Preferential interactions of trehalose, L-arginine.HCl and sodium chloride with therapeutically relevant IgG1 monoclonal antibodies.

Preferential interactions of weakly interacting formulation excipients govern their effect on the equilibrium and kinetics of several reactions of protein molecules in solution. Using vapor pressure osmometry, we characterized the preferential interactions of commonly used excipients trehalose, L-arginine.HCl and NaCl with three therapeutically-relevant, IgG1 monoclonal antibodies that have similar size and shape, but differ in their surface hydrophobicity and net charge.

Utility of High Throughput Screening Techniques to Predict Stability of Monoclonal Antibody Formulations During Early Stage Development.

Selecting optimal formulation conditions for monoclonal antibodies for first time in human clinical trials is challenging due to short timelines and reliance on predictive assays to ensure product quality and adequate long-term stability. Accelerated stability studies are considered to be the gold standard for excipient screening, but they are relatively low throughput and time consuming. High throughput screening (HTS) techniques allow for large amounts of data to be collected quickly and easily, and can be used to screen solution conditions for early formulation development.

Molecular Investigation of the Mechanism of Non-Enzymatic Hydrolysis of Proteins and the Predictive Algorithm for Susceptibility.

A number of potential degradation routes can limit the shelf life of a biotherapeutic. While these are experimentally measurable, the tests to do so require a substantial investment in both time and material, resources rarely available early in the drug development process. To address the potential degradation route of non-enzymatic hydrolysis, we performed a molecular modeling analysis, together with an experimental study, to gain detailed insight into the reaction.

Computational tool for the early screening of monoclonal antibodies for their viscosities.

Highly concentrated antibody solutions often exhibit high viscosities, which present a number of challenges for antibody-drug development, manufacturing and administration. The antibody sequence is a key determinant for high viscosity of highly concentrated solutions; therefore, a sequence- or structure-based tool that can identify highly viscous antibodies from their sequence would be effective in ensuring that only antibodies with low viscosity progress to the development phase. Here, we present a spatial charge map (SCM) tool that can accurately identify highly viscous antibodies from their sequence alone (using homology modeling to determine the 3-dimensional structures).

Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody.

There is a need for new analytical approaches to better characterize the nature of the concentration-dependent, reversible self-association (RSA) of monoclonal antibodies (mAbs) directly, and with high resolution, when these proteins are formulated as highly concentrated solutions. In the work reported here, hydrogen exchange mass spectrometry (HX-MS) was used to define the concentration-dependent RSA interface, and to characterize the effects of association on the backbone dynamics of an IgG1 mAb (mAb-C). Dynamic light scattering, chemical cross-linking, and solution viscosity measurements were used to determine conditions that caused the RSA of mAb-C.

Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life.

This study compares the local conformational dynamics and physical stability of an IgG1 mAb (mAb-A) with its corresponding YTE (M255Y/S257T/T259E) mutant (mAb-E), which was engineered for extended half-life in vivo. Structural dynamics was measured using hydrogen/deuterium (H/D) exchange mass spectrometry while protein stability was measured with differential scanning calorimetry (DSC) and size exclusion chromatography (SEC). The YTE mutation induced differences in H/D exchange kinetics at both pH 6.

A systematic multitechnique approach for detection and characterization of reversible self-association during formulation development of therapeutic antibodies.

In addition to controlling typical instabilities such as physical and chemical degradations, understanding monoclonal antibodies' (mAbs) solution behavior is a key step in designing and developing process and formulation controls during their development. Reversible self-association (RSA), a unique solution property in which native, reversible oligomeric species are formed as a result of the noncovalent intermolecular interactions has been recognized as a developability risk with the potential to negatively impact manufacturing, storage stability, and delivery of mAbs. Therefore, its identification, characterization, and mitigation are key requirements during formulation development.

Correlating excipient effects on conformational and storage stability of an IgG1 monoclonal antibody with local dynamics as measured by hydrogen/deuterium-exchange mass spectrometry.

The effects of sucrose and arginine on the conformational and storage stability of an IgG1 monoclonal antibody (mAb) were monitored by differential scanning calorimetry (DSC) and size-exclusion chromatography (SEC), respectively. Excipient effects on protein physical stability were then compared with their effects on the local flexibility of the mAb in solution at pH 6, 25°C using hydrogen/deuterium-exchange mass spectrometry (H/D-MS). Compared with a 0.

Effects of salts from the Hofmeister series on the conformational stability, aggregation propensity, and local flexibility of an IgG1 monoclonal antibody.

This work examines the effect of three anions from the Hofmeister series (sulfate, chloride, and thiocyanate) on the conformational stability and aggregation rate of an IgG1 monoclonal antibody (mAb) and corresponding changes in the mAb's backbone flexibility (at pH 6 and 25 °C). Compared to a 0.1 M NaCl control, thiocyanate (0.

A systematic multitechnique approach for detection and characterization of reversible self-association during formulation development of therapeutic antibodies.

In addition to controlling typical instabilities such as physical and chemical degradations, understanding monoclonal antibodies' (mAbs) solution behavior is a key step in designing and developing process and formulation controls during their development. Reversible self-association (RSA), a unique solution property in which native, reversible oligomeric species are formed as a result of the noncovalent intermolecular interactions has been recognized as a developability risk with the potential to negatively impact manufacturing, storage stability, and delivery of mAbs. Therefore, its identification, characterization, and mitigation are key requirements during formulation development.

