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1.
Bioanalysis ; 14(15): 1067-1079, 2022 Aug.
Article in English | MEDLINE | ID: mdl-36125040

ABSTRACT

Background: Respiratory syncytial virus (RSV) vaccine is an unmet medical need. The virus reduction neutralization test (VRNT) was developed to replace the LI-COR microneutralization assay to measure RSV neutralization titers. Methods: A bridging study using selected V171 phase I samples and calibration studies using the WHO international standard antiserum to RSV were performed to compare VRNT and LI-COR. Results: From the bridging study, we showed good concordance between VRNT and LI-COR titers, and similar post-/prevaccination titer ratios. From the calibration studies, we can convert VRNT and LI-COR titers into similar IU/ml. Conclusion: The VRNT and LI-COR microneutralization assay correlate well and the titers can be standardized as similar IU/ml, enabling direct comparison of titers from different assays.


Subject(s)
Respiratory Syncytial Viruses , Vaccines , Antibodies, Neutralizing , Calibration , Neutralization Tests , World Health Organization
2.
Biologicals ; 74: 1-9, 2021 Nov.
Article in English | MEDLINE | ID: mdl-34716091

ABSTRACT

There is an increasing demand for monoclonal antibody (mAb) therapies to confer passive immunity against viral diseases. Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis, lower respiratory tract infections, and hospitalization in infants. Currently, there is no RSV vaccine but a humanized mAb available for high risk infants. MK-1654 is a fully human mAb with YTE mutation in the fragment crystallizable (Fc) region to extend the half-life in circulation. It binds to a highly conserved epitope of RSV Fusion protein with high affinity and neutralizes RSV infection. A functional cell-based assay is a regulatory requirement for clinical development, commercial release, and stability testing of MK-1654. In this study, we have evaluated three RSV neutralization assays to test the potency of MK-1654, including an imaging-based virus reduction neutralization test (VRNT) and two reporter virus-based assays (RSV-GFP and RSV-NLucP). All three methods showed good dose response curves of MK-1654 with similar EC50 values. RSV-NLucP method was chosen for further development because it is simple and can be easily adapted to quality control testing laboratories. After optimization, the RSV-NLucP assay was pre-qualified with good linearity, relative accuracy, intermediate precision, and specificity, therefore suitable for a cell-based potency assay.


Subject(s)
Antibodies, Monoclonal , Antibodies, Viral , Respiratory Syncytial Virus Infections , Respiratory Syncytial Virus, Human , Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/pharmacology , Humans , Neutralization Tests , Respiratory Syncytial Virus Infections/drug therapy , Viral Fusion Proteins/immunology
3.
J Immunol Methods ; 494: 113054, 2021 07.
Article in English | MEDLINE | ID: mdl-33845088

ABSTRACT

Respiratory syncytial virus (RSV) is a common pathogen causing severe respiratory illness in infants and elder adults. The development of an effective RSV vaccine is an important unmet medical need and an area of active research. The traditional method for testing neutralizing antibodies against RSV in clinical trials is the plaque reduction neutralization test (PRNT), which uses 24-well plates and needs several days post infection to develop viral plaques. In this study, we have developed a virus reduction neutralization test (VRNT), which allows the number of RSV infected cells to be automatically counted by an imaging cytometer at one day post infection in 96-well plates. VRNT was found robust to cell seeding density, detection antibody concentration, virus input and infection time. By testing twenty human sera, we have shown good correlation between VRNT50 and PRNT50 titers for multiple RSV strains: A2, Long and 18537 (serotype B). To understand the VRNT performance, eight human serum samples with high, medium and low neutralization titers were selected for VRNT qualification. We have demonstrated that VRNT had good specificity, precision, linearity and relative accuracy. In conclusion, VRNT is a better alternative to PRNT in serum neutralization test for RSV vaccine candidates.


