Comprehensive Analysis of Photoreceptor Outer Segments: Flow Cytometry Characterization and Stress-Driven Impact on Retinal Pigment Epithelium Phagocytosis.

Phagocytosis is one of the key functions of retinal pigment epithelium (RPE) cells, which maintain photoreceptor health by removing photoreceptor outer segments (POSs) that are regularly shed. A deficiency in RPE function to phagocytose POSs may lead to vision loss in inherited retinal diseases and eventually to age-related macular degeneration (AMD) with geographic atrophy. Significant progress has been made in the field of cell replacement therapy for AMD using stem-cell-derived RPE. To test their function, RPE cells are incubated with purified bovine POSs for the demonstration of efficient binding, internalization, and digestion of POSs. Here, we present an image-based method to measure phagocytosis activity by using POSs labeled with a pH-sensitive fluorescent dye, which has low fluorescence at neutral pH outside of the cell and high fluorescence at low pH inside the phagosome. Further, we introduce a unique flow-cytometry-based method for the characterization of POSs by measuring specific markers for POSs such as rhodopsin and opsin. Using this method, we demonstrated a comparable quality of several bovine POS isolation batches and a reliable assessment of POS quality on RPE phagocytosis assay performance when subjected to different stress conditions. This work provides new tools to characterize POSs and insight into RPE phagocytosis assay development for the functional evaluation of RPE cells in the field of cell replacement therapy.

Global intercompany assessment of ICIEF platform comparability for the characterization of therapeutic proteins.

An international team spanning 19 sites across 18 biopharmaceutical and in vitro diagnostics companies in the United States, Europe, and China, along with one regulatory agency, was formed to compare the precision and robustness of imaged CIEF (ICIEF) for the charge heterogeneity analysis of the National Institute of Standards and Technology (NIST) mAb and a rhPD-L1-Fc fusion protein on the iCE3 and the Maurice instruments. This information has been requested to help companies better understand how these instruments compare and how to transition ICIEF methods from iCE3 to the Maurice instrument. The different laboratories performed ICIEF on the NIST mAb and rhPD-L1-Fc with both the iCE3 and Maurice using analytical methods specifically developed for each of the molecules. After processing the electropherograms, statistical evaluation of the data was performed to determine consistencies within and between laboratory and outlying information. The apparent isoelectric point (pI) data generated, based on two-point calibration, for the main isoform of the NIST mAb showed high precision between laboratories, with RSD values of less than 0.3% on both instruments. The SDs for the NIST mAb and the rhPD-L1-Fc charged variants percent peak area values for both instruments are less than 1.02% across different laboratories. These results validate the appropriate use of both the iCE3 and Maurice for ICIEF in the biopharmaceutical industry in support of process development and regulatory submissions of biotherapeutic molecules. Further, the data comparability between the iCE3 and Maurice illustrates that the Maurice platform is a next-generation replacement for the iCE3 that provides comparable data.

Multi-dimensional LC-MS: the next generation characterization of antibody-based therapeutics by unified online bottom-up, middle-up and intact approaches.

Accelerated development of new therapeutics in an increasingly competitive landscape requires the use of high throughput analytical platforms. In addition, the complexity of novel biotherapeutic formats (e.g. fusion proteins, protein-polymer conjugates, co-formulations, etc.) reinforces the need to improve the selectivity and resolution of conventional one-dimensional (1D) liquid chromatography (LC). Liquid chromatography-mass spectrometry (LC-MS)-based technologies such as native LC-MS for intact mass analysis or peptide mapping (also called bottom-up approach)-based multi-attribute methods (MAM) have already demonstrated their potential to complement the conventional analytical toolbox for monoclonal antibody (mAb) characterization. Two-dimensional liquid-chromatography (2D-LC-MS) methods have emerged in the last ten years as promising approaches to address the increasing analytical challenges faced with novel antibody formats. However, off-line sample preparation procedures are still required for conventional 1D and 2D-LC-MS methods for the in-depth variant characterization at the peptide level. Multi-dimensional LC-MS (mD-LC-MS) combine sample preparation and multi-level (i.e. intact, reduced, middle-up and peptide) analysis within the same chromatographic set-up. This review presents an overview of the benefits and limitations of mD-LC-MS approaches in comparison to conventional chromatographic methods (i.e. 1D-LC-UV methods at intact protein level and 1D-LC-MS methods at peptide level). The current analytical trends in antibody characterization by mD-LC-MS approaches, beyond the 2D-LC-MS workhorse, are also reviewed, and our vision on a more integrated multi-level mD-LC-MS characterization platform is shared.

