Technology development to evaluate the effectiveness of viscosity reducing excipients.

Addition of pharmaceutical excipients is a commonly used approach to decrease the viscosity of highly concentrated protein formulations, which otherwise could not be subcutaneously injected or processed. The variety of protein-protein interactions, which are responsible for increased viscosities, makes a portfolio approach necessary. Screening of several excipients to develop such a portfolio is time and money consuming in industrial settings.

The Effect of Low Ionic Strength on Diffusion and Viscosity of Monoclonal Antibodies.

Purpose: To determine the effect of solution conditions, especially low ionic strength, on the dynamics of molecular diffusion and protein-protein interactions in monoclonal antibody solutions.

Methods: The interaction parameter, k, was calculated from diffusion data obtained from dynamic light scattering (DLS) measurements performed using a Zetasizer. Theoretical considerations were utilized to evaluate the hard sphere and electrostatic contribution to molecular interactions.

Effect of Aggregation on the Hydrodynamic Properties of Bovine Serum Albumin.

Purpose: To systematically analyze shape and size of soluble irreversible aggregates and the effect of aggregate formation on viscosity.

Methods: Online light scattering, refractive index and viscosity detectors attached to HPLC (Viscotek®) were used to study aggregation, molecular weight and intrinsic viscosity of bovine serum albumin (BSA). Irreversible aggregates were generated by heat stress.

Challenges in Determining Intrinsic Viscosity Under Low Ionic Strength Solution Conditions.

Purpose: To determine the intrinsic viscosity of several monoclonal antibodies (mAbs) under varying pH and ionic strength solution conditions.

Methods: An online viscosity detector attached to HPLC (Viscotek®) was used to determine the intrinsic viscosity of mAbs. The Ross and Minton equation was used for viscosity prediction at high protein concentrations.

Real-time shape approximation and fingerprinting of single proteins using a nanopore.

Established methods for characterizing proteins typically require physical or chemical modification steps or cannot be used to examine individual molecules in solution. Ionic current measurements through electrolyte-filled nanopores can characterize single native proteins in an aqueous environment, but currently offer only limited capabilities. Here we show that the zeptolitre sensing volume of bilayer-coated solid-state nanopores can be used to determine the approximate shape, volume, charge, rotational diffusion coefficient and dipole moment of individual proteins.

Pharmaceutical Perspective on Opalescence and Liquid-Liquid Phase Separation in Protein Solutions.

Opalescence in protein solutions reduces aesthetic appeal of a formulation and can be an indicator of the presence of aggregates or precursor to phase separation in solution signifying reduced product stability. Liquid-liquid phase separation of a protein solution into a protein-rich and a protein-poor phase has been well-documented for globular proteins and recently observed for monoclonal antibody solutions, resulting in physical instability of the formulation. The present review discusses opalescence and liquid-liquid phase separation (LLPS) for therapeutic protein formulations.

Effect of Excipients on Liquid-Liquid Phase Separation and Aggregation in Dual Variable Domain Immunoglobulin Protein Solutions.

Liquid-liquid phase separation (LLPS) and aggregation can reduce the physical stability of therapeutic protein formulations. On undergoing LLPS, the protein-rich phase can promote aggregation during storage due to high concentration of the protein. Effect of different excipients on aggregation in protein solution is well documented; however data on the effect of excipients on LLPS is scarce in the literature.

Viscosity Analysis of Dual Variable Domain Immunoglobulin Protein Solutions: Role of Size, Electroviscous Effect and Protein-Protein Interactions.

Purpose: Increased solution viscosity results in difficulties in manufacturing and delivery of therapeutic protein formulations, increasing both the time and production costs, and leading to patient inconvenience. The solution viscosity is affected by the molecular properties of both the solute and the solvent. The purpose of this work was to investigate the effect of size, charge and protein-protein interactions on the viscosity of Dual Variable Domain Immunoglobulin (DVD-Ig(TM)) protein solutions.

