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

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

Characterization of antigen presentation capability for neoantigen-based products using targeted LC-MS/MS method.

The generation of an immune response in neoantigen-based products relies on antigen presentation, which is closely analyzed by bioassays for T-cell functions such as tetramer or cytokine release. Mass spectrometry (MS) has the potential to directly assess the antigen-presenting capability of antigen-presenting cells (APCs), offering advantages such as speed, multi-target analysis, robustness, and ease of transferability. However, it has not been used for quality control of these products due to challenges in sensitivity, including the number of cells and peptide diversity. In this study, we describe the development and validation of an improved targeted LC-MS/MS method with high sensitivity for characterizing antigen presentation, which could be applied in the quality control of neoantigen-based products. The parameters for the extraction were carefully optimized by different short peptides. Highly sensitive targeted triple quadrupole mass spectrometry combined with ultra-high performance liquid chromatography (UHPLC) was employed using a selective ion monitoring mode (Multiple Reaction Monitoring, MRM). Besides, we successfully implemented robust quality control peptides to ensure the reliability and consistency of this method, which proved invaluable for different APCs. With reference to the guidelines from ICH Q2 (R2), M10, as well as considering the specific attributes of the product itself, we validated the method for selectivity, specificity, sensitivity, limit of detection (LOD), recovery rate, matrix effect, repeatability, and application in dendritic cells (DCs) associated with neoantigen-based products. The validation process yields satisfactory results. Combining this approach with T cell assays will comprehensively assess cell product quality attributes from physicochemical and biological perspectives.

A Highly Sensitive LC-MS/MS Method for Targeted Quantitation of Lipase Host Cell Proteins in Biotherapeutics.

Identification and accurate quantitation of host cell proteins (HCPs) in biotherapeutic drugs has become increasingly important due to the negative impact of certain HCPs on the safety, stability, and other product quality of biotherapeutics. Recently, several lipase HCPs have been identified to potentially cause the enzymatic degradation of polysorbate, a widely used excipient in the formulation of biotherapeutics, which can severely impact the stability and product quality of drug products. In this study, we identified three lipase HCPs that were frequently detected in Chinese hamster ovary (CHO) cell cultures using shotgun proteomics, including phospholipase B-like 2 (PLBL2), lipoprotein lipase (LPL), and lysosomal acid lipase (LIPA). A targeted quantitation method for these three lipase HCPs was developed utilizing liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) with high-resolution multiple-reaction-monitoring (MRMhr) quantitation. The method demonstrated good sensitivity with low limit of quantitation (LLOQ) around 1 ng/mL, and linear dynamic range of three orders of magnitude for the three lipase HCPs. It has been applied for the characterization of process intermediates from various in-house monoclonal antibody (mAb) production. In addition, the method has also been used to evaluate the robustness of clearance for one of the lipase HCPs, PLBL2, under different column purification process conditions.

Benchmarking and optimization of a high-throughput sequencing based method for transgene sequence variant analysis in biotherapeutic cell line development.

In recent years, High-Throughput Sequencing (HTS) based methods to detect mutations in biotherapeutic transgene products have become a key quality step deployed during the development of manufacturing cell line clones. Previously we reported on a higher throughput, rapid mutation detection method based on amplicon sequencing (targeting transgene RNA) and detailed its implementation to facilitate cell line clone selection. By gaining experience with our assay in a diverse set of cell line development programs, we improved the computational analysis as well as experimental protocols. Here we report on these improvements as well as on a comprehensive benchmarking of our assay. We evaluated assay performance by mixing amplicon samples of a verified mutated antibody clone with a non-mutated antibody clone to generate spike-in mutations from ∼60% down to ∼0.3% frequencies. We subsequently tested the effect of 16 different sample and HTS library preparation protocols on the assay's ability to quantify mutations and on the occurrence of false-positive background error mutations (artifacts). Our evaluation confirmed assay robustness, established a high confidence limit of detection of ∼0.6%, and identified protocols that reduce error levels thereby significantly reducing a source of false positives that bottlenecked the identification of low-level true mutations.

The potential markers of NK-92 associated to cytotoxicity against K562 cells.

Markers associated to NK cytolytic activity are in a great need to regulate NK cell immunotherapy products. We assume that biomarkers which response to cytolysis will change their transcription, expression or secretion. To find NK-92 indicator to cytolytic activity, we have evaluated the potential markers by quantifying the expression of well-known cytotoxicity functional molecules (cytokine IFN-γ, Granzyme B, perforin, CD69 and CD107a), and explored candidate markers by a sweeping transcription picture of NK-92 using a direct cytolysis model (incubation with K562). We found that IFN-γ secretion was highly correlated to cytotoxicity of NK-92, neither Granzyme B, perforin secretion, nor CD69, CD107a positive population were upregulated by K562 stimulation. RNAseq revealed 432 genes expression changed during cytolysis, several genes (BIRC3, CSF2, VCAM1 and TNFRSF9) mRNA expression were validated by real time RT-PCR under K562 being killed or protected from being killed conditions. Results suggested IFN-γ secretion, BIRC3 and TNFRSF9 transcription in NK-92 were responsive to K562 cytolysis. In a word, our results confirmed one marker and reveal an array of novel candidate markers associated with NK-92 cytotoxicity. Further studies are greatly needed to determine the roles these new makers play in NK-92 cytolysis process.

