Browse
Submit Data
Databases
API
Help

Dataset Information

15 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Identification, characterization and control of a sequence variant in monoclonal antibody drug product: a case study.

ABSTRACT: Sequence variants (SV) in protein bio therapeutics can be categorized as unwanted impurities and may raise serious concerns in efficacy and safety of the product. Early detection of specific sequence modifications, that can result in altered physicochemical and or biological properties, is therefore desirable in product manufacturing. Because of their low abundance, and finite resolving power of conventional analytical techniques, they are often overlooked in early drug development. Here, we present a case study where trace amount of a sequence variant is identified in a monoclonal antibody (mAb) based therapeutic protein by LC-MS/MS and the structural and functional features of the SV containing mAb is assessed using appropriate analytical techniques. Further, a very sensitive selected reaction monitoring (SRM) technique is developed to quantify the SV which revealed both prominent and inconspicuous nature of the variant in process chromatography. We present the extensive characterization of a sequence variant in protein biopharmaceutical and first report on control of sequence variants to < 0.05% in final drug product by utilizing SRM based mass spectrometry method during the purification steps.

SUBMITTER: Thakur A

PROVIDER: S-EPMC8225904 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Identification and characterization of host cell protein product-associated impurities in monoclonal antibody bioprocessing.

Project description:Downstream processing of monoclonal antibodies (mAbs) has evolved to allow the specific process for a new product to be developed largely by empirical specialization of a platform process that enables removal of impurities of different kinds. A more complete characterization of impurities and the product itself would provide insights into the rational design of efficient downstream processes. This work identifies and characterizes host cell protein (HCP) product-associated impurities, that is, HCP species carried through the downstream processes via direct interactions with the mAb. Interactions between HCPs and mAbs are characterized using cross-interaction chromatography under solution conditions typical of those used in downstream processing. The interacting species are then identified by two-dimensional gel electrophoresis and mass spectrometry. This methodology has been applied to identify product-associated impurities in one particular purification step, namely protein A affinity chromatography, for four therapeutic mAbs as well as the Fab and Fc domains of one of these mAbs. The results show both the differences in HCP-mAb interactions among different mAbs, and the relative importance of product association compared to co-elution in protein A affinity chromatography.

| S-EPMC5815507 | biostudies-literature

Identification and characterization of M6903, an antagonistic anti-TIM-3 monoclonal antibody.

Project description:T cell immunoglobulin and mucin domain-3 (TIM-3) is an immune checkpoint that regulates normal immune responses but can be exploited by tumor cells to evade immune surveillance. TIM-3 is primarily expressed on immune cells, particularly on dysfunctional and exhausted T cells, and engagement of TIM-3 with its ligands promotes TIM-3-mediated T cell inhibition. Antagonistic ligand-blocking anti-TIM-3 antibodies have the potential to abrogate T cell inhibition, activate antigen-specific T cells, and enhance anti-tumor immunity. Here we describe M6903, a fully human anti-TIM-3 antibody without effector function and with high affinity and selectivity to TIM-3. We demonstrate that M6903 blocks the binding of TIM-3 to three of its ligands, phosphatidylserine (PtdSer), carcinoembryonic antigen cell adhesion-related molecule 1 (CEACAM1), and galectin 9 (Gal-9). These results are supported by an atomic resolution crystal structure and functional assays, which demonstrate that M6903 monotherapy enhanced T cell activation. This activation was further enhanced by the combination of M6903 with bintrafusp alfa, a bifunctional fusion protein that simultaneously blocks the transforming growth factor-β (TGF-β) and programmed death ligand 1 (PD-L1) pathways. M6903 and bintrafusp alfa combination therapy also enhanced anti-tumor efficacy in huTIM-3 knock-in mice, relative to either monotherapy. These in vitro and in vivo data, along with favorable pharmacokinetics in marmoset monkeys, suggest that M6903 as a monotherapy warrants further pre-clinical assessment and that M6903 and bintrafusp alfa may be a promising combination therapy in the clinic.

| S-EPMC7153820 | biostudies-literature

Selection, identification, and characterization of SARS-CoV-2 monoclonal antibody resistant mutants.

