dimensional liquid chromatography coupled with mass spectrometry (mD-LC-MS), covering aspects such as inter-laboratory studies, digestion strategy, trapping column, and multi-level analysis. The shift from an offline to an online workflow reduces sample processing artifacts, analytical variability, analysis time, and the labor required for data acquisition. Over the past few years, this technique has demonstrated sufficient maturity for application across a diverse range of complex products. Moreover, there is potential for this strategy to evolve into an integrated process analytical technology tool for the real-time monitoring of monoclonal antibody quality. This review also identifies emerging trends, including its application to new modalities, the possibility of evaluating biological activity within the mD-LC set-up, and the consideration of multi-dimensional capillary electrophoresis as an alternative to mD-LC. As mD-LC-MS continues to evolve and integrate emerging trends, it holds the potential to shape the next generation of analytical tools, offering exciting possibilities for enhanced characterization and monitoring of complex biopharmaceutical products.