Subject(s)
Neutralization Tests/methods , Respiratory Syncytial Virus Infections/diagnosis , Respiratory Syncytial Virus Vaccines/immunology , Respiratory Syncytial Viruses/physiology , Aged, 80 and over , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Chlorocebus aethiops , Diagnostic Imaging , High-Throughput Screening Assays , Humans , Infant , Infant, Newborn , Miniaturization , Reproducibility of Results , Sensitivity and Specificity , Time Factors , Vero Cells , Viral Plaque Assay
4.
ACS Pharmacol Transl Sci ; 3(6): 1310-1317, 2020 Dec 11.
Article in English | MEDLINE | ID: mdl-33344904

ABSTRACT

The robustness of good laboratory practice and clinical data is reliant upon a clear understanding of the bioanalytical assays. One of the most important components of ligand-binding based assays is critical reagents used to directly or indirectly measure biologic markers or signals. High quality, reproducible, sustainable critical reagents through the development lifecycle could avoid unnecessary rework, multiple validations, cross-validations, and ensure consistency of the data. Numerous analytical methods (UPLC-size exclusion chromatography, cation exchange chromatography, biacore/octet, and high-resolution mass spectrometry) have been evaluated by using current critical reagents. A comprehensive analytical toolbox of biochemical and biophysical methods has been employed to evaluate the quality of critical reagents and explore potential issues if there are any. Moving forward, this "tiered approach" of critical reagents characterization will be used not only to establish critical quality attributes for new reagents but also to evaluate stability in support of reagents recertification.

5.
MAbs ; 10(4): 513-538, 2018.
Article in English | MEDLINE | ID: mdl-29513619

ABSTRACT

Process changes are inevitable in the life cycle of recombinant monoclonal antibody therapeutics. Products made using pre- and post-change processes are required to be comparable as demonstrated by comparability studies to qualify for continuous development and commercial supply. Establishment of comparability is a systematic process of gathering and evaluating data based on scientific understanding and clinical experience of the relationship between product quality attributes and their impact on safety and efficacy. This review summarizes the current understanding of various modifications of recombinant monoclonal antibodies. It further outlines the critical steps in designing and executing successful comparability studies to support process changes at different stages of a product's lifecycle.


Subject(s)
Antibodies, Monoclonal/chemistry , Chemistry, Pharmaceutical/methods , Chemistry, Pharmaceutical/standards , Quality Assurance, Health Care/methods , Recombinant Proteins/chemistry , Drug Discovery , Humans , Protein Processing, Post-Translational
6.
MAbs ; 9(8): 1217-1230, 2017.
Article in English | MEDLINE | ID: mdl-28853987

ABSTRACT

Forced degradation studies have become integral to the development of recombinant monoclonal antibody therapeutics by serving a variety of objectives from early stage manufacturability evaluation to supporting comparability assessments both pre- and post- marketing approval. This review summarizes the regulatory guidance scattered throughout different documents to highlight the expectations from various agencies such as the Food and Drug Administration and European Medicines Agency. The various purposes for forced degradation studies, commonly used conditions and the major degradation pathways under each condition are also discussed.


Subject(s)
Antibodies, Monoclonal/metabolism , Chemistry, Pharmaceutical/methods , Recombinant Proteins/metabolism , Technology, Pharmaceutical/methods , Antibodies, Monoclonal/genetics , Guidelines as Topic , Humans , Hydrogen-Ion Concentration , Protein Stability , Proteolysis , Temperature
7.
Langmuir ; 25(5): 2994-3002, 2009 Mar 03.
Article in English | MEDLINE | ID: mdl-19437769