Project description:The use of monoclonal neutralizing antibodies (mNAbs) is being actively pursued as a viable intervention for the treatment of Severe Acute Respiratory Syndrome CoV-2 (SARS-CoV-2) infection and associated coronavirus disease 2019 (COVID-19). While highly potent mNAbs have great therapeutic potential, the ability of the virus to mutate and escape recognition and neutralization of mNAbs represents a potential problem in their use for the therapeutic management of SARS-CoV-2. Studies investigating natural or mNAb-induced antigenic variability in the receptor binding domain (RBD) of SARS-CoV-2 Spike (S) glycoprotein, and their effects on viral fitness are still rudimentary. In this manuscript we described experimental approaches for the selection, identification, and characterization of SARS-CoV-2 monoclonal antibody resistant mutants (MARMs) in cultured cells. The ability to study SARS-CoV-2 antigenic drift under selective immune pressure by mNAbs is important for the optimal implementation of mNAbs for the therapeutic management of COVID-19. This will help to identify essential amino acid residues in the viral S glycoprotein required for mNAb-mediated inhibition of viral infection, to predict potential natural drift variants that could emerge upon implementation of therapeutic mNAbs, as well as vaccine prophylactic treatments for SARS-CoV-2 infection. Additionally, it will also enable the assessment of MARM viral fitness and its potential to induce severe infection and associated COVID-19 disease.

| S-EPMC7837211 | biostudies-literature

Determination of ultra-trace metal-protein interactions in co-formulated monoclonal antibody drug product by SEC-ICP-MS.

Project description:Transition metals can be introduced in therapeutic protein drugs at various steps of the manufacturing process (e.g. manufacturing raw materials, formulation, storage), and can cause a variety of modifications on the protein. These modifications can potentially influence the efficacy, safety, and stability of the therapeutic protein, especially if critical quality attributes (CQAs) are affected. Therefore, it is meaningful to understand the interactions between proteins and metals that can occur during the manufacturing process, formulation, and storage of biotherapeutics. Here, we describe a novel strategy to differentiate between ultra-trace levels of transition metals (cobalt, chromium, copper, iron, and nickel) interacting with therapeutic proteins and free metal in solution in the drug formulation using size exclusion chromatography coupled to inductively coupled plasma mass spectrometry (SEC-ICP-MS). Two monoclonal antibodies (mAbs) were coformulated and stored up to nine days in a scaled down model to mimic metal exposure from manufacturing tanks. The samples containing the mAbs were first analyzed by ICP-MS for bulk metal analysis, then studied using SEC-ICP-MS to measure the extent of metal-protein interactions. The SEC separation was used to differentiate metal associated with the mAbs from free metal in solution. Relative quantitation of metal-protein interaction was then calculated using the relative peak areas of protein-associated metal to free metal in solution and weighting it to the total metal concentration in the mixture as measured by bulk metal analysis by ICP-MS. The SEC-ICP-MS method offers an informative means of measuring metal-protein interactions during drug development.

| S-EPMC10085571 | biostudies-literature

Identification and characterization of a multispecific monoclonal antibody G2 against chicken prion protein.

Project description:We previously generated a monoclonal antibody (mAb), G2, by immunizing mice with Residues 174-247 of the chicken prion protein (ChPrP(C) ). In this study, we found that G2 possessed an extremely unusual characteristic for a mAb; in particular, it could react with at least three proteins other than ChPrP(C) , the original antigenic protein. We immunoscreened a complementary DNA library from chicken brain DNA and found three proteins (SEPT3, ATP6V1C1, and C6H10orf76) that reacts with G2. There were no regions of amino acid sequence similarity between ChPrP(C) and SEPT3, ATP6V1C1, or C6H10orf76. We selected ATP6V1C1 as a representative of the three proteins and identified the epitope within ATP6V1C1 that reacts with G2. The amino acid sequence of the G2 epitope within ATP6V1C1 (Pep8) was not related to the G2 epitope within ChPrP(C) (Pep18mer). However, enzyme-linked immunosorbent assay, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) experiments indicated that these two peptides have similar binding affinity for G2. The apparent KD values of Pep18mer and Pep8 obtained from SPR experiments were 2.9 × 10(-8) and 1.6 × 10(-8) M, respectively. Antibody inhibition test using each peptide indicated that the binding sites of the two different peptides overlapped each other. We observed that these two peptides substantially differed in several binding characteristics. Based on the SPR experiments, the association and dissociation rate constants of Pep18mer were higher than those of Pep8. A clear difference was also observed in ITC experiments. These differences may be explained by G2 adopting different binding conformations and undergoing different binding pathways.

| S-EPMC4116654 | biostudies-literature

KM55 Monoclonal Antibody and IgA Variant of Proliferative Glomerulonephritis With Monoclonal Ig Deposits.

Project description: Not available

| S-EPMC7270977 | biostudies-literature

Identification and characterization of a residual host cell protein hexosaminidase B associated with N-glycan degradation during the stability study of a therapeutic recombinant monoclonal antibody product.