Local dynamics and their alteration by excipients modulate the global conformational stability of an lgG1 monoclonal antibody.

A molecular understanding of excipient effects on the interrelationship(s) between dynamics and conformational stability of proteins, such as monoclonal antibodies (mAbs), can be important for their pharmaceutical development. The current study examines stabilizing and destabilizing effects of excipients on the conformational stability and local dynamics of distinct solvent-exposed regions within an IgG1 monoclonal antibody (mAb-B). The principles of site-selective photoselection upon red-edge excitation, accompanied by acrylamide quenching of tryptophan fluorescence were employed in this study.

Advancements in high throughput biophysical technologies: applications for characterization and screening during early formulation development of monoclonal antibodies.

The formulation development of monoclonal antibodies is extremely challenging, due to the diversity and complexity contained within this class of molecules. The physical and chemical properties of a monoclonal antibody dictate the behavior of the protein drug during manufacturing, storage and clinical administration. In the past few years, the use of high throughput technologies has been widely adapted to delineate unique properties of individual immunoglobulin G's (IgG's) important for their development.

The effects of substituted cyclodextrins on the colloidal and conformational stability of selected proteins.

The effects of various types of substituted and nonsubstituted cyclodextrins (CDs) on the physical and colloidal stability of three different proteins were studied to further ascertain the mechanism by which cyclodextrins stabilize proteins. The three proteins examined in this study are the Clostridium difficile Toxoid A, Yersinia pestis low-calcium-response V or V antigen (LcrV), and fibroblast growth factor 10 (FGF-10). These three pharmaceutically relevant proteins differ in molecular weight, pI, as well as in their secondary and tertiary structure.

Biophysical and stabilization studies of the Chlamydia trachomatis mouse pneumonitis major outer membrane protein.

Native Chlamydia trachomatis mouse pneumonitis major outer membrane protein (nMOMP) induces effective protection against genital infection in a mouse challenge model. The conformation of nMOMP is crucial to confer this protective immunity. To achieve a better understanding of the conformational behavior and stability of nMOMP, a number of spectroscopic techniques are employed to characterize the secondary structure (circular dichroism), tertiary structure (intrinsic fluorescence) and aggregation properties (static light scattering and optical density) as a function of pH (3-8) and temperature (10-87.

The role of specific beta-gamma subunit interactions in oxyanion stimulation of the MgATP hydrolysis of a hybrid photosynthetic F1-ATPase.

Pairs of cysteine residues were introduced into the twisted N- and C-terminal helices of the gamma subunit of the chloroplast F1-ATPase to test, via disulfide cross-linking, potential inter-helical movements involved in catalysis of ATP hydrolysis. The extent of disulfide cross-linking was determined by estimating the amount of free sulfhydryl available for labeling with fluoresceinyl maleimide before and after cross-linking. Significant disulfide formation (50-75%) was observed between cysteines introduced at positions 30 and 31 in the N-terminal helix and 276 and 278 in the C-terminal helix.

C-Terminal mutations in the chloroplast ATP synthase gamma subunit impair ATP synthesis and stimulate ATP hydrolysis.

Two highly conserved amino acid residues, an arginine and a glutamine, located near the C-terminal end of the gamma subunit, form a "catch" by hydrogen bonding with residues in an anionic loop on one of the three catalytic beta subunits of the bovine mitochondrial F1-ATPase [Abrahams, J. P., Leslie, A.

Mutations within the C-terminus of the gamma subunit of the photosynthetic F1-ATPase activate MgATP hydrolysis and attenuate the stimulatory oxyanion effect.

Two highly conserved amino acid residues near the C-terminus within the gamma subunit of the mitochondrial ATP synthase form a "catch" with an anionic loop on one of the three beta subunits within the catalytic alphabeta hexamer of the F1 segment [Abrahams, J. P., Leslie, A.

Structural analysis of the regulatory dithiol-containing domain of the chloroplast ATP synthase gamma subunit.

The gamma subunit of the F1 portion of the chloroplast ATP synthase contains a critically placed dithiol that provides a redox switch converting the enzyme from a latent to an active ATPase. The switch prevents depletion of intracellular ATP pools in the dark when photophosphorylation is inactive. The dithiol is located in a special regulatory segment of about 40 amino acids that is absent from the gamma subunits of the eubacterial and mitochondrial enzymes.

Ectoadenylate kinase and plasma membrane ATP synthase activities of human vascular endothelial cells.

Formation of ATP from ADP on the external surface of vascular endothelial cells has been attributed to plasma membrane ATP synthase, ectoadenylate kinase (ecto-AK), and/or ectonucleoside diphosphokinase. These enzymes or their catalytic products have been causatively linked to the elaboration of vascular networks and the regulation of capillary function. The amount of ATP generated extracellularly is small, requiring sensitive analytical methods for quantification.

Coupling proton movement to ATP synthesis in the chloroplast ATP synthase.

The chloroplast F(0)F(1)-ATP synthase-ATPase is a tiny rotary motor responsible for coupling ATP synthesis and hydrolysis to the light-driven electrochemical proton gradient. Reversible oxidation/reduction of a dithiol, located within a special regulatory domain of the gamma subunit of the chloroplast F(1) enzyme, switches the enzyme between an inactive and an active state. This regulatory mechanism is unique to the ATP synthases of higher plants and its physiological significance lies in preventing nonproductive depletion of essential ATP pools in the dark.