A5064723795- Monoclonal and Polyclonal Antibodies Research
- Protein purification and stability
- Microfluidic and Capillary Electrophoresis Applications
- Viral Infectious Diseases and Gene Expression in Insects
- Glycosylation and Glycoproteins Research
- Mass Spectrometry Techniques and Applications
- Advanced Proteomics Techniques and Applications
- Myasthenia Gravis and Thymoma
- SARS-CoV-2 and COVID-19 Research
- Peripheral Neuropathies and Disorders
- Influenza Virus Research Studies
- Salivary Gland Tumors Diagnosis and Treatment
- Animal Virus Infections Studies
- Immune Cell Function and Interaction
- Peptidase Inhibition and Analysis
- Metabolomics and Mass Spectrometry Studies
- Pneumonia and Respiratory Infections
- Virus-based gene therapy research
- Enzyme Production and Characterization
- Complement system in diseases
- Enzyme Structure and Function
- Neonatal Health and Biochemistry
- Advanced Glycation End Products research
- Neonatal and Maternal Infections
- Viral gastroenteritis research and epidemiology
Netherlands Metabolomics Centre
2019-2024
Leiden University Medical Center
2019-2024
Roche (Switzerland)
2017
Characterization of unknown monoclonal antibody (mAb) variants is important in order to identify their potential impact on safety, potency, and stability. Ion exchange chromatography (IEC) coupled with UV detection frequently used separate quantify mAb routine quality control (QC). However, characterization the chromatographic peaks resulting from an IEC separation extremely time-consuming process, involving many cumbersome steps. Presented here online four-dimensional high performance...
As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is still ongoing and dramatically influences our life, need for recombinant viral proteins diagnostics, vaccine development, research very high. The spike (S) protein, particularly its receptor-binding domain (RBD), mediates interaction with angiotensin-converting enzyme (ACE2) receptor on host cells may be modulated by structural features. Therefore, well-characterized RBDs are essential. We have performed an...
Significance An expanding group of autoimmune diseases is now recognized to be hallmarked by pathogenic IgG4 autoantibodies. has the unique ability exchange Fab-arms, rendering it bispecific and functionally monovalent. Here we show that autoantibody functional monovalency significantly amplifies pathogenicity autoantibodies using patient-derived monoclonal antibodies in an vivo model MuSK myasthenia gravis. Therefore, subclass switching predominant a critical step development This new...
Bispecific monoclonal antibodies (BsAbs) are engineered proteins with multiple functionalities and properties. The "bi-specificity" of these complex biopharmaceuticals is a key characteristic for the development novel more effective therapeutic strategies. high structural complexity BsAbs poses challenge to analytical methods needed their characterization. Modifications BsAb structure, resulting from enzymatic non-enzymatic processes, further complicate analysis. An important example latter...
Characterization of post-translational modifications (PTMs) therapeutic antibodies is commonly performed by bottom-up approaches, involving sample preparation and peptide analysis liquid chromatography-mass spectrometry (LC-MS). Conventional requires extensive hands-on time can increase the risk inducing artificial as many off-line steps - denaturation, disulfide-reduction, alkylation tryptic digestion are performed. In this study, we developed an on-line multidimensional (mD)-LC-MS approach...
Assessment of critical quality attributes the biopharmaceutical erythropoietin (EPO) prior to product release requires use several analytical methods. We developed an MS-compatible anion exchange (AEX) method for monitoring multiple EPO biopharmaceuticals. AEX was performed using a stationary phase with quaternary ammonium functional groups and pH gradient elution. Baseline separation charge variants high-quality MS data were achieved 30 mM formate 5.5 formic acid 2.5 as mobile phases. In...
Biotherapeutics may contain a multitude of different post-translational modifications (PTMs) that need to be assessed and possibly monitored controlled ensure reproducible product quality. During early development biotherapeutics, unexpected PTMs might prevented by in silico identification characterization together with further molecular engineering. Mass determinations human IgG1 (mAb1) bispecific IgG-ligand fusion protein (BsAbA) demonstrated the presence unusual resulting major +80 Da,...
Monoclonal antibody (mAb) pharmaceuticals consist of a plethora different proteoforms with functional characteristics, including pharmacokinetics and pharmacodynamics, requiring their individual assessment. Current binding techniques do not distinguish between coexisting tedious production enriched proteoforms. Here, we have developed an approach based on mobility shift-affinity capillary electrophoresis–mass spectrometry (ACE–MS), which permitted us to determine the mAb Fc receptors (FcRs)....