Targeted Bottom-up Characterization of Recombinant Monoclonal Antibodies by Multidimensional LC/MS.

On-line bottom-up approaches have recently emerged as promising alternatives to standard off-line processes for characterizing post-translational modifications (PTMs) of therapeutic monoclonal antibodies (mAbs). The benefits of on-line processing include reductions in required sample amount and sample handling, as well as reducing the overall turnaround time. However, shortening digestion time for the on-line approach of an intact mAb can cause incomplete peptide cleavages, leading to low sequence coverage and poor repeatability of analyses. For the first time, we describe a novel, automated targeted bottom-up strategy consisting of reducing the complexity of intact mAb by digesting the product into small ∼25 kDa fragments, followed by an on-line peptide mapping analysis of each fragment. For this purpose, a four-dimensional-liquid chromatography/mass spectrometry (4D-LC/MS) method was developed using an immobilized IdeS-high-performance liquid chromatography (HPLC) column as a first dimension (1D) for on-line digestion, followed by a (2D) on-column reversed-phase liquid chromatography (RPLC) for reduction and fragments separation. Then, only one fragment was selected for digestion using a (3D) immobilized trypsin cartridge and, finally, the obtained peptides were analyzed by (4D) RPLC-MS. This strategy considerably improved the on-line digestion efficiency with higher sequence coverages (LC and HC >97%), thus allowing various PTMs including oxidation, deamidation, and isomerization located in the complementarity-determining regions (CDRs), as well as N-glycans present on the Fc/2 fragment, to be monitored with similar sensitivity to those obtained with standard off-line approaches. Additional investigations at a middle-up level were also performed via a three-dimensional-LC/MS (3D-LC/MS) approach within the same system, demonstrating the feasibility to achieve a multilevel comprehensive characterization of mAbs.

Development of a 3D-LC/MS Workflow for Fast, Automated, and Effective Characterization of Glycosylation Patterns of Biotherapeutic Products.

Glycosylation is a common post-translational modification of therapeutic monoclonal antibodies produced in mammalian cells and is considered an important critical quality attribute (CQA), as it is known to impact efficacy, stability, half-life, and immunogenicity. For these reasons, glycosylation requires characterization and close monitoring during the manufacturing process. Due to the complexity of the glycosylation patterns, sophisticated analytical tools with high resolving power are required for the characterization of the glycoforms. This study describes, for the first time, the development and use of an online three-dimensional high-performance liquid chromatography/mass spectrometry (3D-HPLC/MS) approach for the monitoring of glycosylation patterns at the middle-up level. An immobilized IdeS-enzyme column was used in the first dimension for the digestion of mAbs in 10 min. Then, following an online reversed phase liquid chromatography (RPLC) column reduction, the ≈25 kDa proteolytic fragments were analyzed using hydrophilic interaction chromatography (HILIC) coupled to MS. This novel analytical workflow demonstrated the ability to accurately profile glycosylated variants within a total run time of 95 min. To compare the performance of this analytical strategy with a conventional offline procedure (IdeS digestion x reduction-HILIC/MS), a proof of concept study using two mAbs is described here.

Fast and Automated Characterization of Monoclonal Antibody Minor Variants from Cell Cultures by Combined Protein-A and Multidimensional LC/MS Methodologies.

