Intermolecular Dearomative 1,2-Amination/Carbonylation via Nucleophilic Addition of Simple Amines to Arene π-Bonds.

The incorporation of the privileged amino functionality is of paramount importance in organic synthesis. In contrast to the well-developed amination methods for alkenes, the dearomative amination of arenes is largely underexplored due to the inherently inert reactivity of arene π-bonds and selectivity challenges. Herein, we report an intermolecular dearomative aminofunctionalization via direct nucleophilic addition of simple amines to chromium-bound arenes. This multicomponent 1,2-amination/carbonylation reaction enables rapid access to complicated alicyclic compounds containing amino and amide functionalities from benzene derivatives under CO-gas-free conditions, which also represents the first application of nitrogen-based nucleophiles in η6 -coordination-induced arene dearomatizations.

Mechanistic insights into inter-chain disulfide bond reduction of IgG1 and IgG4 antibodies.

Therapeutic monoclonal antibodies (mAbs), primarily immunoglobin G1 (IgG1) and IgG4 with an engineered CPPC motif in its hinge region, are predominant biologics. Inter-chain disulfide bonds of IgG mAbs are crucial to maintaining IgG integrity. Inter-chain disulfide bond-reduced low molecular weight (LMW) is considered as one of quality attributes of IgG drug substance and is observed in drug substance manufacturing. In this study, we demonstrate that IgG1 and IgG4 are susceptible to the reducing agent TCEP differently and they follow different pathways to form LMWs. Our study shows that IgG1 is more sensitive to TCEP than IgG4. Both therapeutic IgG1 and human blood plasma IgG1 follow a heavy-heavy-light chain (HHL) pathway, featured with HHL and HH as intermediate species. Human blood plasma IgG4 with a CPSC motif in its hinge region follows heavy-light chain (HL) pathway, featured with HL as the intermediate species. However, therapeutic IgG4 follows a hybrid pathway with the HL pathway as the primary and the HHL pathway as the secondary. These experimental observations are further explained using solvent accessibility of inter-chain disulfide bonds obtained from computational modeling and molecular dynamics simulations. Findings from this study provide mechanistic insights of LMW formation of IgG1 and IgG4, which suggest selection of IgG1 or IgG4 for bispecific antibodies and cysteine-based antibody-drug conjugates. KEY POINTS: • Experimentally discovered preferable disulfide bond reduction pathways between IgG1 and IgG4 antibodies, driven by the different solvent accessibilities of these disulfide bonds. • Computationally explained the solvent accessibility aided by molecular dynamics simulations. • Provided insights in developing robust biologics process and designing bispecific antibodies and cysteine-based antibody-drug conjugates.

Antibody disulfide bond reduction and recovery during biopharmaceutical process development-A review.

Antibody disulfide bond reduction has been a challenging issue in monoclonal antibody manufacturing. It could lead to a decrease of product purity and failure to meet the targeted product profile and/or specifications. More importantly, disulfide bond reduction could also impact drug safety and efficacy. Scientists across the industry have been examining the root causes and developing mitigation strategies to address the challenge. In recent years, with the development of high titer mammalian cell culture processes to meet the rapidly growing demand for antibody biopharmaceuticals, disulfide bond reduction has been observed more frequently. Thus, it is necessary to continue evolving the disulfide reduction mitigation strategies and developing novel approaches to maintain high product quality. Additionally, in recent years as more complex molecules (such as bispecific and trispecific antibodies) emerge, the molecular heterogeneity due to incomplete formation of the interchain disulfide bonds becomes a more imperative challenging issue. Given the disulfide reduction challenges that biotech industry is facing, in this review, we provide a comprehensive scientific summary of the root cause analysis of disulfide reduction during process development of antibody therapeutics, mitigation strategies and its potential remediated recovery based on published papers. First, this paper intends to highlight different aspects of the root cause for disulfide reduction. Secondly, to provide a broader understanding of the disulfide bond reduction in downstream process, this paper discusses disulfide bond reduction impact on product stability, associated analytical methods for disulfide bond reduction detection and characterization, process control strategies as well as their manufacturing implementation. In addition, brief perspectives on the development of future mitigation strategies are also reviewed, including platform alignment, mitigation strategy application for the emerging new modalities such as bispecific and trispecific antibodies as well as using machine learning to identify molecule susceptibility of disulfide bond reduction. The data in this review are originated from the published papers.

