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Antibody Solution Viscosity and Intermolecular Interactions: Considerations for Development of Highly Concentrated Formulations
by Elena Krayukhina
U-medico, Inc.; Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan
Susumu Uchiyama
Department of Biotechnology, Graduate School of Engineering, Osaka University; Okazaki Institute for Integrative Bioscience, Japan


In recent years, a dramatic increase in the demand for highly concentrated antibody formulations have been observed. This is due to the increasing interest in reducing burden on patients and medical professionals, which in turn is driving the shift from antibody formulations for intravenous toward subcutaneous administration. While intravenous injections should be performed by a qualified medical professional, formulations for subcutaneous administration can potentially be self-infused. In spite of the simplicity of the subcutaneous infusion, however, the maximum volume of pharmaceutical that can be injected is usually limited to approximately 2 mL. Therefore, high concentration antibody solutions are needed to achieve therapeutic dose requirements. At high concentration, proteins experience molecular crowding and usually demonstrate non-ideal behavior related to the tendency of proteins to self-associate. These factors can contribute to an increase in viscosity of pharmaceutical solution, which is highly undesirable since higher force and longer time are required for subcutaneous injection of viscous formulations relative to the solution of lower viscosity. This can result in enhanced pain upon injection or even make it impossible to administer drug by this route [1,2]. Thus, development of highly concentrated antibody solutions requires careful studies and consideration of the appropriate formulations with preferably low viscosity. Here, the analytical techniques to access the viscosity of highly-concentrated formulations and the general approaches to reducing high viscosity will be introduced.

Prediction of highly concentrated solution viscosity based on the measurements of second virial coefficient in diluted solutions

High viscosity of highly concentrated antibody solutions is primarily caused by reversible self-associations, which are induced in the presence of attractive intermolecular interactions [3]. These associations lead to formation of transient network in the high-concentrated solution, which can be completely dissociated upon dilution [4]. A mechanism underlying the network formation where antibodies were modeled as linear polymers undergoing molecular entanglements through the reversible bonds between the Fab domains was described [5]. Reversible clusters of self-assembled antibody observed by using the combination of small-angle neutron/x-ray scattering (SANS/SAXS), neutron spin echo (NSE), and computer simulations, were suggested to attribute to high viscosity of concentrated antibody solutions [6]. In contrast, irreversible self-association, or physical aggregation results in a decrease of concentrated solution viscosity due to a decrease in excluded volume [4].

In dilute solutions, the intermolecular forces can be conveniently evaluated using the second virial coefficient (B2). The positive B2 values are indicative of repulsive intermolecular interactions, while the negative values imply the presence of attractive forces. B2 can be measured using a variety of techniques including SANS [7], size-exclusion chromatography (SEC) [8,9], and self-interaction chromatography (SIC) [10]. However, the most reliable and commonly used methods are static light scattering (SLS) [11] and sedimentation equilibrium analytical ultracentrifugation (SE-AUC) [12]. By simultaneous SE-AUC measurements performed using antibody solutions below 10 mg/mL and the viscosity measurements of antibody solutions at high concentrations, it was demonstrated that B2 values derived from AUC-SE can yield reliable predictions of antibody behavior at high concentrations [12]. Thus, the increase in the viscosity of high-concentrated antibody solution was observed with decreasing B2 values. In agreements with viscosity measurements, it was shown that B2 depends on solution pH. For the antibodies studied, viscosities of the solutions with similar positive B2 values acquire similar viscosities profile regardless of amino acid sequences, while for the solutions with negative B2 values, correlation between viscosity and amino acid sequence was detected. Overall, it was concluded that in order to achieve lower viscosity in highly concentrated antibody solution, larger positive B2 values should be used as the criteria of selection among the potential formulation assessed [12].

Electrostatic interactions are a major contributor to high viscosity of concentrated antibody solutions

Numerous studies addressed factors that may contribute to the intermolecular interactions and subsequent solution behavior. It was suggested that the attractive inter-molecular interactions causing the increase in antibody solution viscosity at high concentration is a result of electrostatic interactions [3, 13-15]. The possible correlation between electrostatic surface potential distribution and viscosity behavior was evaluated and it was shown that an evenly distributed surface charge would promote repulsions of antibody molecules and prevent long-range order formation with increasing concentration [4]. On the contrary, asymmetry of surface charge distribution favors intermolecular attractions between oppositely charged patches at high concentration where molecules are in a close proximity to each other. This stimulates an increase in self-association and results in high viscosity values. These findings suggest that an attempt to achieve optimal viscosity and self-association properties can be reasonably addressed at the early stage through engineering/design of antibody molecules prior to optimization of solution conditions.

