Viscosity-Lowering Effect of Amino Acids and Salts on Highly Concentrated Solutions of Two IgG1 Monoclonal Antibodies Shujing Wang, † Ning Zhang, ‡ Tao Hu, ‡ Weiguo Dai, § Xiuying Feng, ‡ Xinyi Zhang, † and Feng Qian* ,† † School of Pharmaceutical Sciences and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Tsinghua University, Beijing 100084, China ‡ China R&D and ScientificAffair, Shanghai Discovery Center, Janssen Research & Development, Johnson & Johnson, Shanghai 200030, China § Janssen Research & Development, Johnson & Johnson, Malvern, Pennsylvania 19355, United States * S Supporting Information 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 subcuta- neous 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, Na 2 SO 4 , and NH 4 Cl), 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, Na 2 SO 4 , and NaAc, could potentially serve as viscosity-lowering excipients during high- concentration mAb formulation development. KEYWORDS: monoclonal antibody, viscosity, subcutaneous injection, amino acids, protein-protein interaction, mAb stability ■ INTRODUCTION Over the past 20 years, monoclonal antibodies (mAbs) have rapidly evolved into a major class of highly promising therapeutic molecules against various diseases, such as inflammatory diseases, cancers, and other human disorders, 1-3 largely due to their superb target specificity and safety profile. As biologics with large size and complex structure, the formulation and delivery of mAbs pose substantial challenges. 4 Although current mAb therapeutics are mostly administered through intravenous infusion due to their high doses, subcutaneous injection of mAb solution or suspension endows more patient compliance and thus is more desirable. 5-8 However, subcutaneous delivery of mAbs is often restricted by the complicated solution properties of mAb at high concentrations of above 100 mg/mL. These properties, including solubility, viscosity, and aggregation, are governed by abundant protein-protein interactions. 9 The protein- protein and protein-excipient interaction under various environmental conditions have been investigated to facilitate the formulation design of the biopharmaceuticals. 10 Although protein engineering enables editing and optimiza- tion of the intrinsic biophysical properties of mAbs, formulation optimization is the most cost-efficient and effective approach to improve the mAb solution properties, especially at the product development stage where the amino acid sequence of the mAb has been selected. Excipients, such as sugars/polyols, amino acids (AAs), polymers, and salts, are added to stabilize protein therapeutics in the formulation strategy. 11-13 Previously, investigations were conducted on the effects of excipients (pH-buffering reagents, mannitol, sucrose, arginine, proline, arginine glutamate, urea, guanidine-HCl, NaCl, and other salt Received: August 21, 2015 Revised: October 29, 2015 Accepted: November 3, 2015 Published: November 3, 2015 Article pubs.acs.org/molecularpharmaceutics © 2015 American Chemical Society 4478 DOI: 10.1021/acs.molpharmaceut.5b00643 Mol. Pharmaceutics 2015, 12, 4478-4487