Continued insights into virus clearance validation across continuous capture chromatography.

Multi-column capture chromatography (MCC) has gained increased attention lately due to the significant economic and process-related advantages it offers compared to traditional batch mode chromatography. However, for wide adoption of this technology in the clinical and commercial space, it requires scalable models for viral validation. In this study, additional viral validation studies were conducted under cGLP guidelines to assess retro-(X-MuLV) and parvo-virus (minute virus of mice) clearance across twin-column continuous capture chromatography (CaptureSMB) to supplement work previously performed. A surrogate model was validated using standard batch mode chromatography equipment based on flow path modifications to mimic the loading strategy employed in CaptureSMB. In addition, aged resin was used in this surrogate format to assess the impact of resin lifetime on viral clearance during continuous capture operation. The impact of column loading was also explored to shed light on the viral clearance mechanisms of protein A chromatography in overloading conditions. The proposed approach greatly simplifies MCC virus validation studies, and provides a robust strategy for regulatory filing of continuous biomanufacturing processes.

Productivity improvement and charge variant modulation for intensified cell culture processes by adding a carboxypeptidase B (CpB) treatment step.

The goal of cell culture process intensification is to improve productivity while maintaining acceptable quality attributes. In this report, four processes, namely a conventional manufacturing Process A, and processes intensified by enriched N-1 seed (Process B), by perfusion N-1 seed (Process C), and by perfusion production (Process D) were developed for the production of a monoclonal antibody. The three intensified processes substantially improved productivity, however, the product either failed to meet the specification for charge variant species (main peak) for Process D or the production process required early harvest to meet the specification for charge variant species, Day 10 or Day 6 for Processes B and C, respectively. The lower main peak for the intensified processes was due to higher basic species resulting from higher C-terminal lysine. To resolve this product quality issue, we developed an enzyme treatment method by introducing carboxypeptidase B (CpB) to clip the C-terminal lysine, leading to significantly increased main peak and an acceptable and more homogenous product quality for all the intensified processes. Additionally, Processes B and C with CpB treatment extended bioreactor durations to Day 14 increasing titer by 38% and 108%, respectively. This simple yet effective enzyme treatment strategy could be applicable to other processes that have similar product quality issues.

Understanding the pathway and kinetics of aspartic acid isomerization in peptide mapping methods for monoclonal antibodies.

Isomerization of aspartic acid (Asp) in therapeutic proteins could lead to safety and efficacy concerns. Thus, accurate quantitation of various Asp isomerization along with kinetic understanding of the variant formations is needed to ensure optimal process development and sufficient product quality control. In this study, we first observed Asp-succinimide conversion in complementarity-determining regions (CDRs) Asp-Gly motif of a recombinant mAb through ion exchange chromatography, intact protein analysis by mass spectrometry, and LC-MS/MS. Then, we developed a specific peptide mapping method, with optimized sample digestion conditions, to accurately quantitate Asp-succinimide-isoAsp variants at peptide level without method-induced isomerization. Various kinetics of Asp-succinimide-isoAsp isomerization pathways were elucidated using 18O labeling followed by LC-MS analysis. Molecular modeling and molecular dynamic simulation provide additional insight on the kinetics of Asp-succinimide formation and stability of succinimide intermediate. Findings of this work shed light on the molecular construct and the kinetics of the formation of isoAsp and succinimide in peptides and proteins, which facilitates analytical method development, protein engineering, and late phase development for commercialization of therapeutic proteins.

Selective Capture and Recovery of Monoclonal Antibodies by Self-Assembling Supramolecular Polymers of High Affinity for Protein Binding.

The separation and purification of therapeutic proteins from their biological resources pose a great limitation for industrial manufacturing of biologics in an efficient and cost-effective manner. We report here a supramolecular polymeric system that can undergo multiple reversible processes for efficient capture, precipitation, and recovery of monoclonal antibodies (mAbs). These supramolecular polymers, namely immunofibers (IFs), are formed by coassembly of a mAb-binding peptide amphiphile with a rationally designed filler molecule of varying stoichiometric ratios. Under the optimized conditions, IFs can specifically capture mAbs with a precipitation yield greater than 99%, leading to an overall mAb recovery yield of 94%. We also demonstrated the feasibility of capturing and recovering two mAbs from clarified cell culture harvest. These results showcase the promising potential of peptide-based supramolecular polymers as reversible affinity precipitants for mAb purification.