Monitoring of post-translational modifications (PTMs) in therapeutic monoclonal antibodies (mAbs) is essential during their production in both upstream and downstream processes. However, characterization of PTMs using a conventional peptide mapping procedure requires time-consuming and labor-intensive offline sample preparation steps. This work describes for the first time, the implementation of a Protein-A affinity chromatography column as the first dimension (1D) in a multidimensional LC (3D and 4D) setup for the automated characterization of mAb variants from harvest cell culture fluid (HCCF) materials at different purification/production steps. A 4D-LC/MS method (Protein-A-Reduction-RPLC-Digestion-RPLC/MS) was developed to determine PTM levels including oxidation, deamidation, and succinimide formation by online peptide mapping analysis. To obtain an accurate and comprehensive profiling of mAb glycosylation patterns at the reduced level, a 3D-LC/MS method (Protein-A-Reduction-RPLC-HILIC/MS) was also developed on the same chromatographic system. Overall, the full workflow (data acquisition and analysis) for both 3D and 4D-LC/MS setups can be completed within less than 1-2 days, compared to weeks with the conventional manual approach. This proof of concept study demonstrates that mD-LC/MS has the potential to be used as a powerful tool to perform a fast and reliable monitoring of PTMs during the manufacturing process for both bioreactor control or as a monitoring assay.

Streamlined Characterization of an Antibody-Drug Conjugate by Two-Dimensional and Four-Dimensional Liquid Chromatography/Mass Spectrometry.

This study describes the use of a multidimensional HPLC (2D and 4D) system for a faster and more effective characterization of an antibody-drug conjugate (ADC) product, compared to the standard off-line approach of fraction collection and off-line variant characterization. The size variants of an interchain cysteine-linked ADC were characterized to understand the effect of the different drug-to-antibody ratio (DAR) species on aggregate formation. For this purpose, the ADC product and a full panel of stressed samples were analyzed. The dimeric ADC species were baseline resolved from the main peak (Rs = 2.7) by UHP-SEC (ultra-high-performance size exclusion chromatography) under nondenaturing conditions using a buffered mobile phase containing 5% 2-propanol. A 2D-LC (SEC-HIC) method was then developed to compare the average DAR values of the main peak species vs the aggregates. A 4D-LC/MS method (SEC-reduction-digestion-RPHPLC) was also developed to determine levels of potential critical quality attributes (pCQAs) including aggregation, average DAR, oxidation, and deamidation, in a 2 h run. An average DAR value of 3.5-3.6 was found for the main peak using both 2D-LC and 4D-LC methods, and these values were consistent with DAR determined by the in-house reference hydrophobic interaction chromatography (HIC) method. The multidimensional LC approaches also showed an increase in the content of high-DAR species in the SEC fractions containing the aggregates. Overall the entire workflow of data acquisition is completed within a day using the multidimensional on-line approach, in comparison to multiple days required with the traditional off-line approaches.

Characterization of oxidative carbonylation on recombinant monoclonal antibodies.

In the biotechnology industry, oxidative carbonylation as a post-translational modification of protein pharmaceuticals has not been studied in detail. Using Quality by Design (QbD) principles, understanding the impact of oxidative carbonylation on product quality of protein pharmaceuticals, particularly from a site-specific perspective, is critical. However, comprehensive identification of carbonylation sites has so far remained a very difficult analytical challenge for the industry. In this paper, we report for the first time the identification of specific carbonylation sites on recombinant monoclonal antibodies with a new analytical approach via derivatization with Girard's Reagent T (GRT) and subsequent peptide mapping with high-resolution mass spectrometry. Enhanced ionization efficiency and high quality MS(2) data resulted from GRT derivatization were observed as key benefits of this approach, which enabled direct identification of carbonylation sites without any fractionation or affinity enrichment steps. A simple data filtering process was also incorporated to significantly reduce false positive assignments. Sensitivity and efficiency of this approach were demonstrated by identification of carbonylation sites on both unstressed and oxidized antibody bulk drug substances. The applicability of this approach was further demonstrated by identification of 14 common carbonylation sites on three highly similar IgG1s. Our approach represents a significant improvement to the existing analytical methodologies and facilitates extended characterization of oxidative carbonylation on recombinant monoclonal antibodies and potentially other protein pharmaceuticals in the biotechnology industry.