Biolayer Interferometry-based FcγRIIa binding assay for a therapeutic antibody with strong effector function.

FcγRIIa receptor binding is part of the mechanism of action for many therapeutic antibodies. AlphaScreen® technology and Biolayer Interferometry (BLI) are often used to assess protein-protein interactions. Recently we demonstrated that the presence of aggregates in samples significantly increased binding potency values in AlphaScreen®-based FcRn binding assays, sometimes masking the loss of potency. Even bigger effect of aggregates was observed in an AlphaScreen®-based FcγRIIa binding assay for a monoclonal antibody with strong effector function. To resolve this issue a novel BLI-based FcγRIIa binding assay was developed and qualified. The assay measures association binding responses and calculates the binding potency of the samples relative to the standard using Parallel Line Analysis. The method overcomes interference of aggregates present in the samples, distinguishes different Fc glycosylation patterns, and is stability-indicating. It can be used for sample characterization, drug product release and stability testing.

Sequence Variant and Posttranslational Modification Analysis During Cell Line Selection via High-Throughput Peptide Mapping.

Selection of high-producing lead and backup cell lines with high-fidelity primary structure is a major goal of cell line development of protein therapeutics. Conventional techniques for sequence variant analysis, such as mass spectrometry (MS) and next-generation sequencing (NGS) have limitations on the sample number and turnaround time, thus often are only applied at the final stages of development, where an undesired lead or backup clone could cause a significant delay in project timeline. Here we presented a high-throughput (HT) peptide mapping workflow which can be applied at early stages of cell line selection for testing of a batch of 30-40 clones within 2-week turnaround while reporting valuable information on sequence variants and posttranslational modifications (PTMs). The successful application of this workflow was demonstrated for two mAb programs. Multiple clones were removed from a total of 33 mAb-1 clones using various criteria: nine clones contained at least one >1% upregulated unknown peptide ions, 11 clones contained at least eight >0.1% upregulated unknowns, and six clones contained upregulated critical PTMs. For mAb-2, light chain (LC) sequence extension of approximately 30 amino acids were detected in 6 out of 36 clones at levels up to 11%. Besides, a Q to H mutation at ~30% was detected in the heavy chain (HC) of a single clone. Q to H mutation has mass change of 9.00 Da and failed to be detected by intact mass analysis. Rapid PTM quantitation also facilitated the selection of clones with desirable quality attributes, such as N-glycan profile. Hence, we demonstrated a risk-reducing strategy where abnormal clones could be detected at earlier stages of cell line selection, which should result in reduced and more predictable timeline of cell line development.

Application of a label-free and domain-specific free thiol method in monoclonal antibody characterization.

Characterization of free thiol variants in antibody therapeutics is important for biopharmaceutical development, as the presence of free thiols may have an impact on aggregate formation, structural and thermal stability, as well as antigen-binding potency of monoclonal antibodies. Most current methods for free thiol quantification involve labeling of free thiol groups by different tagging molecules followed by UV, fluorescence or mass spectrometry (MS) detection. Here, we optimized a label-free liquid chromatography (LC)-UV/MS method for free thiol quantification at a subunit level and compared this method with two orthogonal and conventional approaches, Ellman's assay and peptide mapping with differential alkylation. This subunit unit approach was demonstrated to be able to provide domain-specific free thiol quantification and comparable results with labeling approaches, using a relatively simple and efficient workflow.

Latent Membrane Protein 1 of Epstein-Barr Virus Promotes RIG-I Degradation Mediated by Proteasome Pathway.

RIG-I signaling is critical to host innate immune response against RNA virus infection, and also can be activated against many kinds of cancer. Oncogene LMP1 of Epstein-Barr virus (EBV) contributes to various tumors progress. In this study, we have provided strong evidence that LMP1 inhibits Sendai virus mediated type I interferon production and downregulates RIG-I signaling pathway by promotion RIG-I degradation dependent on proteasome. Nineteen kinds of E3 ligase are identified by IP-MS as LMP1-interactors, they are candidate E3s, which are possibly recruited by LMP1 to mediate RIG-I degradation. CHIP is among these E3s, which has been reported to lead RIG-I degradation. Notably, we find C666-1, an EBV-positive nasopharyngeal carcinoma cell line, expresses low level of RIG-I, even treated with IFN-α, RIG-I expression could not be induced. This evidence indicates that EBV employs a unique strategy to evade RIG-I mediated immune responses.