Comprehensive Impurity Profiling of mRNA: Evaluating Current Technologies and Advanced Analytical Techniques.

In vitro transcription (IVT) of mRNA is a versatile platform for a broad range of biotechnological applications. Its rapid, scalable, and cost-effective production makes it a compelling choice for the development of mRNA-based cancer therapies and vaccines against infectious diseases. The impurities generated during mRNA production can potentially impact the safety and efficacy of mRNA therapeutics, but their structural complexity has not been investigated in detail yet. This study pioneers a comprehensive profiling of IVT mRNA impurities, integrating current technologies with innovative analytical tools. We have developed highly reproducible, efficient, and stability-indicating ion-pair reversed-phase liquid chromatography and capillary gel electrophoresis methods to determine the purity of mRNA from different suppliers. Furthermore, we introduced the applicability of microcapillary electrophoresis for high-throughput (<1.5 min analysis time per sample) mRNA impurity profiling. Our findings revealed that impurities are mainly attributed to mRNA variants with different poly(A) tail lengths due to aborted additions or partial hydrolysis and the presence of double-stranded mRNA (dsRNA) byproducts, particularly the dsRNA 3'-loop back form. We also implemented mass photometry and native mass spectrometry for the characterization of mRNA and its related product impurities. Mass photometry enabled the determination of the number of nucleotides of different mRNAs with high accuracy as well as the detection of their size variants [i.e., aggregates and partial and/or total absence of the poly(A) tail], thus providing valuable information on mRNA identity and integrity. In addition, native mass spectrometry provided insights into mRNA intact mass, heterogeneity, and important sequence features such as poly(A) tail length and distribution. This study highlights the existing bottlenecks and opportunities for improvement in the analytical characterization of IVT mRNA, thus contributing to the refinement and streamlining of mRNA production, paving the way for continued advancements in biotechnological applications.

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.

Novel Multidimensional Liquid Chromatography Workflow with In-Loop Enzymatic Digests of Multiple Heart-Cuts for Fast and Flexible Characterization of Biotherapeutic Protein Variants.

Multidimensional liquid chromatography (mD-LC) is becoming a powerful tool for complete characterization of individual peaks and protein variants through separation methods such as nondenaturing ion exchange (IEC) or size-exclusion chromatography coupled to reversed-phase (RP) chromatography. The flexibility of commercially available and customized mD-LC systems is still limited in terms of enzymatic peak processing between chromatographic dimensions. In this regard, only a few column-immobilized proteases are available for detailed peak characterization by mD-LC coupled to mass spectrometry (mD-LC-MS). Here, we present a purpose-built and automated multiple heart-cutting mD-LC design with a novel analytical workflow involving in-loop enzymatic heart-cut digestion between the first-dimensional column and transfer to the second dimension before MS or MS/MS analyses. The setup facilitates the spike-in of any enzyme to multiple heart-cuts for multilevel analysis, for example, for peptide mapping, fragment generation, or deglycosylation, to reduce heterogeneity and provide maximum flexibility in terms of incubation time for optimal peak characterization. We demonstrate the application of IEC coupled to RP-LC-MS and automated in-loop deglycosylation and on-column reduction of an IgG antibody combined with upper hinge region cleavage for Fab generation. We further employ mD-LC-MS and mD-LC-MS/MS to assess post-translational modifications of a bispecific antibody and to support molecule selection by evaluating the best downstream purification strategy. The novel design and automated workflow of the mD-LC system described here offers enhanced flexibility for in-solution processing and real-time monitoring of multiple heart-cuts enabling streamlined characterization of unknown biotherapeutic charge and size variants.

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.

Physicochemical and Functional Characterization of Differential CRISPR-Cas9 Ribonucleoprotein Complexes.

