Viscosity-Lowering Effect of Amino Acids and Salts on Highly Concentrated Solutions of Two IgG1 Monoclonal Antibodies

Abstract

Monoclonal antibodies display complicated solution properties in highly concentrated (>100 mg/mL) formulations, such as high viscosity, high aggregation propensity, and low stability, among others, originating from protein-protein interactions within the colloidal protein solution. These properties severely hinder the successful development of high-concentration mAb solution for subcutaneous injection. We hereby investigated the effects of several small-molecule excipients with diverse biophysical-chemical properties on the viscosity, aggregation propensity, and stability on two model IgG1 (JM1 and JM2) mAb formulations. These excipients include nine amino acids or their salt forms (Ala, Pro, Val, Gly, Ser, HisHCl, LysHCl, ArgHCl, and NaGlu), four representative salts (NaCl, NaAc, Na2SO4, and NH4Cl), and two chaotropic reagents (urea and GdnHCl). With only salts or amino acids in their salt-forms, significant decrease in viscosity was observed for JM1 (by up to 30-40%) and JM2 (by up to 50-80%) formulations, suggesting charge-charge interaction between the mAbs dictates the high viscosity of these mAbs formulations. Most of these viscosity-lowering excipients did not induce substantial protein aggregation or changes in the secondary structure of the mAbs, as evidenced by HPLC-SEC, DSC, and FT-IR analysis, even in the absence of common protein stabilizers such as sugars and surfactants. Therefore, amino acids in their salt-forms and several common salts, such as ArgHCl, HisHCl, LysHCl, NaCl, Na2SO4, and NaAc, could potentially serve as viscosity-lowering excipients during high-concentration mAb formulation development.

Characterization of highly concentrated antibody solution - A toolbox for the description of protein long-term solution stability

“…A decreasing effect of glycine on viscosity had previously been described by Wang et al. 69 Glycine is a hydrophilic amino acid and preferably hydrates molecules in solution, which may reduce mAb-mAb interactions and, hence, viscosity. The increase in viscosity by the addition of PEG4000 can be attributed to 2 effects.…”

Amino Acid‐Based Advanced Liquid Formulation Development for Highly Concentrated Therapeutic Antibodies Balances Physical and Chemical Stability and Low Viscosity

“…Currently, approved therapeutic antibody liquid formulations may comprise a) histidine as buffering agent; b) arginine to avoid aggregation; c) glycine as osmolytic stabilizer, bulking agent and/or tonicity adjusting agent; and d) methionine as an antioxidant. In addition, the amino acid salts arginine*HCl, lysine*HCl, histidine*HCl, and sodium glutamate have recently been reported to reduce viscosity in highly concentrated antibody solutions up to 200 mg mL −1 in conjunction with reduced aggregation …”

“…To date, different formulation strategies have been tried to reduce the viscosity of highly concentrated monoclonal antibody formulations suitable for subcutaneous administration. As for highly concentrated liquid formulations, the addition of special viscosity reducing excipients, for example, salts, amino acids, or sugars to balance repulsion and attractive forces through intermediated ionic strength or the adjustment of pH are well known strategies for viscosity reduction during manufacturing as well as in the drug product of highly concentrated antibody formulations . Moreover, the desired pH for reducing viscosity or the particular desired viscosity reducing excipient can have detrimental effects on the stability of the therapeutic antibody .…”

“…Moreover, the desired pH for reducing viscosity or the particular desired viscosity reducing excipient can have detrimental effects on the stability of the therapeutic antibody . Furthermore, it is well known that particularly amino acid‐based formulations containing single amino acids at high concentrations are beneficial for the stabilization of biopharmaceuticals and the lowering of viscosities on antibody formulations up to and including 200 mg mL −1 . However, the systematic combination of amino acids has not been studied extensively especially at concentrations >200 mg mL −1 .…”

Stress Factors in mAb Drug Substance Production Processes: Critical Assessment of Impact on Product Quality and Control Strategy

“…Nevertheless, depending on temperature, the type of salt (Hofmeister series), salt concentration, and protonation state of the protein (net positive charge at pH < pI; net neutral charge at pHp I; net negative charge at pH > pI), complex scenarios of solubility reduction and solubility enhancement ("salting in") have been reported. [89][90][91][92][93][94] The addition of (chaotropic) salts 86,95,96 or charged excipients, for example, amino acids with a hydrophobic moiety such as histidine and arginine 96,97 are reported to lower the tendency to form associates, which in turn also lowers the viscosity of the solution. Similarly, reversible association that leads to liquidliquid phase separation was recently reported to follow similar trends 92,[98][99][100][101] ; this is seen especially at low temperatures, for example, during intermediate storage during DSP.…”