Development and Qualification of Analytical Methods to Support Low Concentration Drug Product in-use Studies.

The emergence of highly potent therapeutics with low expected clinical doses creates a challenge for analytical characterization of simulated drug product in-use samples. The low expected protein concentration (often µg/mL) and highly charged and sub-optimal sample matrices like 0.9% saline or 5% dextrose make ensuring dose solution stability and characterizing product quality changes difficult. Health authority expectations require analysis of low concentration in-use samples to be completed with suitable assays to ensure little to no changes are occurring during drug product dose preparation and administration, thus ensuring patient safety. However, characterization of these samples for protein concentration, size variants, charge variants and potency often necessitates additional analytical method development to improve sensitivity and compatibility with in-use samples. Here we report the development and qualification of reliable in-use methods to characterize simulated in-use samples to assist during drug product development.

Comprehensive UHPLC- and CE-based methods for engineered Cas9 characterization.

CRISPR (clustered regularly interspaced short palindromic repeats)-associated proteins (Cas) are powerful gene-editing tools used in therapeutic applications. Efforts to minimize off-target cleavage by CRISPR-Cas9 have motivated the development of engineered Cas9 variants. The wild-type (WT) Streptococcus pyogenes (SpCas9) has been engineered into a high-fidelity Cas9 (SpyFi Cas9) that shows promising results in providing high on-target activity (targeting efficiency) while reducing off-target editing (unwanted mutations). This work describes for the first time the development of ultra-high-performance liquid chromatography (UHPLC) and capillary electrophoresis (CE)-based methods for a full characterization of different engineered Cas9 variants, including determination of purity, size variants, isoelectric points (pI), post-translational modifications (PTMs), and functional activities. The purity and size variant characterization were first determined by CE-sodium dodecyl sulfate (SDS). An in vitro DNA cleavage assay using an automated electrophoresis tool was employed to investigate the functional activity of ribonucleoprotein (RNP) complexes derived from Cas9 variants. The pIs of the engineered Cas9 proteins were determined by imaged capillary isoelectric focusing (icIEF), while intact mass measurements were performed by reversed-phase (RP)-UHPLC coupled with high-resolution mass spectrometry (HRMS). A peptide mapping assay based on LC-UV-MS/MS using endoproteinase Lys-C under non-reducing conditions was developed to confirm amino acid sequences, allowing differentiation of SpyFi Cas9 from WT SpCas9. The potential of using a low-resolution MS detector, especially for a GMP environment, as a low-cost and simple method to identify SpyFi Cas9 is discussed.

Monitoring multiple quality attributes of a complex Fc-fusion protein during cell culture production processes by mD-LC-MS peptide mapping.

Fc-fusion proteins represent a successful class of biopharmaceutical products. They are considered highly heterogeneous products due to the common degradation of amino acids that occurs during their production in upstream and downstream processes (e.g., oxidation and deamidation) and, above all, their complex glycosylation profile. Multi-dimensional liquid chromatography-mass spectrometry (mD-LC-MS) has recently gained much interest for process analytical technology, enabling the integration of this analytical technology in production and purification environments. In this study, an online mD-LC-MS/MS peptide mapping method was developed for monitoring multiple quality attributes, including the N-glycosylation state of a complex Fc-fusion protein, which is made by combining two heavily glycosylated cytokines with an Fc domain. This fully automated workflow includes sample purification, reduction, digestion, peptide mapping, and subsequent mass spectrometric analysis. Two immobilized enzyme cartridges based on trypsin and Lys-C protease were employed to generate a detailed glycosylation mapping, as trypsin allowed the identification of only one of four glycosylation sites, while Lys-C was more informative for two other sites. Site-specific glycosylation information such as antennarity, sialyation, and core fucosylation state was also determined. In addition to glycans, other post-translational modifications could be monitored simultaneously during the cell culture production processes by the mD-LC-MS/MS approach. In summary, the generated data demonstrate the applicability of mD-LC-MS for the monitoring and trending of multiple attributes for complex antibody formats over production processes in an automated and fast manner, compared to the complex and time-consuming traditional offline assays.

