A Sensitive and Controlled Data-Independent Acquisition Method for Proteomic Analysis of Cell Therapies.

Mass spectrometry (MS)-based proteomic measurements are uniquely poised to impact the development of cell and gene therapies. With the adoption of rigorous instrumental performance qualifications (PQs), large-scale proteomics can move from a research to a manufacturing control tool. Especially suited, data-independent acquisition (DIA) approaches have distinctive qualities to extend multiattribute method (MAM) principles to characterize the proteome of cell therapies. Here, we describe the development of a DIA method for the sensitive identification and quantification of proteins on a Q-TOF instrument. Using the improved acquisition parameters, we defined a control strategy and highlighted some metrics to improve the reproducibility of SWATH acquisition-based proteomic measurements. Finally, we applied the method to analyze the proteome of Jurkat cells that here serves as a model for human T-cells. Raw and processed data were deposited in PRIDE (PXD029780).

Biomanufacturing readiness levels [BRL]-A shared vocabulary for biopharmaceutical technology development and commercialization.

The Manufacturing Readiness Levels (MRLs) developed by the Department of Defense are well-established tools for describing the maturity of new technologies resulting from government-sponsored Research and Development programs, from the concept phase to commercial deployment. While MRLs are generally applicable to a wide range of industries and technologies, there is significant value in offering an industry-specific view on how the basic principles may be applied to biomanufacturing. This paper describes Biomanufacturing Readiness Levels (BRLs) developed by the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL), a public/private partnership that is part of the Manufacturing USA network. NIIMBL brings together private, federal, nonprofit, and academic stakeholders to accelerate the deployment of innovative technologies for biopharmaceutical production and to educate and train a world-leading biomanufacturing workforce. We anticipate that these BRLs will lay the groundwork for a shared vocabulary for assessment of technology maturity and readiness for commercial biomanufacturing that effectively meets the needs of this critical, specialized, and highly regulated industry.

Bioindustrial manufacturing readiness levels (BioMRLs) as a shared framework for measuring and communicating the maturity of bioproduct manufacturing processes.

Readiness level (RL) frameworks such as technology readiness levels and manufacturing readiness levels describe the status of a technology/manufacturing process on its journey from initial conception to commercial deployment. More importantly, they provide a roadmap to guide technology development and scale-up from a ''totality of system'' approach. Commercialization risks associated with too narrowly focused R&D efforts are mitigated. RLs are defined abstractly so that they can apply to diverse industries and technology sectors. However, differences between technology sectors make necessary the definition of sector specific RL frameworks. Here, we describe bioindustrial manufacturing readiness levels (BioMRLs), a classification system specific to bioindustrial manufacturing. BioMRLs will give program managers, investors, scientists, and engineers a shared vocabulary for prioritizing goals and assessing risks in the development and commercialization of a bioindustrial manufacturing process.

Antigenicity and Immunogenicity of Differentially Glycosylated Hepatitis C Virus E2 Envelope Proteins Expressed in Mammalian and Insect Cells.

