Gruia F.,Analytical science |
Parupudi A.,Analytical science |
Baca M.,Protein Discovery |
Ward C.,Biosuperiors |
And 3 more authors.
Journal of Pharmaceutical Sciences | Year: 2017
This study explores the structural and functional changes associated with a low-temperature thermal transition of 2 engineered bacterial uricase mutants. Uricase has a noncovalent homotetrameric structure, with 4 active sites located at the interface of subunits. Using differential scanning calorimetry, a low-temperature transition was identified at 42°C for mutant A and at 33°C for mutant B. This transition was stabilized by the uricase inhibitor, oxonic acid, suggesting a strong structural relationship to the active site. For mutant B, there was a reversible loss of enzymatic activity above the low-temperature transition. Spectroscopic measurements demonstrated that there was also a reversible loss of secondary and tertiary structures and an increase in surface hydrophobicity. However, the hydrophobic core environment and the tetrameric structure were not altered over the low-temperature transition suggesting that the changes occurred primarily at the surface of the enzyme. The protein became aggregation-prone at temperatures approaching the cluster of higher-temperature melting transitions at 84°C, indicating these transitions represent a global unfolding of the protein. Our findings shed light on the structural changes that affect the uricase mechanism of action and provide new insights into how enzyme therapeutic development may be approached. © 2017 American Pharmacists Association®.
Metz D.C.,University of Pennsylvania |
Pilmer B.L.,Clinical Science |
Han C.,Analytical Science |
Perez M.C.,Clinical Science
American Journal of Gastroenterology | Year: 2011
Objectives: Withdrawal of proton pump inhibitors (PPIs) may induce symptoms in healthy volunteers, suggesting that discontinuing PPI therapy induces acid-peptic disease. Similar assessments in patients with documented acid-related disorders are lacking. Methods: We performed a retrospective analysis of data from 287 Helicobacter pylori-negative erosive esophagitis (EE) patients healed after 4 or 8 weeks of therapy with dexlansoprazole modified release (MR) or lansoprazole, and then randomized to placebo in 6-month maintenance trials. We compared serum gastrin levels and 24-h heartburn severity before enrollment in the healing trials (baseline) and after receiving placebo in the 6-month maintenance trials. Results: Mean gastrin values at maintenance months 1 and 3 were essentially unchanged (median changes, 1.0 and 1.0 pg/ml), showing that gastrin normalized within 1 month of discontinuing PPIs and remained flat. Mean heartburn severity at maintenance month 1 was <1 on a 5-point scale (1=mild) and significantly lower than at baseline (median decrease, 0.41 points; P≤0.001). Heartburn severity in patients healed at week 4 or 8 with either PPI was generally similar, suggesting that neither longer exposure nor more potent therapy was associated with rebound. In those with month 2 data, mean heartburn severity at months 1 and 2 was significantly lower than baseline (median decrease, 0.54 and 0.58 points; both P<0.001), indicating an ongoing symptom response for 2 months after PPI withdrawal. Conclusions: In H. pylori-negative EE patients, there was no indication of recurring heartburn symptom worsening beyond baseline levels within 2 months of discontinuing 4-8 weeks of PPI therapy. © 2011 by the American College of Gastroenterology.
Golden R.,ToxLogic LLC |
Valentini M.,Analytical science
Regulatory Toxicology and Pharmacology | Year: 2014
Due largely to the controversy concerning the potential human health effects from exposure to formaldehyde gas in conjunction with the misunderstanding of the well-established equilibrium relationship with its hydrated reaction product, methylene glycol, the concept of chemical equivalence between these two distinctly different chemicals has been adopted by regulatory authorities. Chemical equivalence implies not only that any concentration of methylene glycol under some condition of use would be nearly or completely converted into formaldehyde gas, but also that these two substances would be toxicologically equivalent as well. A relatively simple worst case experiment using 37% formalin (i.e., concentrated methylene glycol) dispels the concept of chemical equivalence and a review of relevant literature demonstrates that methylene glycol has no inherent toxicity apart from whatever concentration of formaldehyde that might be present in equilibrium with such solutions. © 2014 Elsevier Inc.