Liquid-Liquid Phase Separation in a Dual Variable Domain Immunoglobulin Protein Solution: Effect of Formulation Factors and Protein-Protein Interactions.

Dual variable domain immunoglobulin proteins (DVD-Ig proteins) are large molecules (MW ∼ 200 kDa) with increased asymmetry because of their extended Y-like shape, which results in increased formulation challenges. Liquid-liquid phase separation (LLPS) of protein solutions into protein-rich and protein-poor phases reduces solution stability at intermediate concentrations and lower temperatures, and is a serious concern in formulation development as therapeutic proteins are generally stored at refrigerated conditions. In the current work, LLPS was studied for a DVD-Ig protein molecule as a function of solution conditions by measuring solution opalescence.

Opalescence in monoclonal antibody solutions and its correlation with intermolecular interactions in dilute and concentrated solutions.

Opalescence indicates physical instability of a formulation because of the presence of aggregates or liquid-liquid phase separation in solution and has been reported for monoclonal antibody (mAb) formulations. Increased solution opalescence can be attributed to attractive protein-protein interactions (PPIs). Techniques including light scattering, AUC, or membrane osmometry are routinely employed to measure PPIs in dilute solutions, whereas opalescence is seen at relatively higher concentrations, where both long- and short-range forces contribute to overall PPIs.

Dipole-dipole interaction in antibody solutions: correlation with viscosity behavior at high concentration.

Purpose: The purpose of this study was to investigate the contribution of the dipole moment to overall protein-protein interactions and viscosity of a monoclonal antibody MAb1.

Methods: The dipole moment of MAb1 was measured at various solution pH conditions using dielectric relaxation spectroscopy.

Results: The dipole moment for MAb1 was highest at pH 6.

Weak antibody-cyclodextrin interactions determined by quartz crystal microbalance and dynamic/static light scattering.

In a quest to elucidate the mechanism by which hydroxypropyl β-cyclodextrin (HPβCD) stabilizes antibodies against shaking stress, two heavily debated hypotheses exist, namely that stabilization is due to HPβCD's surface activity, or due to specific interactions with proteins. In a previous study by Serno et al. (Pharm.

Viscosity of high concentration protein formulations of monoclonal antibodies of the IgG1 and IgG4 subclass - prediction of viscosity through protein-protein interaction measurements.

The purpose of this work was to explore the relation between protein-protein interactions (PPIs) and solution viscosity at high protein concentration using three monoclonal antibodies (mAbs), two of the IgG4 subclass and one of the IgG1 subclass. A range of methods was used to quantify the PPI either at low concentration (interaction parameter (kD) obtained from dynamic light scattering, DLS) or at high concentration (solution storage modulus (G') from ultrasonic shear rheology). We also developed a novel method for the determination of PPI using the apparent radius of the protein at either low or high protein concentration determined using DLS.

Characterization of antibody-polyol interactions by static light scattering: implications for physical stability of protein formulations.

In this study, the nature of interactions between monoclonal antibodies and polyols was studied using static light scattering. Solutions of mAb-U and mAb-P (4-12 mg/mL) were analyzed using static light scattering in buffer, 10% w/v trehalose and ethylene glycol solutions at pH 5.0, 7.

Protein-silicone oil interactions: comparative effect of nonionic surfactants on the interfacial behavior of a fusion protein.

Purpose: To study the effect of three nonionic surfactants on the protein-silicone oil interactions.

Methods: The adsorption of Tween® 80, Pluronic® F68 and Tween® 20 at the silicone oil/water interface (using shifts in frequency (ΔF) and resistance (ΔR) with quartz crystal microbalance) was compared to the adsorption at air/water interface (using surface tension). Effect of surfactants on protein adsorption to the silicone oil/water interface was studied in sequential- and co-adsorption modes.

Application of second-derivative fluorescence spectroscopy to monitor subtle changes in a monoclonal antibody structure.