The development of a prophylactic vaccine for hepatitis C virus (HCV) remains a global health challenge. Cumulative evidence supports the importance of antibodies targeting the HCV E2 envelope glycoprotein to facilitate viral clearance. However, a significant challenge for a B cell-based vaccine is focusing the immune response on conserved E2 epitopes capable of eliciting neutralizing antibodies not associated with viral escape. We hypothesized that glycosylation might influence the antigenicity and immunogenicity of E2. Accordingly, we performed head-to-head molecular, antigenic, and immunogenic comparisons of soluble E2 (sE2) produced in (i) mammalian (HEK293) cells, which confer mostly complex- and high-mannose-type glycans; and (ii) insect (Sf9) cells, which impart mainly paucimannose-type glycans. Mass spectrometry demonstrated that all 11 predicted N-glycosylation sites were utilized in both HEK293- and Sf9-derived sE2, but that N-glycans in insect sE2 were on average smaller and less complex. Both proteins bound CD81 and were recognized by conformation-dependent antibodies. Mouse immunogenicity studies revealed that similar polyclonal antibody responses were generated against antigenic domains A to E of E2. Although neutralizing antibody titers showed that Sf9-derived sE2 induced moderately stronger responses than did HEK293-derived sE2 against the homologous HCV H77c isolate, the two proteins elicited comparable neutralization titers against heterologous isolates. Given that global alteration of HCV E2 glycosylation by expression in different hosts did not appreciably affect antigenicity or overall immunogenicity, a more productive approach to increasing the antibody response to neutralizing epitopes may be complete deletion, rather than just modification, of specific N-glycans proximal to these epitopes.IMPORTANCE The development of a vaccine for hepatitis C virus (HCV) remains a global health challenge. A major challenge for vaccine development is focusing the immune response on conserved regions of the HCV envelope protein, E2, capable of eliciting neutralizing antibodies. Modification of E2 by glycosylation might influence the immunogenicity of E2. Accordingly, we performed molecular and immunogenic comparisons of E2 produced in mammalian and insect cells. Mass spectrometry demonstrated that the predicted glycosylation sites were utilized in both mammalian and insect cell E2, although the glycan types in insect cell E2 were smaller and less complex. Mouse immunogenicity studies revealed similar polyclonal antibody responses. However, insect cell E2 induced stronger neutralizing antibody responses against the homologous isolate used in the vaccine, albeit the two proteins elicited comparable neutralization titers against heterologous isolates. A more productive approach for vaccine development may be complete deletion of specific glycans in the E2 protein.

Mass Spectrometry Advances and Perspectives for the Characterization of Emerging Adoptive Cell Therapies.

Adoptive cell therapy is an emerging anti-cancer modality, whereby the patient's own immune cells are engineered to express T-cell receptor (TCR) or chimeric antigen receptor (CAR). CAR-T cell therapies have advanced the furthest, with recent approvals of two treatments by the Food and Drug Administration of Kymriah (trisagenlecleucel) and Yescarta (axicabtagene ciloleucel). Recent developments in proteomic analysis by mass spectrometry (MS) make this technology uniquely suited to enable the comprehensive identification and quantification of the relevant biochemical architecture of CAR-T cell therapies and fulfill current unmet needs for CAR-T product knowledge. These advances include improved sample preparation methods, enhanced separation technologies, and extension of MS-based proteomic to single cells. Innovative technologies such as proteomic analysis of raw material quality attributes (MQA) and final product quality attributes (PQA) may provide insights that could ultimately fuel development strategies and lead to broad implementation.

The NISTmAb Reference Material 8671 lifecycle management and quality plan.

Comprehensive analysis of monoclonal antibody therapeutics involves an ever expanding cadre of technologies. Lifecycle-appropriate application of current and emerging techniques requires rigorous testing followed by discussion between industry and regulators in a pre-competitive space, an effort that may be facilitated by a widely available test metric. Biopharmaceutical quality materials, however, are often difficult to access and/or are protected by intellectual property rights. The NISTmAb, humanized IgG1κ Reference Material 8671 (RM 8671), has been established with the intent of filling that void. The NISTmAb embodies the quality and characteristics of a typical biopharmaceutical product, is widely available to the biopharmaceutical community, and is an open innovation tool for development and dissemination of results. The NISTmAb lifecyle management plan described herein provides a hierarchical strategy for maintenance of quality over time through rigorous method qualification detailed in additional submissions in the current publication series. The NISTmAb RM 8671 is a representative monoclonal antibody material and provides a means to continually evaluate current best practices, promote innovative approaches, and inform regulatory paradigms as technology advances. Graphical abstract The NISTmAb Reference Material (RM) 8671 is intended to be an industry standard monoclonal antibody for pre-competitive harmonization of best practices and designing next generation characterization technologies for identity, quality, and stability testing.

Qualification of NISTmAb charge heterogeneity control assays.