ABSTRACT

Mimicking the in vivo stem cell niche to increase stem cell expansion will likely require the presentation of multiple ligands. Presenting ligands in fluid-supported lipid monolayers (SLMs) or bilayers (SLBs) allows for ligand diffusion to complement the arrangement of cell receptors as well as cell-mediated ligand rearrangement and clustering. Cells in tissues interact with ligands presented by other cells and the extracellular matrix (ECM), so it will likely be beneficial to present both cell-associated and ECM-derived ligands. A number of investigators have incorporated cell-membrane-associated ligands within fluid surfaces, and several groups have shown that these ligands cluster beneath the cells. However, few studies have investigated cell adhesion to ECM-derived ligands in fluid surfaces. Fibronectin is an important ECM component in many tissues, including the hematopoietic stem cell niche. We examined the adhesion of the M07e and THP-1 hematopoietic progenitor cell lines to fibronectin-derived peptide ligands for the alpha5beta1 (cyclic and linear RGD) and alpha4beta1 (cyclic LDV) integrins as well as the heparin-binding domain (HBD) presented as lipopeptides in fluid and gel SLMs. M07e cells adhered more avidly than THP-1 cells to all of the lipopeptides in fluid and gel surfaces. The adhesion of both cell lines to all peptides was less avid in fluid versus gel SLMs. Adhesion to cyclic LDV (cLDV) and cRGD was similar on gel SLMs for both cell lines. In contrast, adhesion to cLDV was less extensive than to cRGD in fluid SLMs, especially for M07e cells. Adhesion to linear RGD was less avid than to cRGD or cLDV and decreased to a greater extent in fluid SLMs. Human aortic endothelial cells adhered to cRGD in fluid SLMs and remained viable for at least 24 h but did not spread. We also showed additive THP-1 cell adhesion to cLDV and linear RGD lipopeptides presented in a fluid SLM. Although DOPC (dioleoyl phosphatidyl choline) SLMs are not sufficiently stable for long-term cell culture studies, our results and those of others suggest that fluid SLMs are likely to be useful for presenting multiple ligands and for mimicking short-term interactions in the stem cell niche.


Subject(s)
Fibronectins/chemistry , Hematopoietic Stem Cells/metabolism , Integrin alpha4beta1/chemistry , Integrin alpha5beta1/chemistry , Lipid Bilayers/chemistry , Peptides/chemistry , Cell Adhesion , Cell Line, Tumor , Extracellular Matrix/metabolism , Heparin/chemistry , Humans , Ligands , Lipopeptides/chemistry , Silanes/chemistry , Stem Cells/metabolism
8.
Curr Opin Biotechnol ; 19(5): 534-40, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18725291

ABSTRACT

Niches regulate lineage-specific stem cell self-renewal versus differentiation in vivo and are composed of supportive cells and extracellular matrix components arranged in a three-dimensional topography of controlled stiffness in the presence of oxygen and growth factor gradients. Mimicking stem cell niches in a defined manner will facilitate production of the large numbers of stem cells needed to realize the promise of regenerative medicine and gene therapy. Progress has been made in mimicking components of the niche. Immobilizing cell-associated Notch ligands increased the self-renewal of hematopoietic (blood) stem cells. Culture on a fibrous scaffold that mimics basement membrane texture increased the expansion of hematopoietic and embryonic stem cells. Finally, researchers have created intricate patterns of cell-binding domains and complex oxygen gradients.


Subject(s)
Biomimetics/methods , Biotechnology/methods , Cell Culture Techniques/methods , Stem Cell Niche/physiology , Stem Cells/cytology , Stem Cells/physiology , Tissue Engineering/methods
9.
Science ; 318(5849): 426-30, 2007 Oct 19.
Article in English | MEDLINE | ID: mdl-17947576

ABSTRACT

We report a method to form multifunctional polymer coatings through simple dip-coating of objects in an aqueous solution of dopamine. Inspired by the composition of adhesive proteins in mussels, we used dopamine self-polymerization to form thin, surface-adherent polydopamine films onto a wide range of inorganic and organic materials, including noble metals, oxides, polymers, semiconductors, and ceramics. Secondary reactions can be used to create a variety of ad-layers, including self-assembled monolayers through deposition of long-chain molecular building blocks, metal films by electroless metallization, and bioinert and bioactive surfaces via grafting of macromolecules.


Subject(s)
Dopamine/chemistry , Polymers/chemistry , Adhesiveness , Animals , Biopolymers/chemistry , Cell Adhesion , Cell Adhesion Molecules/chemistry , Cell Line , Ceramics/chemistry , Dihydroxyphenylalanine/chemistry , Fibroblasts/physiology , Humans , Hyaluronic Acid/chemistry , Hydrogen-Ion Concentration , Metals/chemistry , Mytilus edulis/chemistry , Mytilus edulis/physiology , Oxidation-Reduction , Oxides/chemistry , Proteins/chemistry , Semiconductors , Surface Properties
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