Group B Streptococcus (GBS) is a Gram-positive bacterium and the most common cause of neonatal blood brain infections. At least 10 different serotypes exist, that are characterized by their capsular polysaccharides. The carbohydrate (GBC) shared all therefore attractive be used in glycoconjugate vaccine. GBC highly complex multiantennary structure, composed rhamnose rich oligosaccharides interspaced with glucitol phosphates. We here report development convergent approach to assemble...
The antibody- FcγRIIIa interaction triggers key immunological responses such as antibody dependent cellular cytotoxicity (ADCC), making it highly important for therapeutic mAbs. Due to the direct glycan-glycan with receptor, differences in glycosylation can drastically influence binding affinity. Understanding differential of mAb glycoforms is a very important, yet challenging task due co-existence multiple sample. Affinity liquid chromatography (AC) and affinity capillary electrophoresis...
The impact of antibody glycoforms on FcγRIIa activation and immune responses is poorly understood. Yet, glycoform binding assessment remains one the major analytical challenges requiring long enrichment or glycoengineering steps. Here, we developed applied an affinity capillary electrophoresis-mass spectrometry approach to selectively assess different FcγIIa receptor without need glycoengineering. required only low microgram amounts enables assessing high low-abundance glycoforms. indicated...
Bispecific monoclonal antibodies (BsAbs) are receiving great attention due to their extensive benefits as biopharmaceuticals and involvement in IgG4 mediated autoimmune diseases. While the production of BsAbs is getting more accessible, analytical characterization remains challenging. We explored potential sheathless CE-MS for monitoring exchange efficiency stability in-house produced bispecific antibodies. Two with different molecular characteristics were prepared using controlled Fragment...
Complement activation protects against infection but also contributes to pathological mechanisms in a range of clinical conditions such as autoimmune diseases and transplant rejection. Complement-inhibitory drugs, either approved or development, usually act systemically, thereby increasing the risk for infections. We therefore envisioned novel class bispecific antibodies (bsAbs) which are capable site-directed complement inhibition by bringing endogenous regulators vicinity defined cell...
The conserved region (Fc) of IgG antibodies dictates the interactions with designated receptors thus defining immunological effector functions IgG. Amino acid sequence variations in Fc, recognized as subclasses and allotypes, well post-translational modifications (PTMs) modulate these interactions. Yet, high similarity Fc sequences hinders allotype-specific PTM analysis by state-of-the-art bottom-up methods current subunit approaches lack sensitivity face co-elution near-isobaric allotypes....
Abstract Chemically defined media are reconstituted batchwise and stored in hold tanks until use. To avoid large production of media, we developed continuous on‐demand reconstitutions directly from solids consisting a hopper screw conveyor capable feeding dry powdered with the required precision ±5% at low dosing rates 0.171 g min −1 . A commercially available cell culture medium was continuously fed over duration 12 h into mixer which connected to UV‐cell for monitoring were compared...
After decades of research, gene therapy products have reached market maturity in recent years. Recombinant adeno-associated viruses (rAAVs) are one the most promising delivery vehicles and currently under intense scientific investigation. These next-generation medicines remain very challenging when it comes to designing appropriate analytical techniques for quality control. One critical attribute is integrity ssDNA incorporated these vectors. The genome active compound driving rAAV therefore...
Abstract As the SARS-CoV-2 pandemic is still ongoing and dramatically influences our life, need for recombinant proteins diagnostics, vaccine development, research very high. The spike (S) protein, particularly its receptor binding domain (RBD), mediates interaction with ACE2 on host cells may be modulated by structural features. Therefore, well characterized RBDs are essential. We have performed an in-depth functional characterization of expressed in Chinese hamster ovary (CHO) human...
Abstract Human IgG4 usually displays anti-inflammatory activity, and observations of autoantibodies causing severe autoimmune disorders are therefore poorly understood. In blood, antibodies naturally engage in a stochastic process termed Fab-arm exchange which unrelated IgG4s half-molecules continuously. The resulting composed two different binding sites, thereby acquiring monovalent inability to cross-link for each antigen recognized. Here, we demonstrate this amplifies autoantibody...