The NISTmAb is a monoclonal antibody Reference Material from the National Institute of Standards and Technology; it is a class-representative IgG1κ intended serve as a pre-competitive platform for harmonization and technology development in the biopharmaceutical industry. The publication series of which this paper is a part describes NIST's overall control strategy to ensure NISTmAb quality and availability over its lifecycle. In this paper, the development and qualification of methods for monitoring NISTmAb charge heterogeneity are described. Capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF) assays were optimized and evaluated as candidate assays for NISTmAb quality control. CIEF was found to be suitable as a structural characterization assay yielding information on the apparent pI of the NISTmAb. CZE was found to be better suited for routine monitoring of NISTmAb charge heterogeneity and was qualified for this purpose. This paper is intended to provide relevant details of NIST's charge heterogeneity control strategy to facilitate implementation of the NISTmAb as a test molecule in the end user's laboratory. Graphical Abstract Representative capillary zone electropherogram of the NIST monoclonal antibody (NISTmAb). The NISTmAb is a publicly available research tool intended to facilitate advancement of biopharmaceutical analytics.

Development of an LC-MS/MS peptide mapping protocol for the NISTmAb.

Peptide mapping is a component of the analytical toolbox used within the biopharmaceutical industry to aid in the identity confirmation of a protein therapeutic and to monitor degradative events such as oxidation or deamidation. These methods offer the advantage of providing site-specific information regarding post-translational and chemical modifications that may arise during production, processing or storage. A number of such variations may also be induced by the sample preparation methods themselves which may confound the ability to accurately evaluate the true modification levels. One important focus when developing a peptide mapping method should therefore be the use of sample preparation conditions that will minimize the degree of artificial modifications induced. Unfortunately, the conditions that are amenable to effective reduction, alkylation and digestion are often the same conditions that promote unwanted modifications. Here we describe the optimization of a tryptic digestion protocol used for peptide mapping of the NISTmAb IgG1κ which addresses the challenge of balancing maximum digestion efficiency with minimum artificial modifications. The parameters on which we focused include buffer concentration, digestion time and temperature, as well as the source and type of trypsin (recombinant vs. pancreatic; bovine vs porcine) used. Using the optimized protocol we generated a peptide map of the NISTmAb which allowed us to confirm its identity at the level of primary structure. Graphical abstract Peptide map of the NISTmAb RM 8671 monoclonal antibody. Tryptic digestion was performed using an optimized protocol and followed by LC-UV-MS analysis. The trace represents the total ion chromatogram. Each peak was mapped to peptides identified using mass spectrometry data.

The NISTmAb Reference Material 8671 value assignment, homogeneity, and stability.

The NISTmAb Reference Material (RM) 8671 is intended to be an industry standard monoclonal antibody for pre-competitive harmonization of best practices and designing next generation characterization technologies for identity, quality, and stability testing. It must therefore embody the quality and characteristics of a typical biopharmaceutical product and be available long-term in a stable format with consistent product quality attributes. A stratified sampling and analysis plan using a series of qualified analytical and biophysical methods is described that assures RM 8671 meets these criteria. Results for the first three lots of RM 8671 highlight the consistency of material attributes with respect to size, charge, and identity. RM 8671 was verified to be homogeneous both within and between vialing lots, demonstrating the robustness of the lifecycle management plan. It was analyzed in concert with the in-house primary sample 8670 (PS 8670) to provide a historical link to this seminal material. RM 8671 was verified to be fit for its intended purpose as a technology innovation tool, external system suitability control, and cross-industry harmonization platform. Graphical abstract The NISTmAb Reference Material (RM) 8671 is intended to be an industry standard monoclonal antibody for pre-competitive harmonization of best practices and designing next generation characterization technologies for identity, quality, and stability testing.

Development of orthogonal NISTmAb size heterogeneity control methods.