Minimization of artifact protein aggregation using tetradecyl sulfate and hexadecyl sulfate in capillary gel electrophoresis under reducing conditions.

In the biopharmaceutical industry, CE-SDS assesses the purity, heterogeneity, and stability of therapeutic proteins. However, for mAb-1 and mAb-2, typical CE-SDS under reducing conditions produced atypical protein peak profiles, which led to biased purity results, thus were not acceptable for biologics manufacturing. This bias was caused by the formation of method-induced higher molecular weight artifacts, the levels of which correlated with protein concentration. Here we show that adding sodium tetradecyl and hexadecyl sulfates to the sample and the sieving gel buffer solutions was required to prevent formation of aggregate artifacts and to maintain detergent:protein uniformity, suggesting their importance during the sample preparation steps of heat denaturation and subsequent cooling as well as during capillary migration. For these proteins, we show that this uniformity was likely due to the ability of these detergents to bind proteins with markedly higher affinities compared to SDS. "CE-SCX S" methods (where CE-SCX S is CGE using detergent composed of a sodium sulfate head group and a hydrocarbon tail, with "CX " representing various tail lengths), were developed with a sodium tetradecyl sulfate sample buffer and a sodium hexadecyl sulfate containing sieving gel buffer that minimized artifacts and provided robust characterization and release results for mAb-1 and mAb-2.

Kinetic modeling of Chinese hamster ovary cell culture: factors and principles.

As a host for therapeutic protein expression, Chinese hamster ovary (CHO) cells are widely utilized in the mainstream biopharmaceutical industry. Cell culture process development plays an important role in transitioning laboratory research to manufacturing. Among different mathematic tools, kinetic modeling is commonly achieved through analyzing cell culture data to design process parameters, optimize media, and scale up bioreactors. In this review, we examine key factors for upstream process development, and summarize currently used kinetic modeling strategies. In addition, two original examples of kinetic modeling application optimizing cell culture performance are presented. A comprehensive understanding is provided for the kinetic modeling and its applications in cell culture process development.

Strategies for high-concentration drug substance manufacturing to facilitate subcutaneous administration: A review.

To achieve the high protein concentrations required for subcutaneous administration of biologic therapeutics, numerous manufacturing process challenges are often encountered. From an operational perspective, high protein concentrations result in highly viscous solutions, which can cause pressure increases during ultrafiltration. This can also lead to low flux during ultrafiltration and sterile filtration, resulting in long processing times. In addition, there is a greater risk of product loss from the hold-up volumes during filtration operations. From a formulation perspective, higher protein concentrations present the risk of higher aggregation rates as the closer proximity of the constituent species results in stronger attractive intermolecular interactions and higher frequency of self-association events. There are also challenges in achieving pH and excipient concentration targets in the ultrafiltration/diafiltration (UF/DF) step due to volume exclusion and Donnan equilibrium effects, which are exacerbated at higher protein concentrations. This paper highlights strategies to address these challenges, including the use of viscosity-lowering excipients, appropriate selection of UF/DF cassettes with modified membranes and/or improved flow channel design, and increased understanding of pH and excipient behavior during UF/DF. Additional considerations for high-concentration drug substance manufacturing, such as appearance attributes, stability, and freezing and handling are also discussed. These strategies can be employed to overcome the manufacturing process challenges and streamline process development efforts for high-concentration drug substance manufacturing.

A CFD model for predicting protein aggregation in low-pH virial inactivation for mAb production.