High-throughput analysis to select solutions with desired viscoelastic properties

It was demonstrated that combination of kD and ks parameters, reflecting concentration-dependence of the diffusion coefficient and sedimentation coefficient, derived from dynamic light scattering (DLS) and sedimentation velocity analytical ultracentrifugation (SV-AUC) measurements, respectively, can be successfully used to estimate the B2 [16]. Nevertheless, due to relatively low throughput of the AUC analysis, this approach has a limited utility for the large-scale screening. Weak protein-protein interactions were suggested to be responsible for the viscoelastic properties of concentrated antibody solutions, and a simplified alternative approach based solely on the measurement of kD was described. The results showed good correlation between the estimated kD values and viscosity of concentrated monoclonal antibody solutions, suggesting suitability of the approach for high-throughput screening [12, 17].

Managing viscosity behavior by modulating the solution conditions

Viscosity behavior of highly concentrated antibody solutions can be modulated through optimization of solution conditions such as ionic strength, pH, or addition of excipients. Since the nature and extent of intermolecular interactions occurring in solution can vary significantly for different antibodies even in the case of more than 90% sequence identity, there are no general rules and each formulation requires particular considerations. For instance, it was demonstrated that viscosity of monoclonal antibody solution at concentration near 100 mg/mL can be minimized through balance of the repulsive and attractive forces using intermediate ionic strengths [18]. However, in another study, it was shown that antibody solution with a clear appearance in an isotonic ionic strength condition turned opalescent in a low ionic strength condition [19].

The technique of ultrasonic shear rheometry was applied to measure storage modulus of IgG2 antibody solution at a concentration of 120 mg/mL over the pH range, and the observed rheological profile was different from the typical pattern of protein solutions as a function of pH [20]. At high concentrations, charge–charge and charge–dipole interactions at the surface in the Fab regions arising from proton fluctuations invoke the specific attractive intermolecular interactions which can explain the unusual pH dependency observed in the rheological profile [21].

By using a high throughput dynamic light scattering method to measure the viscosity, effect of sugar molecules on the viscosity of high concentration monoclonal antibody solutions was evaluated [22]. The addition of either trehalose, sucrose, sorbitol, glucose, fructose, xylose or galactose resulted in an increase of antibody solution viscosity at high concentration, and disaccharides had a more pronounced effect than monosaccharides.

Packaging material

Careful investigations of compatibility of pharmaceutical solutions with the container material are of a great importance for the development of safe and stable formulations. Recently, numbers of studies aimed at elucidating the effects of packaging material surfaces have been conducted increasingly. Silicone oil lubrication can affect aggregates and particulate formation, and likely lead to the increase of viscosity. The results of accelerated stress studies suggest that recently developed silicone oil-free polymer-based prefillable syringes have a potential to improve stability of pharmaceutical antibody solutions upon storage and shipping [23].


High viscosity is a major obstacle to successful development of highly concentrated antibody formulations. A number of steps should be taken in order to achieve formulations with minimized viscosities, starting from rational engineering of antibody molecules to compensate surface charge distribution and diminish Fab-Fab interactions, followed by high-throughput screening of solutions with different ionic strengths and at different pH to optimize solution conditions. At a later developmental stage, to improve the stability of the finished product, the compatibility of antibody with various packaging materials should be evaluated.

About the Authors

Dr. Elena Krayukhina is an appointed researcher at Graduate School of Engineering, Osaka University and a group leader at U-medico Inc. - company providing contract research services and consulting in the field of protein biophysical characterization. In addition to biopharmaceutical formulation development and quantitative evaluation of protein aggregates and particles, her major area of expertise includes analysis of protein-protein and protein-ligand interaction using a variety of biophysical methods such as analytical ultracentrifugation, size-exclusion chromatography, and dynamic flow-imaging analyses. Her recent efforts have been aimed at elucidating the mechanism of antigen/pharmaceutical antibody complex formation in human serum.

Dr. Susumu Uchiyama is an associate professor at Graduate School of Engineering, Osaka University and is also an appointed visiting associate professor at Okazaki Institute for Integrative Biosciences and at South China University of Technology. He has been studying solution properties and structures of proteins for 20 years using various biophysical methods such as analytical ultracentrifugation, differential scanning calorimetry and mass spectrometry. Factors that govern biophysical properties have been uncovered based on the results with different angles. So far, he has more than 150 publications in international journals. Recent scope of his study focuses on the rational formulation developments of therapeutic proteins and quantitative analysis of protein aggregates.


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