The NISTmAb is a monoclonal antibody Reference Material from the National Institute of Standards and Technology; it is a class-representative IgG1κ intended to serve as a pre-competitive platform for harmonization and technology development in the biopharmaceutical industry. The publication series of which this paper is a part describes NIST's overall control strategy to ensure NISTmAb quality and availability over its lifecycle. In this paper, the development of a control strategy for monitoring NISTmAb size heterogeneity is described. Optimization and qualification of size heterogeneity measurement spanning a broad size range are described, including capillary electrophoresis-sodium dodecyl sulfate (CE-SDS), size exclusion chromatography (SEC), dynamic light scattering (DLS), and flow imaging analysis. This paper is intended to provide relevant details of NIST's size heterogeneity control strategy to facilitate implementation of the NISTmAb as a test molecule in the end user's laboratory. Graphical abstract Representative size exclusion chromatogram of the NIST monoclonal antibody (NISTmAb). The NISTmAb is a publicly available research tool intended to facilitate advancement of biopharmaceutical analytics. HMW = high molecular weight (trimer and dimer), LMW = low molecular weight (2 fragment peaks). Peak labeled buffer is void volume of the column from L-histidine background buffer.

Multivariate Analysis of Two-Dimensional 1H, 13C Methyl NMR Spectra of Monoclonal Antibody Therapeutics To Facilitate Assessment of Higher Order Structure.

Two-dimensional (2D) 1H-13C methyl NMR provides a powerful tool to probe the higher order structure (HOS) of monoclonal antibodies (mAbs), since spectra can readily be acquired on intact mAbs at natural isotopic abundance, and small changes in chemical environment and structure give rise to observable changes in corresponding spectra, which can be interpreted at atomic resolution. This makes it possible to apply 2D NMR spectral fingerprinting approaches directly to drug products in order to systematically characterize structure and excipient effects. Systematic collections of NMR spectra are often analyzed in terms of the changes in specifically identified peak positions, as well as changes in peak height and line widths. A complementary approach is to apply principal component analysis (PCA) directly to the matrix of spectral data, correlating spectra according to similarities and differences in their overall shapes, rather than according to parameters of individually identified peaks. This is particularly well-suited for spectra of mAbs, where some of the individual peaks might not be well resolved. Here we demonstrate the performance of the PCA method for discriminating structural variation among systematic sets of 2D NMR fingerprint spectra using the NISTmAb and illustrate how spectral variability identified by PCA may be correlated to structure.

Characterizing Adeno-Associated Virus Capsids with Both Denaturing and Intact Analysis Methods.

Adeno-associated virus (AAV) capsids are among the leading gene delivery platforms used to treat a vast array of human diseases and conditions. AAVs exist in a variety of serotypes due to differences in viral protein (VP) sequences with distinct serotypes targeting specific cells and tissues. As the utility of AAVs in gene therapy increases, ensuring their specific composition is imperative for the correct targeting and gene delivery. From a quality control perspective, current analytical tools are limited in their selectivity for viral protein (VP) subunits due to their sequence similarities, instrumental difficulties in assessing the large molecular weights of intact capsids, and the uncertainty in distinguishing empty and filled capsids. To address these challenges, we combined two distinct analytical workflows that assess the intact capsids and VP subunits separately. First, a selective temporal overview of resonant ion (STORI)-based charge detection-mass spectrometry (CD-MS) was applied for characterization of the intact capsids. Liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) separations were then used for the capsid denaturing measurements. This multimethod combination was applied to three AAV serotypes (AAV2, AAV6, and AAV8) to evaluate their intact empty and filled capsid ratios and then examine the distinct VP sequences and modifications present.

Glycoprotein analysis using mass spectrometry: unraveling the layers of complexity.

A glycoprotein exists as a heterogeneous mixture of forms due to differential glycosylation, each of which may confer different functionality and/or serve as a biochemical marker for disease. The complex structure of glycans make them a bioanalytical challenge requiring multiple mass spectrometry based approaches to gain different types of information. The following article will briefly describe recently utilized mass spectrometry methods to identify glycosylation sites and measure glycan composition, sequence, branching, and relative quantities. Potential metrological developments are discussed in light of current trends toward complete, reliable glycoanalytical characterization in a high-throughput manner.