Project description:Host cell proteins (HCPs) are process-related impurities derived from host organisms, which need to be controlled to ensure adequate product quality and safety. In this study, product quality attributes were tracked for several monoclonal antibodies (mAbs) under the intended storage and accelerated stability conditions. One product quality attribute not expected to be stability indicating is the N-glycan heterogeneity profile. However, significant N-glycan degradation was observed for one mAb under accelerated and stressed stability conditions. The root cause for this instability was attributed to hexosaminidase B (HEXB), an enzyme known to remove terminal N-acetylglucosamine (GlcNAc). HEXB was identified by liquid chromatography-mass spectrometry (LC-MS)-based proteomics approach to be enriched in the impacted stability batches from mAb-1. Subsequently, enzymatic and targeted multiple reaction monitoring (MRM) MS assays were developed to support process and product characterization. A potential interaction between HEXB and mAb-1 was initially observed from the analysis of process intermediates by proteomics among several mAbs and later supported by computational modeling. An improved bioprocess was developed to significantly reduce HEXB levels in the final drug substance. A risk assessment was conducted by evaluating the in silico immunogenicity risk and the impact on product quality. To the best of our knowledge, HEXB is the first residual HCP reported to have impact on the glycan profile of a formulated drug product. The combination of different analytical tools, mass spectrometry, and computational modeling provides a general strategy on how to study residual HCP for biotherapeutics development.

| S-EPMC8365702 | biostudies-literature

Identification and characterization of a neutralizing monoclonal antibody that provides complete protection against Yersinia pestis.

Project description:Yersinia pestis (Y. pestis) has caused an alarming number of deaths throughout recorded human history, and novel prophylactics and therapeutics are necessary given its potential as a bioweapon. Only one monoclonal antibody has been identified to date that provides complete protection against Y. pestis. Here, we describe a second novel murine monoclonal antibody (F2H5) that provided complete protection against Y. pestis 141 infection when administered prophylactically to Balb/c mice (100 μg intravenously). We humanized F2H5, characterized its ability to bind to the Y. pestis F1 protein and further characterized the neutralizing epitope using computational and experimental approaches. While Western blot results suggested a linear epitope, peptide mapping using ELISA failed to identify an epitope, suggesting a conformational epitope instead. We adopted a computational approach based on Residue Contact Frequency to predict the site of antigen-antibody interaction and defined the F2H5/F1 binding site computationally. Based on computational approach, we determined that residues G104E105N106 in F1 were critical to F2H5 binding and that CDRH2 and CDRH3 of F2H5 interacted with F1. Our results show that combining computational approach and experimental approach can effectively identify epitopes.

| S-EPMC5423616 | biostudies-literature

Unambiguous Sequence Characterization of a Monoclonal Antibody in a Single Analysis Using a Nonspecific Immobilized Enzyme Reactor.

Project description:Accurate sequence characterization is essential for the development of therapeutic antibodies by the pharmaceutical industry. Presented here is a methodology to obtain comprehensive sequence analysis of a monoclonal antibody. An enzyme reactor of immobilized Aspergillopepsin I, a highly stable nonspecific protease, was used to cleave reduced antibody subunits into a peptide profile ranging from 1 to 20 kDa. Utilizing the Thermo Orbitrap Fusion's unique instrument architecture combined with state-of-the-art instrument control software allowed for dynamic instrument methods that optimally characterize eluting peptides based on their size and charge density. Using a data-dependent instrument method, both collisional dissociation and electron transfer dissociation were used to fragment the appropriate charge state of analyte peptides. The instrument layout also allowed for scans to be taken in parallel using both the ion trap and Orbitrap concurrently, thus allowing larger peptides to be analyzed in high resolution using the Orbitrap while simultaneously analyzing tryptic-like peptides using the ion trap. We harnessed these capabilities to develop a custom method to optimally fragment the eluting peptides based on their mass and charge density. Using this approach, we obtained 100% sequence coverage of the total antibody in a single chromatographic analysis, enabling unambiguous sequence assignment of all residues.

| S-EPMC8092443 | biostudies-literature

Monoclonal Antibodies and Antibody Drug Conjugates in Multiple Myeloma.

Project description:Multiple myeloma is the second most common hematologic malignancy. Current treatment strategies are mainly based on immunomodulatory drugs, proteasome inhibitors or combination of both. Novel agents added to these backbone treatments represent a promising strategy in treatment of newly diagnosed as well as relapsed and refractory multiple myeloma patients. In this respect, the incorporation of monoclonal antibodies into standard-of-care regimens markedly improved prognosis of myeloma patients during the last years. More specifically, monoclonal anti-CD38 antibodies, daratumumab and isatuximab, have been implemented into treatment strategies from first-line treatment to refractory disease. In addition, the monoclonal anti-SLAM-F7 antibody elotuzumab in combination with immunomodulatory drugs has improved the clinical outcomes of patients with relapsed/refractory disease. Belantamab mafodotin is the first approved antibody drug conjugate directed against B cell maturation antigen and is currently used as a monotherapy for patients with advanced disease. This review focuses on clinical efficacy and safety of monoclonal antibodies as well as antibody drug conjugates in multiple myeloma.

| S-EPMC8037134 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data