Real-time monitoring of antibody quality attributes for cell culture production processes in bioreactors via integration of an automated sampling technology with multi-dimensional liquid chromatography mass spectrometry.

Online monitoring of quality attributes (QAs) directly within the bioreactor can provide the basis for advanced modes of protein production including process intensification, smart manufacturing, and real-time release testing. The development of technologies to enable monitoring of QAs has been highly challenging due to the relative immaturity of commercial technologies for online analysis, generally low abundance of the attributes requiring highly specialized analytics not always amenable to automation, and the significant burden on development organizations to demonstrate the comparability and suitability of the online technologies resulting in low investment interest. In this study, we present for the first time a fully automated and highly flexible method for direct monitoring of QAs from the bioreactor. The method combines an automated sampling system and multi-dimensional (mD) LC-MS/MS technology to provide a means of quantifying post-translational modifications (PTMs) during the cell culture process and making real-time process decisions based on the resulting peptide mapping data. In doing so, a wide variety of PTMs can be identified and quantified including, but not limited to, oxidation, succinimidation, deamidation, isomerization, and glycosylation. The potential of this analytical workflow for the monitoring and trending of multiple attributes during cell culture production processes was first demonstrated with a standard IgG1 antibody over the production process. Then, the online workflow was applied to a complex format Fc-fusion protein to monitor sialylation. The ability to monitor sialylation offers a unique opportunity to develop process control schemes to ensure the final product meets quality specifications, showing the potential of this workflow in the context of online process analytical technology (PAT).

Extending the performance of FcRn and FcγRIIIa affinity liquid chromatography for protein biopharmaceuticals.

Affinity liquid chromatography using FcRn and FcγRIIIa columns can provide important information on the drug effector functions and the unique PK/PD properties of therapeutic mAbs. In this study, we propose a unique strategy to improve the performance of affinity chromatography by applying pH-gradient programs that incorporate multi-isocratic and negative gradient segments. These alternative gradient programs are known to greatly improve the separation of large solutes that follow a "bind-and-elute" type retention behavior. First, judicious optimization of the mobile phase compositions was performed to obtain a linear pH response. Then, with the developed strategy using multi-isocratic analysis conditions, the FcRn affinity separation selectivity for the analysis of oxidized mAb species was greatly improved. Furthermore, the introduction of negative gradient segments after each eluted peak improved the resolution between multiple glycosylated mAb species on the FcγRIIIa column. Therefore, this work provides a new strategy to improve the performance of affinity chromatography with mAb species, and could assist in the development of more accurate binding assays for important critical quality attributes related to FcRn and FcγRIIIa binding.

Identification of human urinary biomarkers of cruciferous vegetable consumption by metabonomic profiling.

Consumption of cruciferous vegetables (CVs) is inversely correlated to many human diseases including cancer (breast, lung, and bladder), diabetes, and cardiovascular and neurological disease. Presently, there are no readily measurable biomarkers of CV consumption and intake of CVs has relied on dietary recall. Here, biomarkers of CV intake were identified in the urine of 20 healthy Caucasian adult males using (1)H NMR spectroscopy with multivariate statistical modeling. The study was separated into three phases of 14 days: a run-in period with restricted CV consumption (phase I); a high CV phase where participants consumed 250 g/day of both broccoli and Brussels sprouts (phase II); a wash-out phase with a return to restricted CV consumption (phase III). Each study participant provided a complete cumulative urine collection over 48 h at the end of each phase; a spot urine (U0), 0-10 h (U0-10), 10-24 h (U10-24), and 24-48 h (U24-48) urine samples. Urine samples obtained after consumption of CVs were differentiated from low CV diet samples by four singlet (1)H NMR spectroscopic peaks, one of which was identified as S-methyl-l-cysteine sulfoxide (SMCSO) and the three other peaks were tentatively identified as other metabolites structurally related to SMCSO. These stable urinary biomarkers of CV consumption will facilitate future assessment of CVs in nutritional population screening and dietary intervention studies and may correlate to population health outcomes.