Significant amounts of soluble product aggregates were observed in the low-pH viral inactivation (VI) operation during an initial scale-up run for an immunoglobulin-G 4 (IgG4) monoclonal antibody (mAb IgG4-N1). Being earlier in development, a scale-down model did not exist, nor was it practical to use costly Protein A eluate (PAE) for testing the VI process at scale, thus, a computational fluid dynamics (CFD)-based high-molecular weight (HMW) prediction model was developed for troubleshooting and risk mitigation. It was previously reported that the IgG4-N1 molecules upon exposure to low pH tend to change into transient and partially unfolded monomers during VI acidification (i.e., VIA) and form aggregates after neutralization (i.e., VIN). Therefore, the CFD model reported here focuses on the VIA step. The model mimics the continuous addition of acid to PAE and tracks acid distribution during VIA. Based on the simulated low-pH zone (≤pH 3.3) profiles and PAE properties, the integrated low-pH zone (ILPZ) value was obtained to predict HMW level at the VI step. The simulations were performed to examine the operating parameters, such as agitation speed, acid addition rate, and protein concentration of PAE, of the pilot scale (50-200 L) runs. The conditions with predictions of no product aggregation risk were recommended to the real scale-up runs, resulted in 100% success rate of the consecutive 12 pilot-scale runs. This study demonstrated that the CFD-based HMW prediction model could be used as a tool to facilitate the scale up of the low-pH VI process directly from bench to pilot/production scale.

Technology outlook for real-time quality attribute and process parameter monitoring in biopharmaceutical development-A review.

Real-time monitoring of bioprocesses by the integration of analytics at critical unit operations is one of the paramount necessities for quality by design manufacturing and real-time release (RTR) of biopharmaceuticals. A well-defined process analytical technology (PAT) roadmap enables the monitoring of critical process parameters and quality attributes at appropriate unit operations to develop an analytical paradigm that is capable of providing real-time data. We believe a comprehensive PAT roadmap should entail not only integration of analytical tools into the bioprocess but also should address automated-data piping, analysis, aggregation, visualization, and smart utility of data for advanced-data analytics such as machine and deep learning for holistic process understanding. In this review, we discuss a broad spectrum of PAT technologies spanning from vibrational spectroscopy, multivariate data analysis, multiattribute chromatography, mass spectrometry, sensors, and automated-sampling technologies. We also provide insights, based on our experience in clinical and commercial manufacturing, into data automation, data visualization, and smart utility of data for advanced-analytics in PAT. This review is catered for a broad audience, including those new to the field to those well versed in applying these technologies. The article is also intended to give some insight into the strategies we have undertaken to implement PAT tools in biologics process development with the vision of realizing RTR testing in biomanufacturing and to meet regulatory expectations.

Real-time monitoring of quality attributes by in-line Fourier transform infrared spectroscopic sensors at ultrafiltration and diafiltration of bioprocess.

Technologies capable of monitoring product quality attributes and process parameters in real time are becoming popular due to the endorsement of regulatory agencies and also to support the agile development of biotherapeutic pipelines. The utility of vibrational spectroscopic techniques such as Fourier transform mid-infrared (Mid-IR) and multivariate data analysis (MVDA) models allows the prediction of multiple critical attributes simultaneously in real time. This study reports the use of Mid-IR and MVDA model sensors for monitoring of multiple attributes (excipients and protein concentrations) in real time (measurement frequency of every 40 s) at ultrafiltration and diafiltration (UF/DF) unit operation of biologics manufacturing. The platform features integration of fiber optic Mid-IR probe sensors to UF/DF set up at the bulk solution and through a flow cell at the retentate line followed by automated Mid-IR data piping into a process monitoring software platform with pre-loaded partial least square regression (PLS) chemometric models. Data visualization infrastructure is also built-in to the platform so that upon automated PLS prediction of excipients and protein concentrations, the results were projected in a graphical or numerical format in real time. The Mid-IR predicted concentrations of excipients and protein show excellent correlation with the offline measurements by traditional analytical methods. Absolute percent difference values between Mid-IR predicted results and offline reference assay results were ≤5% across all the excipients and the protein of interest; which shows a great promise as a reliable process analytical technology tool.

Metabolic understanding of disulfide reduction during monoclonal antibody production.