Worsley G.J.,Analytical Science |
Worsley G.J.,DNA Electronics Ltd |
Kumarswami N.,Analytical Science |
Minelli C.,Analytical Science |
Noble J.E.,Analytical Science
Analytical Methods | Year: 2015
The batch-to-batch assay performance 'activity' of antibody conjugated particles is often variable, leading to poor reproducibility between different production batches. We therefore sought to quantify the properties of such particles using differential centrifugal sedimentation (DCS) to see how they influence assay performance, with the aim to improve the reproducibility of the conjugation reaction. The DCS high resolution size distributions of the antibody conjugated particles allowed us to examine the thickness of the antibody corona on the particles and to quantify the amount of 'contaminating' particle oligomers produced via carbodiimide chemistry. The DCS data was correlated with the assay response of the resulting conjugate using an interleukin 6 (IL6) lateral flow assay developed in house. We prepared a series of antibody conjugates using various carbodiimide reaction conditions and analysed the size, antibody corona and oligomerisation profile of the resulting particles using both dynamic light scattering (DLS) and DCS. Both the amount of antibody bound to the particle and the presence of higher order particle oligomers produced conjugates that when applied in an IL6 lateral flow assay were associated with an enhanced fluorescent signal. Both the amount of particle oligomers and antibody bound to the particle was found to be positively correlated with increased assay response in the lateral flow. The DCS estimation of protein corona thickness for each carbodiimide condition tested was found to correlate with the amount of antibody coupled to the particles, as assessed using the bicinchoninic acid (BCA) assay. We have shown the novel application of DCS for the analysis of antibody-particle conjugates. DCS analysis provides a quantitative method to characterise particles and provides a rationale for variable assay performance observed from batch-to-batch production. © 2015 The Royal Society of Chemistry.
Noble J.E.,Analytical Science
Methods in Enzymology | Year: 2014
The measurement of a solubilized protein concentration in solution is an important assay in biochemistry research and development labs for applications ranging from enzymatic studies to providing data for biopharmaceutical lot release. Spectrophotometric protein quantification assays are methods that use UV and visible spectroscopy to rapidly determine the concentration of protein, relative to a standard, or using an assigned extinction coefficient. Where multiple samples need measurement, and/or the sample volume and concentration is limited, preparations of the Coomassie dye commonly known as the Bradford assay can be used. © 2014 Elsevier Inc. All rights reserved.
PubMed | Analytical science, Purification Process science and Protein Discovery
Type: | Journal: Biotechnology progress | Year: 2016
Recombinant therapeutic monoclonal antibodies (mAbs) must be purified from host cell proteins (HCPs), DNA, and other impurities present in Chinese hamster ovary (CHO) cell culture media. HCPs can potentially result in adverse clinical responses in patients and, in specific cases, have caused degradation of the final mAb product. As reported previously, residual traces of cathepsin D caused particle formation in the final product of mAb-1. The current work was focused on identification of a primary sequence in mAb-1 responsible for the binding and consequent co-purification of trace levels of CHO cathepsin D. Surface plasmon resonance (SPR) was used to detect binding between immobilized CHO cathepsin D and a panel of mAbs. Out of 13 mAbs tested, only mAb-1 and mAb-6 bound to cathepsin D. An LYY motif in the HC CDR2 was common, yet unique, to only these two mAbs. Mutation of LYY to AAA eliminated binding of mAb-1 to cathepsin D providing confirmation that this sequence motif was involved in the binding to CHO cathepsin D. Interestingly, the binding between mAb-1 and cathepsin D was weaker than that of mAb-6, which may be related to the fact that two aspartic acid residues near the LYY motif in mAb-1 are replaced with neutral serine residues in mAb-6. 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016.
News Article | December 9, 2015
Citizen scientists are an underrated source of observations on medical conditions. They frequently offer researchers a head start in the hunt for biomarkers (see, for example, the tentative identification of volatile indicators of early Parkinson's disease: go.nature.com/wggoss). The precision and high-throughput capability of analytical technology drives most advances in clinical diagnostics ( et al. Nature 502, 317–320; 2013). Analytical science and its subdiscipline metabolomics (the study of chemical fingerprints left by cellular processes) are also crucial for guiding clinical decisions (see go.nature.com/l8pcde). These tools are set to be valuable for investigating and tapping into citizen scientists' previously unreported medical phenomena.