Inter-laboratory study to evaluate the performance of automated online characterization of antibody charge variants by multi-dimensional LC-MS/MS.

An international study was conducted to evaluate the performance and reliability of an online multi-dimensional (mD)-LC-MS/MS approach for the characterization of antibody charge variants. The characterization of antibody charge variants is traditionally performed by time-consuming, offline isolation of charge variant fractions by ion exchange chromatography (IEC) that are subsequently subjected individually to LC-MS/MS peptide mapping. This newly developed mD-LC-MS/MS approach enables automated and rapid characterization of charge variants using much lower sample requirements. This online workflow includes sample reduction, digestion, peptide mapping, and subsequent mass spectrometric analysis within a single, fully-automated procedure. The benefits of using online mD-LC-MS/MS for variant characterization include fewer handling steps, a more than 10-fold reduction in required sample amount, reduced sample hold time as well as a shortening of the overall turnaround time from weeks to few days compared to standard offline procedures. In this site-to-site comparison study, we evaluated the online peptide mapping data collected from charge variants of trastuzumab (Herceptin®) across three international laboratories. The purpose of this study was to compare the overall performance of the online mD-LC-MS/MS approach for antibody charge variant characterization, with all participating sites employing different mD-LC-MS/MS setups (e.g., instrument vendors, modules, columns, CDS software). The high sequence coverage (95%-97%) obtained in each laboratory, enabled a reproducible generation of tryptic peptides and the comparison of values of the charge variants. Results obtained at all three participating sites were in good agreement, highlighting the reliability and performance of this approach, and correspond with data gained by the standard offline procedure. Overall, our results underscore of the benefit mD-LC-MS/MS technology for therapeutic antibody characterization, confirming its potential to become an important tool in the toolbox of protein characterization scientists.

Discovery of a dual pathway aggregation mechanism for a therapeutic constrained peptide.

Constrained peptides (CPs) have emerged as attractive candidates for drug discovery and development. To fully unlock the therapeutic potential of CPs, it is crucial to understand their physical stability and minimize the formation of aggregates that could induce immune responses. Although amyloid like aggregates have been researched extensively, few studies have focused on aggregates from other peptide scaffolds (e.g., CPs). In this work, a streamlined approach to effectively profile the nature and formation pathway of CP aggregates was demonstrated. Aggregates of various sizes were detected and shown to be amorphous. Though no major changes were found in peptide structure upon aggregation, these aggregates appeared to have mixed natures, consisting of primarily non-covalent aggregates with a low level of covalent species. This co-existence phenomenon was also supported by two kinetic pathways observed in time- and temperature-dependent aggregation studies. Furthermore, a stability study with 8 additional peptide variants exhibited good correlation between aggregation propensity and peptide hydrophobicity. Therefore, a dual aggregation pathway was proposed, with the non-covalent aggregates driven by hydrophobic interactions, whereas the covalent ones formed through disulfide scrambling. Overall, the workflow presented here provides a powerful strategy for comprehensive characterization of peptide aggregates and understanding their mechanisms of formation.

Liquid chromatography-mass spectrometry methods for urinary biomarker detection in metabonomic studies with application to nutritional studies.