In this study, the tertiary structure of a monoclonal antibody was analyzed under thermal and chemical stresses using second-derivative fluorescence spectroscopy. The effect of polyols, sucrose, and ethylene glycol on the tertiary structure of monoclonal antibody-U (mAb-U) (pH 7.0) was studied under thermal stress (25°C-75°C).

Application of maximum bubble pressure surface tensiometer to study protein-surfactant interactions.

Binding of a surfactant to proteins can affect their physicochemical stability and solubility in a formulation. The extent of the effect depends on the binding stoichiometry. In this study, we have utilized the technique of maximum bubble pressure surface tensiometry to characterize the binding between human serum albumin (HSA) and surfactants (sodium dodecyl sulfate (SDS) and polysorbate 80) by dynamic surface tension measurements.

The effect of Tween(®) 20 on silicone oil-fusion protein interactions.

There is evidence in the literature that silicone oil, a lubricant, can induce aggregation in protein formulations delivered through prefilled syringes. Surfactants are commonly used to minimize protein-silicone oil and protein-container interactions; however, these interactions are not well characterized and understood. The purpose of this manuscript was to understand the competitive interactions of a fusion protein with the silicone oil in the presence of Tween(®) 20.

The influence of charge distribution on self-association and viscosity behavior of monoclonal antibody solutions.

The present work investigates the influence of electrostatic surface potential distribution of monoclonal antibodies (MAbs) on intermolecular interactions and viscosity. Electrostatic models suggest MAb-1 has a less uniform surface charge distribution than MAb-2. The patches of positive and negative potential on MAb-1 are predicted to favor intermolecular attraction, even in the presence of a small net positive charge.

Determination of the dipole moments of RNAse SA wild type and a basic mutant.

In this study, we report the effects of acidic to basic residue point mutations (5K) on the dipole moment of RNAse SA at different pHs. Dipole moments were determined by measuring solution capacitance of the wild type (WT) and the 5K mutant with an impedance analyzer. The dipole moments were then (1) compared with theoretically calculated dipole moments, (2) analyzed to determine the effect of the point mutations, and (3) analyzed for their contribution to overall protein-protein interactions (PPI) in solution as quantitated by experimentally derived second virial coefficients.

Viscosity behavior of high-concentration monoclonal antibody solutions: correlation with interaction parameter and electroviscous effects.

The purpose of this work was to understand the viscosity behavior of high-concentration monoclonal antibody (mAb) solutions in a wide range of solution conditions and generate guidelines helpful to formulate products with manageable viscosity. The zeta potential and effective isoelectric point (pI) were determined from electrophoretic mobility measurements. High-frequency rheology studies characterized viscoelasticity at high concentrations.

Opposite effects of polyols on antibody aggregation: thermal versus mechanical stresses.

Purpose: To investigate the physical stability of antibody-polyol formulations under thermal and mechanical stresses.

Methods: mAb-U was analyzed in buffer, trehalose, sucrose, glycerol and ethylene glycol solutions at pH 7.0.

Individual second virial coefficient determination of monomer and oligomers in heat-stressed protein samples using size-exclusion chromatography-light scattering.

The objective of this work was to determine the second virial coefficient (B(22)) of monomer and oligomer protein species in heat-stressed samples individually and simultaneously. A high-performance size-exclusion chromatography equipped with a flow-mode detector system enabling measurement of light scattering (LS) and ultraviolet transmission in the same cell was used to separate and analyze different species. The folded/unfolded nature of the protein was analyzed by extrinsic fluorescence spectroscopy using 4,4'-Bis(1-anilinonaphthalene 8-sulfonate).

Establishing a link between amino acid sequences and self-associating and viscoelastic behavior of two closely related monoclonal antibodies.

Purpose: To investigate the underlying cause for the observed differences in self-associating and viscoelastic behavior between two monoclonal antibodies, MAb1, and MAb2.

Methods: Several mutants were designed by swapping charged residues in MAb1 with those present in MAb2 at their respective positions and vice versa. Rheological analysis was done at low and high shear rates.