The disulfide reduction of intact monoclonal antibodies (mAbs) and subsequent formation of low molecular weight (LMW) species pose a direct risk to product stability, potency, and patient safety. Although enzymatic mechanisms of reduction are well established, an understanding of the cellular mechanisms during the bioreactor process leading to increased risk of disulfide reduction after harvest remains elusive. In this study, we examined bench, pilot, and manufacturing-scale batches of two mAbs expressed in Chinese hamster ovary (CHO) cells, where harvested cell culture fluid (HCCF) occasionally demonstrated disulfide reduction. Comparative proteomics highlighted a significant elevation in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels in a highly reducing batch of HCCF, compared to a non-reducing batch. Analysis during production cell culture showed that increased GAPDH gene and protein expression correlated to disulfide reduction risk in HCCF in every case examined. Additionally, glucose 6-phosphate dehydrogenase (G6PD) activity and an increased (≥ 300%) lactate/pyruvate molar ratio (lac/pyr) during production cell culture correlated to disulfide reduction risk, suggesting a metabolic shift to the pentose phosphate pathway (PPP). In all, these results suggest that metabolic alterations during cell culture lead to changes in protein expression and enzyme activity that in turn increase the risk of disulfide reduction in HCCF. KEY POINTS: • Bioreactor conditions resulted in reduction susceptible harvest material. • GAPDH expression, G6PD activity, and lac/pyr ratio correlated with mAb reduction. • Demonstrated role for cell metabolic changes in post-harvest mAb reduction. Graphical abstract.

Control of antibody high and low molecular weight species by depth filtration-based cell culture harvesting.

Depth filtration-based harvesting is widely used in mAb manufacturing to remove cell and process-related impurities. However, it has not been studied on control of product-related impurities, which are very critical for product quality. In this article, we studied the interactions of depth filter with high and low molecular weight species (HMWs and LMWs) for their direct removal from cell culture. The process parameters (filter, loading, temperature, and flux) were evaluated for adsorption of HMWs and LMWs by depth filters. The adsorption is significantly dependent on filter media and loading capacity and is mainly on the basis of hydrophobic interaction during harvesting. The HMW and LMW species were characterized as HMW1, HMW2, LMW1, and LMW2. The increasing binding from LMW2 to LMW1, HMW1, and HMW2 is correlated with their increasing hydrophobicity score. Adsorption using enriched HMW sample demonstrated similar total protein binding capacity (36-40 g/m2 ) between depth filters D0HC and X0HC. However, X0HC has stronger HMW binding than D0HC (71% vs 43% of bound protein), indicating more hydrophobic interaction in X0HC. HMW2 DBC on X0HC reached 12 g/m2 , similar to protein binding on hydrophobic interaction membrane adsorbers. Further study showed LMW can induce HMW formation. This study provides a critical understanding of HMW and LMW interaction with depth filters. The strategy of HMW and LMW control by depth filtration-based harvesting was implemented successfully in mAb manufacturing.

Model-assisted process characterization and validation for a continuous two-column protein A capture process.

In this study we introduce three process characterization approaches toward validation of continuous twin-column capture chromatography (CaptureSMB), referred to as "standard," "model assisted," and "hybrid." They are all based on a traditional risk-based approach, using process description, risk analysis, design-of-experiments (DoE), and statistical analysis as essential elements. The first approach, the "standard" approach uses a traditional experimental DoE to explore the design space of the high-ranked process parameters for the continuous process. Due to the larger number of process parameters in the continuous process, the DoE is extensive and includes a larger number of experiments than an equivalent DoE of a single column batch capture process. In the investigated case, many of the operating conditions were practically infeasible, indicating that the design space boundaries had been chosen inappropriately. To reduce experimental burden and at the same time enhance process understanding, an alternative "model assisted" approach was developed in parallel, employing a chromatographic process model to substitute experimental runs by computer simulations. Using the "model assisted" approach only experimental conditions that were feasible in terms of process yield constraints (>90%) were considered for statistical analysis. The "model assisted" approach included an optimization part that identified potential boundaries of the design space automatically. In summary, the "model assisted" approach contributed to increased process understanding compared to the "standard" approach. In this study, a "hybrid" approach was also used containing the general concepts of the "standard" approach but substituting a number of its experiments by computer simulations. The presented approaches contain essential elements of the Food and Drug Administration's process validation guideline.

Assessment of CE-based baseline disturbances using simulation and targeted experimental evaluation-impact on the purity determination of therapeutic proteins.