Advances in gene-editing technology enable efficient, targeted ex vivo engineering of different cell types, which offer a potential therapeutic platform for most challenging disease areas. CRISPR-Cas9 is a widely used gene-editing tool in therapeutic applications. The quality of gene-editing reagents (i.e., Cas9 nuclease, single guide (sg)RNA) is associated with the final cellular product quality as they can impact the gene-editing accuracy and efficiency. To assess the impact of the quality of Cas9 protein and sgRNA in the formation of a Cas9 ribonucleoprotein (RNP) complex, stability, and functional activities, we developed a size exclusion chromatography method that utilizes multiple detectors and an in vitro DNA cleavage assay using anion-exchange chromatography. Using these methods, we characterized the formation and stability of Cas9 RNP complexes associated with Cas9 and sgRNA characteristics as well as their functional activities. Multi-angle light scattering characterization showed different types and levels of aggregates in different source sgRNA materials, which contribute to form different Cas9 RNP complexes. The aggregations irreversibly dissociated at high temperatures. When the Cas9 RNP complexes derived from non-heated and heated sgRNAs were characterized, the data showed that specific RNP peaks were impacted. The Cas9 RNP complexes derived from the heated sgRNA retained their biological function and cleaved the double-strand target DNA at a higher rate. This work provides new tools to characterize the Cas9 RNP complex formation, stability, and functional activity and provides insights into sgRNA properties and handling procedures to better control the Cas9 RNP complex formation.

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.

Therapeutic Fc-fusion proteins: Current analytical strategies.

Fc-Fusion proteins represent a successful class of biopharmaceutical products, with already 13 drugs approved in the European Union and United States as well as three biosimilar versions of etanercept. Fc-Fusion products combine tailored pharmacological properties of biological ligands, together with multiple functions of the fragment crystallizable domain of immunoglobulins. There is a great diversity in terms of possible biological ligands, including the extracellular domains of natural receptors, functionally active peptides, recombinant enzymes, and genetically engineered binding constructs acting as cytokine traps. Due to their highly diverse structures, the analytical characterization of Fc-Fusion proteins is far more complex than that of monoclonal antibodies and requires the use and development of additional product-specific methods over conventional generic/platform methods. This can be explained, for example, by the presence of numerous sialic acids, leading to high diversity in terms of isoelectric points and complex glycosylation profiles including multiple N- and O-linked glycosylation sites. In this review, we highlight the wide range of analytical strategies used to fully characterize Fc-fusion proteins. We also present case studies on the structural assessment of all commercially available Fc-fusion proteins, based on the features and critical quality attributes of their ligand-binding domains.

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.

Analysis of the human chorionic gonadotropin protein at the intact level by HILIC-MS and comparison with RPLC-MS.

In the present work, the human chorionic gonadotropin (hCG) hormone was characterized for the first time by hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution (HR) quadrupole/time-of-flight (qTOF) mass spectrometry (MS) at the intact level. This heterodimeric protein, consisting of two subunits (hCGα and hCGß), possesses 8 potential glycosylation sites leading to a high number of glycoforms and has a molecular weight of about 35 kDa. The HILIC conditions optimized in a first paper but using UV detection were applied here with MS for the analysis of two hCG-based drugs, a recombinant hCG and a hCG isolated from the urine of pregnant women. An amide column (150 × 2.1 mm, 2.6 µm, 150 Å), a mobile phase composed of acetonitrile and water both containing 0.1% of trifluoroacetic acid, and a temperature of 60 °C were used. The gradient was from 85 to 40% ACN in 30 min. The use of TFA that had been shown to be necessary for the separation of glycoforms caused, as expected, an ion suppression effect in MS that was partially overcome by increasing the amount of protein injected (2 µL at 1 mg mL-1) and reducing the detection m/z range (from 1500 to 300). These conditions allowed the detection of different glycoforms of hCGα. The performance of the HILIC-HRMS method was compared with that previously obtained in RPLC-HRMS in terms of the number of detected glycoforms, selectivity, and sensitivity. The complementarity and orthogonality of the HILIC and RP modes for the analysis of hCG at the intact level were demonstrated.

Identification and semi-relative quantification of intact glycoforms by nano-LC-(Orbitrap)MS: application to the α-subunit of human chorionic gonadotropin and follicle-stimulating hormone.