LC-MS/MS biopharmaceutical glycoanalysis: identification of desirable reference material characteristics.

Glycosylation, the enzymatic addition of carbohydrates to a protein, is one of the most abundant post-translational modifications found in nature. There is variability in the number, location, and identity of glycans attached. As a result, a glycoprotein consists of a number of glycoforms with different combinations of glycans, potentially resulting in different stability, toxicity, and activity. This is especially important in the biopharmaceutical industry where product consistency and safety are vital. Glycoprotein analysis involves numerous mass spectrometry based techniques, each of which provides various aspects of characterization. The current paper describes two commonly used analytical techniques for glycoprotein characterization. In one experiment, nonspecific proteolysis is combined with a two-tiered mass spectrometry approach (MALDI-TOF and LC-MS/MS) to gain glycosylation site and glycan identity. In a second approach, glycans were enzymatically released, labeled with a fluorescent dye, and analyzed using LC-Fluorescence-MS/MS to give glycan identification and relative quantification. The type and degree of information yielded by each method is assessed in an effort to identify desired reference material characteristics for improving biopharmaceutical glycoanalysis.

Ligand-Bound Forced Degradation as a Strategy to Generate Functionally Relevant Analytical Challenge Materials for Assessment of CQAs.

Therapeutic monoclonal antibodies (mAbs) contain a variety of amino acids that are susceptible to enzymatic, chemical, and physical modifications. These modifications can happen throughout production, purification, formulation, and storage and many are known to affect the biological activity of a mAb. Methods that are able to characterize and evaluate these attributes are critical in order to understand how they might alter biological activity. Methods capable of site-specific monitoring of these critical quality attributes are extremely valuable to biopharmaceutical research but also require well-defined materials with site-specific attribute modifications. Here, we describe the development and application of a strategy to generate functionally relevant analytical challenge materials that have unique site-specific attributes. This method involves the use of a ligand that is bound to the mAb during oxidative stress resulting in unique oxidation patterns with some methionine residues protected while others are exposed to oxidation. These unique materials were used to develop a rapid surface plasmon resonance (SPR) assay that could detect methionine oxidation in both the Fab and Fc regions using specific molecular probes. The addition of uniquely oxidized materials to our data set enabled us to determine specific methionine residues vital to binding. Further analysis showed that antibody oxidation could also be rapidly detected in multiple domains from qualitative thermal melting using intrinsic tryptophan fluorescence. Methionine oxidation of an antibody was explored in this study, but we envision this method could be useful to explore structure function relationships of a variety of antibody modifications and modifications to other biologically relevant protein drugs.

Interlaboratory Studies Using the NISTmAb to Advance Biopharmaceutical Structural Analytics.

Biopharmaceuticals such as monoclonal antibodies are required to be rigorously characterized using a wide range of analytical methods. Various material properties must be characterized and well controlled to assure that clinically relevant features and critical quality attributes are maintained. A thorough understanding of analytical method performance metrics, particularly emerging methods designed to address measurement gaps, is required to assure methods are appropriate for their intended use in assuring drug safety, stability, and functional activity. To this end, a series of interlaboratory studies have been conducted using NISTmAb, a biopharmaceutical-representative and publicly available monoclonal antibody test material, to report on state-of-the-art method performance, harmonize best practices, and inform on potential gaps in the analytical measurement infrastructure. Reported here is a summary of the study designs, results, and future perspectives revealed from these interlaboratory studies which focused on primary structure, post-translational modifications, and higher order structure measurements currently employed during biopharmaceutical development.

Attribute Analytics Performance Metrics from the MAM Consortium Interlaboratory Study.

