High-Throughput Screening and Analysis of Charge Variants of Monoclonal Antibodies in Multiple Formulations.

Among different biopharmaceutical products, monoclonal antibodies (mAbs) show a high level of complexity, including heterogeneity due to differences in size, hydrophobicity, charge, and so forth. Such heterogeneity can be related to both cell-based production and any of the stages of purification, storage, and delivery that the mAb is subjected to. Choosing the right formulation composition providing both physical and chemical stabilities can be a very challenging process, especially when done in the limited time frame required for a typical drug development cycle.

Effects of sucrose and benzyl alcohol on GCSF conformational dynamics revealed by hydrogen deuterium exchange mass spectrometry.

Protein stability, one of the major concerns for therapeutic protein development, can be optimized during process development by evaluating multiple formulation conditions. This can be a costly and lengthy procedure where different excipients and storage conditions are tested for their impact on protein stability. A better understanding of the effects of different formulation conditions at the molecular level will provide information on the local interactions within the protein leading to a more rational design of stable and efficacious formulations.

Accelerated formulation development of monoclonal antibodies (mAbs) and mAb-based modalities: review of methods and tools.

More therapeutic monoclonal antibodies and antibody-based modalities are in development today than ever before, and a faster and more accurate drug discovery process will ensure that the number of candidates coming to the biopharmaceutical pipeline will increase in the future. The process of drug product development and, specifically, formulation development is a critical bottleneck on the way from candidate selection to fully commercialized medicines. This article reviews the latest advances in methods of formulation screening, which allow not only the high-throughput selection of the most suitable formulation but also the prediction of stability properties under manufacturing and long-term storage conditions.

High-throughput screening and stability optimization of anti-streptavidin IgG1 and IgG2 formulations.

Selection of a suitable formulation that provides adequate product stability is an important aspect of the development of biopharmaceutical products. Stability of proteins includes not only resistance to chemical modifications but also conformational and colloidal stabilities. While chemical degradation of antibodies is relatively easy to detect and control, propensity for conformational changes and/or aggregation during manufacturing or long-term storage is difficult to predict.

Methods of high throughput biophysical characterization in biopharmaceutical development.

Discovery and successful development of biopharmaceutical products depend on a thorough characterization of the molecule both before and after formulation. Characterization of a formulated biotherapeutic, typically a protein or large peptide, requires a rigorous assessment of the molecule's physical stability. Stability of a biotherapeutic includes not only chemical stability, i.

High-throughput assessment of thermal and colloidal stability parameters for monoclonal antibody formulations.

Design of experiment and statistical analyses were applied to evaluate the effects of several formulation components on the thermal and colloidal stability for a series of monoclonal antibody (mAb) formulations. The high-throughput assessment of the protein stability was performed by measuring the temperature of hydrophobic exposure (T(h) , thermal stability) and the diffusion interaction parameter (k(D) , colloidal stability). To correlate the measured parameters with protein stability, the propensity to aggregate was tested by exposing the mAb samples to two types of stress: mechanical stress caused by shaking agitation and thermal stress.

Screening of monoclonal antibody formulations based on high-throughput thermostability and viscosity measurements: design of experiment and statistical analysis.

The purpose of this study was to demonstrate the utility of combining a design of experiment (DOE) approach with high-throughput formulation screening to identify the main factors affecting protein thermostability and solution viscosity. The optimization of buffer compositions was guided by statistical analysis of the data to obtain the targeted combination of low viscosity and high thermostability. Different monoclonal antibody (mAb) formulation variables were evaluated in the study to achieve optimization of two parameters: (i) thermostability characterized by temperature of hydrophobic exposure and (ii) viscosity.

Detection of IgG aggregation by a high throughput method based on extrinsic fluorescence.

The utility of extrinsic fluorescence as a tool for high throughput detection of monoclonal antibody aggregates was explored. Several IgG molecules were thermally stressed and the high molecular weight species were fractionated using size-exclusion chromatography (SEC). The isolated aggregates and monomers were studied by following the fluorescence of an extrinsic probe, SYPRO Orange.

High-throughput dynamic light scattering method for measuring viscosity of concentrated protein solutions.

We propose a new method to measure the viscosity of concentrated protein solutions in a high-throughput format. This method measures the apparent hydrodynamic radius of polystyrene beads with known sizes using a dynamic light scattering (DLS) system with a microplate reader. Glycerol solution viscosities obtained by the DLS method were in good agreement with those reported in the literature.

Stable isotopic labeling of proteins for quantitative proteomic applications.

Straightforward methods for the introduction of stable isotopes into proteins with subsequent isolation and purification of the proteins will greatly aid the field of quantitative proteomics. Proteins containing amino acids with one or more of the stable isotopes of deuterium, 13C, 15N or 18O can be used as internal standards by addition at an early stage of analysis of a complex protein sample and subsequent measurement using mass spectrometry. There are two approaches for introducing a stable isotope into a protein without chemically modifying that protein, metabolic labeling using whole cells grown in culture, or cell-free labeling using a lysate of either Escherichia coli or wheat germ.

Characterization of the enzymatic activity of human kallikrein 6: Autoactivation, substrate specificity, and regulation by inhibitors.

Human kallikrein 6 (hK6) is a trypsin-like serine protease, member of the human kallikrein gene family. Studies suggested a potential involvement of hK6 in the development and progression of Alzheimer's disease. The serum levels of hK6 might be used as a biomarker for ovarian cancer.

Decoy receptor 3 (DcR3) is proteolytically processed to a metabolic fragment having differential activities against Fas ligand and LIGHT.

Fas ligand (FasL) and Fas receptor are members of the tumor necrosis factor (TNF) receptor and ligand family that play an important role in regulating apoptosis in normal physiology. Decoy receptor 3 (DcR3) is a novel member of the TNF receptor superfamily, which binds to and blocks the activities of the ligands FasL and LIGHT. We have demonstrated that DcR3 was degraded rapidly to a major circulating metabolic fragment after subcutaneous administration in primates and mice.

Prostate-specific antigen (PSA)-mediated proliferation, androgenic regulation and inhibitory effects of LY312340 in HOS-TE85 (TE85) human osteosarcoma cells.

Background: PSA mediates growth factor responses that stimulate proliferation of prostatic and other cellular types. Androgen-sensitive TE85 human osteosarcoma cells were used to study PSA as a potential mediator of prostatic cancer growth and osseous metastasis.

Materials And Methods: TE85 cells were probed for PSA mRNA and protein levels under testosterone (T)-replete and--depleted conditions.

Alloxan is an inhibitor of the enzyme O-linked N-acetylglucosamine transferase.

We have previously shown that diabetogenic antibiotic streptozotocin (STZ), an analog of N-acetylglucosamine (GlcNAc), inhibits the enzyme O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase (O-GlcNAcase) which is responsible for the removal of O-GlcNAc from proteins. Alloxan, another beta-cell toxin is a uracil analog. Since the O-GlcNAc transferase (OGT) uses UDP-GlcNAc as a substrate, we investigated whether alloxan might interfere with the process of protein O-glycosylation by blocking OGT, a very abundant enzyme in beta-cells.

Characterization of a novel endocannabinoid, virodhamine, with antagonist activity at the CB1 receptor.

The first endocannabinoid, anandamide, was discovered in 1992. Since then, two other endocannabinoid agonists have been identified, 2-arachidonyl glycerol and, more recently, noladin ether. Here, we report the identification and pharmacological characterization of a novel endocannabinoid, virodhamine, with antagonist properties at the CB1 cannabinoid receptor.