Human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH) belong to the family of glycoprotein polypeptide hormones called gonadotropins. They are heterodimers sharing the α-subunit structure that has 2 N-glycosylation sites. A method based on nano-reversed-phase liquid chromatography coupled to high-resolution mass spectrometry with an Orbitrap analyzer was developed for the first time to characterize the glycosylation state of the α-subunit at the intact level. A recombinant hCG-based drug, Ovitrelle®, was analyzed. This method combined with an appropriate data treatment allowed the detection of not only the major isoforms but also the minority ones with a high mass accuracy. More than 30 hCGα glycoforms were detected without overlapping of the isotopic patterns. The figures of merit of the method were assessed. The relative standard deviations (RSDs) of the retention time ranged between 0.1 and 6.08% (n = 3), with an average of 0.4%. The RSDs of the peak area measured on the extracted ion chromatogram of each glycoform are below 38% (n = 3), with an average of 16%, thus allowing semi-relative quantification. The ability to accurately profile glycosylated variants of hCGα was next demonstrated by comparing qualitatively and semi-quantitatively 3 batches of Ovitrelle®. The method was also used to analyze 3 batches of a recombinant FSH-based drug, Puregon®, and 30 FSHα glycoforms were detected and semi-quantified. This demonstrates the high potential of this method for fast quality control or comparison of the glycosylation of glycoprotein-based pharmaceutical preparations. Graphical abstract.

Assessing TCR identity, knock-in efficiency, and potency for individualized TCR-T cell therapy.

Advances in mass spectrometry, genome sequencing techniques, and bioinformatic strategies have accelerated the discovery of cancer-specific neoantigens. Tumors express multiple immunogenic neoantigens, and neoantigen-specific T cell receptors (TCRs) can be identified in peripheral blood's mononuclear cells in cancer patients. Therefore, individualized TCR-based therapies are a promising approach whereby multiple neoantigen-specific TCRs can be selected in each patient, potentially leading to a highly effective treatment for cancer patients. We developed three multiplex analytical assays to determine the quality attributes of the TCR-T cell drug product with a mixture of five engineered TCRs. The identity of each TCR was determined by two NGS-based methods, Illumina MiSeq and PacBio platforms. This approach not only confirms the expected TCR sequences but also differentiates them by their variable regions. The five individual TCR and total TCR knock-in efficiencies were measured by droplet digital PCR using specific reverse primers. A potency assay based on transfection of antigen-encoding-RNA was developed to assess the dose-dependent activation of T cells for each TCR by measuring the surface activation marker CD137 expression and cytokine secretion. This work provides new assays to characterize individualized TCR-T cell products and insights into quality attributes for the control strategy.

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.

Challenges and Strategies for a Thorough Characterization of Antibody Acidic Charge Variants.

Heterogeneity of therapeutic Monoclonal antibody (mAb) drugs are due to protein variants generated during the manufacturing process. These protein variants can be critical quality attributes (CQAs) depending on their potential impact on drug safety and/or efficacy. To identify CQAs and ensure the drug product qualities, a thorough characterization is required but challenging due to the complex structure of biotherapeutics. Past characterization studies for basic and acidic variants revealed that full characterizations were limited to the basic charge variants, while the quantitative measurements of acidic variants left gaps. Consequently, the characterization and quantitation of acidic variants are more challenging. A case study of a therapeutic mAb1 accounted for two-thirds of the enriched acidic variants in the initial characterization study. This led to additional investigations, closing the quantification gaps of mAb1 acidic variants. This work demonstrates that a well-designed study with the right choices of analytical methods can play a key role in characterization studies. Thus, the updated strategies for more complete antibody charge variant characterization are recommended.

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.

Fc galactosylation follows consecutive reaction kinetics and enhances immunoglobulin G hexamerization for complement activation.

Fc galactosylation is a critical quality attribute for anti-tumor recombinant immunoglobulin G (IgG)-based monoclonal antibody (mAb) therapeutics with complement-dependent cytotoxicity (CDC) as the mechanism of action. Although the correlation between galactosylation and CDC has been known, the underlying structure-function relationship is unclear. Heterogeneity of the Fc N-glycosylation produced by Chinese hamster ovary (CHO) cell culture biomanufacturing process leads to variable CDC potency. Here, we derived a kinetic model of galactose transfer reaction in the Golgi apparatus and used this model to determine the correlation between differently galactosylated species from CHO cell culture process. The model was validated by a retrospective data analysis of more than 800 historical samples from small-scale and large-scale CHO cell cultures. Furthermore, using various analytical technologies, we discovered the molecular basis for Fc glycan terminal galactosylation changing the three-dimensional conformation of the Fc, which facilitates the IgG1 hexamerization, thus enhancing C1q avidity and subsequent complement activation. Our study offers insight into the formation of galactosylated species, as well as a novel three-dimensional understanding of the structure-function relationship of terminal galactose to complement activation in mAb therapeutics.