The baseline instability for capillary electrophoretic analysis is an intrinsic feature of the technique, which has not been thoroughly examined for its impact on therapeutic protein purity analysis with the capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) applications. For the particular CE-SDS application, this phenomenon was manifested through peak migration time shifts and sliding of the superimposed baseline profile. These dual phenomena are closely associated so that experimental assessment alone may not shed enough light to the underlying drivers. In the current study, both experimental and simulation approaches were employed to assess the systematic drifts. Computer simulation was used to decipher the two underlying factors and test their contributions toward purity and impurity peak determination inaccuracies. The data generated in this study demonstrated that the electrophoretic baseline disturbance had more pronounced impact on the purity data than the migration time shift. In addition, the potential contributing factors to the baseline disturbances were assessed experimentally which indicated that the source is related to thermal disruption during a sample run and the unique baseline patterns came from the background electrolytes. To improve data reproducibility for drug purity testing in the industrial setting and quality control (QC) environment, it is recommended to run shorter injection sequences including fewer samples and closely monitor the baseline drift for accurate integration. Those methods would help reduce the impact of systematic drift and disturbances. Graphical abstract.

Therapeutic protein purity and fragmented species characterization by capillary electrophoresis sodium dodecyl sulfate using systematic hybrid cleavage and forced degradation.

Positive identification of capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) electropherogram peaks provides information to understand protein molecular characteristics at the structural level. It is critical in the design of a robust assay that can accurately resolve, differentiate, and quantify all therapeutic protein components including fragmented species, which are considered as product related impurities. However, direct identification of the impurity peaks observed in CE-SDS is a challenging and oftentimes an ambiguous task. This paper proposed a systematic workflow for characterizing CE-SDS fragmentation peaks. Forced degradation of monoclonal antibody (mAb) by multiple stress methods was utilized to induce fragmentation and species enrichment. The characteristics, such as size and the clipped region of sequence, were then evaluated based on multiple enzymatic treatment and particle reduction. The identified fragments were further confirmed using tryptic digestion and liquid chromatography coupled with mass spectrometry (LC-MS) analysis. Common fragment sizes and clipping locations are identified after evaluating multiple IgG molecules. The methodology and procedure described in this article are readily deployable and will provide necessary information for method, process, and product characterizations. Graphical abstract.

DNA RETENTION ON DEPTH FILTERS.

Depth filtration is a commonly-used bioprocessing unit operation for harvest clarification that reduces the levels of process- and product-related impurities such as cell debris, host-cell proteins, nucleic acids and protein aggregates. Since depth filters comprise multiple components, different functionalities may contribute to such retention, making the mechanisms by which different impurities are removed difficult to decouple. Here we probe the mechanisms by which double-stranded DNA (dsDNA) is retained on depth filter media by visualizing the distribution of fluorescently-labeled retained DNA on spent depth filter discs using confocal fluorescence microscopy. The extent of DNA displacement into the depth filter was found to increase with decreasing DNA length with increasing operational parameters such as wash volume and buffer ionic strength. Finally, using 5ethynyl-2'-deoxyuridine (EdU) to label DNA in dividing CHO cells, we showed that Chinese hamster ovary (CHO) cellular DNA in the lysate supernatant migrates deeper into the depth filter than the lysate re-suspended pellet, elucidating the role of the size of the DNA in its form as an impurity. Apart from aiding DNA purification and removal, our experimental approaches and findings can be leveraged in studying the transport and retention of nucleic acids and other impurities on depth filters at a small scale.

Purity Determination by Capillary Electrophoresis Sodium Hexadecyl Sulfate (CE-SHS): A Novel Application For Therapeutic Protein Characterization.

Capillary gel electrophoresis using sodium dodecyl sulfate (CE-SDS) is used commercially to provide quantitative purity data for therapeutic protein characterization and release. In CE-SDS, proteins are denatured under reducing or nonreducing conditions in the presence of SDS and electrophoretically separated by molecular weight and hydrodynamic radius through a sieving polymer matrix. Acceptable performance of this method would yield protein peaks that are baseline resolved and symmetrical. Nominal CE-SDS conditions and parameters are not optimal for all therapeutic proteins, specifically for Recombinant Therapeutic Protein-1 (RTP-1), where acceptable resolution and peak symmetry were not achieved. The application of longer alkyl chain detergents in the running buffer matrix substantially improved assay performance. Matrix running buffer containing sodium hexadecyl sulfate (SHS) increased peak resolution and plate count 3- and 8-fold, respectively, compared to a traditional SDS-based running gel matrix. At Bristol-Myers Squibb (BMS), we developed and qualified a viable method for the characterization and release of RTP-1 using an SHS-containing running buffer matrix. This work underscores the potential of detergents other than SDS to enhance the resolution and separation power of CE-based separation methods.