The multi-attribute method (MAM) was conceived as a single assay to potentially replace multiple single-attribute assays that have long been used in process development and quality control (QC) for protein therapeutics. MAM is rooted in traditional peptide mapping methods; it leverages mass spectrometry (MS) detection for confident identification and quantitation of many types of protein attributes that may be targeted for monitoring. While MAM has been widely explored across the industry, it has yet to gain a strong foothold within QC laboratories as a replacement method for established orthogonal platforms. Members of the MAM consortium recently undertook an interlaboratory study to evaluate the industry-wide status of MAM. Here we present the results of this study as they pertain to the targeted attribute analytics component of MAM, including investigation into the sources of variability between laboratories and comparison of MAM data to orthogonal methods. These results are made available with an eye toward aiding the community in further optimizing the method to enable its more frequent use in the QC environment.

Development of a flow-based ultrafast immunoextraction and reverse displacement immunoassay: analysis of free drug fractions.

A flow-based method employing a reverse displacement immunoassay was combined with ultrafast immunoextraction and near-infrared fluorescence detection for the analysis of free drug fractions, using phenytoin as a model analyte. Factors considered in the design of this method included the sample application conditions, the design of the immobilized drug analog column, the utilization of antibodies or F(ab) fragments as labeled binding agents, and the label application and column regeneration conditions. In the final method, sample injections led to the displacement of labeled binding agents from an immobilized phenytoin analog column. This displacement peak appeared within 20-30 s of sample injection and was proportional in size to the free phenytoin concentration in the sample. It was possible with this method to regenerate the column using only the application of additional label between sample injections. This method was used to measure clinically relevant concentrations of free phenytoin in serum and drug/protein mixtures and gave good correlation with ultrafiltration, while also being faster to perform and requiring significantly less sample. This technique was not limited to free phenytoin measurements but could be adapted for other drugs or analytes through the use of appropriate columns and binding agents.

New Peak Detection Performance Metrics from the MAM Consortium Interlaboratory Study.

The Multi-Attribute Method (MAM) Consortium was initially formed as a venue to harmonize best practices, share experiences, and generate innovative methodologies to facilitate widespread integration of the MAM platform, which is an emerging ultra-high-performance liquid chromatography-mass spectrometry application. Successful implementation of MAM as a purity-indicating assay requires new peak detection (NPD) of potential process- and/or product-related impurities. The NPD interlaboratory study described herein was carried out by the MAM Consortium to report on the industry-wide performance of NPD using predigested samples of the NISTmAb Reference Material 8671. Results from 28 participating laboratories show that the NPD parameters being utilized across the industry are representative of high-resolution MS performance capabilities. Certain elements of NPD, including common sources of variability in the number of new peaks detected, that are critical to the performance of the purity function of MAM were identified in this study and are reported here as a means to further refine the methodology and accelerate adoption into manufacturer-specific protein therapeutic product life cycles.

Measurement of drug-protein dissociation rates by high-performance affinity chromatography and peak profiling.

The rate at which a drug or other small solute interacts with a protein is important in understanding the biological and pharmacokinetic behavior of these agents. One approach that has been developed for examining these rates involves the use of high-performance affinity chromatography (HPAC) and estimates of band-broadening through peak profiling. Previous work with this method has been based on a comparison of the statistical moments for a retained analyte versus nonretained species at a single, high flow rate to obtain information on stationary phase mass transfer. In this study an alternative approach was created that allows a broad range of flow rates to be used for examining solute-protein dissociation rates. Chromatographic theory was employed to derive equations that could be used with this approach on a single column, as well as with multiple columns to evaluate and correct for the impact of stagnant mobile phase mass transfer. The interaction of L-tryptophan with human serum albumin was used as a model system to test this method. A dissociation rate constant of 2.7 (+/-0.2) s(-1) was obtained by this approach at pH 7.4 and 37 degrees C, which was in good agreement with previous values determined by other methods. The techniques described in this report can be applied to other biomolecular systems and should be valuable for the determination of drug-protein dissociation rates.