The effects of sample preparation and chromatographic method differences on the classification and recovery of metabolic biomarkers from UPLC-MS measurements on urine samples of humans exposed to different dietary interventions have been investigated. Eight volunteers consumed three high-fat meals (rich in saturated, monounsaturated and polyunsaturated fatty acids, respectively) in randomized order with a washout period in between. For each participant, urine samples were obtained prior to and at three timed intervals after each meal. Samples were processed either by dilution (1 : 4) or by liquid-liquid extraction and then run under two different gradient conditions. For each analysis method, a total of 96 observations (eight participants, four time points, three diets) were measured. The total ion count chromatograms were analyzed using partial-least-squares discriminant analysis. All three dietary classes could be discriminated irrespective of sample preparation and chromatographic method. However, the main discriminating metabolites varied according to sample preparation, indicating that sample treatment and chromatographic conditions influence the ability to extract biomolecular information. Diluted samples showed higher m/z compounds (ca 400 u) while liquid-liquid extraction samples showed low m/z at the same retention time span. Optimized methods for metabolite identification (e.g. organic acids) were statistically inferior to global screening for mixed compound identification, confirming that multiple compound class-based metabolic profiles are likely to give superior metabonomic (diagnostic) classification, although great care has to be taken in the interpretation in relation to matrix effects.

Automated middle-up approach for the characterization of biotherapeutic products by combining on-line hinge-specific digestion with RPLC-HRMS analysis.

The development of biotherapeutic proteins requires the use of efficient analytical methods to support their manufacturing process and quality control (QC). Analytical approaches at intact and middle-up levels emerged as promising alternatives to bottom-up strategies for protein characterization as they require less sample amount and simplified sample handling, thus reducing the overall turn-around time. This study describes, for the first time, the development of an automated liquid chromatography-mass spectrometry (LC-MS) workflow comprised of an immobilized IdeS-HPLC column for on-line sample digestion, followed by a reversed-phase liquid chromatography (RPLC) for protein subunit separation, and a high-resolution MS for molecular weight analysis. A proof of concept study was described here for the characterization of a therapeutic mAb and a bsAb. For the mAb, this automated workflow enabled rapid characterization of post-translational modifications (PTMs) such as N-glycosylation, glycation and N-terminal lysine. For the bsAb, the same workflow was successfully employed to identify product impurities due to chain pairing. The sample analysis using this workflow can be accomplished within less than one day. This workflow demonstrated its potential as a multi-attribute method for characterization of therapeutic proteins.

Development of an innovative salt-mediated pH gradient cation exchange chromatography method for the characterization of therapeutic antibodies.

The successful application of monoclonal antibodies (mAb) in oncology and autoimmune diseases paved the way for the development of therapeutic antibodies with a wider range of structural and physico-chemical properties. A pH-gradient combining 2-(N-morpholino)ethanesulfonic (MES) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffers and mediated with potassium chloride was developed to sufficiently retain acidic mAbs (pI < 7) in cation exchange chromatography (CEX), while keeping suitable separation performance for basic mAbs (pI > 7). Firstly, the MES and HEPES buffers were individually evaluated in their useful pH range by applying a salt gradient. The performance of a salt-mediated pH gradient combining the MES and HEPES buffers was then compared to a commercial pH gradient kit. The developed conditions were found superior to the salt-gradient approaches and provided a useful alternative to commercial pH gradient kits. In this study, the developed conditions were applied to separate a bispecific antibody (BsAb) from its two parental mAbs.

Immunoaffinity LC-MS/MS is more suitable than ELISA to quantify a PEGylated molecule in cynomolgus monkey serum.

Background: Polyethylene glycolylation (PEGylation) technology is a long-acting delivery platform used to increase the half-life of protein therapeutics. Quantitation of PEGylated anti-Factor D Fab (PEG-aFD) poses bioanalytical challenges. Results: An ELISA was developed to determine total Fab concentration in cynomolgus monkey serum following intravitreal administration of PEG-aFD. However, assay characterization showed a low recovery of about 25% for free unconjugated Fab whereas recovery for PEG-conjugated Fab was within 80-120%. To overcome this challenge, an immunoaffinity liquid chromatography tandem mass spectrometry (IA LC-MS/MS) assay was developed, achieving recovery within 80-120% for both free and conjugated Fab. Conclusion: Immunoaffinity LC-MS/MS is more suitable than ELISA to accurately quantify the total protein concentration of PEG-aFD in cynomolgus monkey serum.