Orthogonal Techniques to Study the Effect of pH, Sucrose, and Arginine Salts on Monoclonal Antibody Physical Stability and Aggregation During Long-Term Storage

Abstract

Keywords:monoclonal antibody(s) pH sucrose arginine physical stability protein aggregation protein formulation fluorescence spectroscopy light scattering (dynamic) nuclear magnetic resonance (NMR) spectroscopy a b s t r a c tUnderstanding the effects of additives on therapeutic protein stability is of paramount importance for obtaining stable formulations. In this work, we apply several high-and medium-throughput methods to study the physical stability of a model monoclonal antibody at pH 5.0 and 6.5 in the presence of sucrose, arginine hydrochloride, and arginine glutamate. In low ionic strength buffer, the addition of salts reduces the antibody colloidal and thermal stability, attributed to screening of electrostatic interactions. The presence of glutamate ion in the arginine salt partially reduces the damaging effect of ionic strength increase. The addition of 280 mM sucrose shifts the thermal protein unfolding to a higher temperature. Arginine salts in the used concentration reduce the relative monomer yield after refolding from urea, whereas sucrose has a favorable effect on antibody refolding. In addition, we show 12-month long-term stability data and observe correlations between thermal protein stability, relative monomer yield after refolding, and monomer loss during storage. The monomer loss during storage is related to protein aggregation and formation of subvisible particles in some of the formulations. This study shows that the effect of commonly used additives on the long-term antibody physical stability can be predicted using orthogonal biophysical measurements. IntroductionOne fundamental aim during the development of therapeutic proteins is finding formulations that provide sufficient protein stability during long-term storage. Some critical variables of these formulations are solution pH, ionic strength, and the presence of additives. The additives usually belong to the group of sugars, polyols, amino acids, or surfactants. 1,2 Among these, sucrose is the most frequently used in marketed therapeutic protein formulations. 1 From the amino acids, arginine is of considerable interest, as in some cases, it can suppress protein aggregation or reduce the viscosity of highly concentrated protein solutions. 3,4 In addition, the use of different arginine salts is a topic of intense research because the arginine counterion can determine the effect on protein stability. [4][5][6][7] Sucrose and arginine salts can affect the thermal protein unfolding and aggregation differently depending on the protein molecule. The presence of other buffer components, such as NaCl, Abbreviations used: A 2 , second virial coefficient; ACF, autocorrelation function; AF STD , protein-additive saturation transfer difference amplification factors from NMR; D, mutual diffusion coefficient from DLS; DLS, dynamic light scattering; D max , maximum dimension from SAXS; FI350/FI330, intrinsic protein fluorescence intensity ratio 350nm/330nm; IP1, inflection point of the first thermal unfolding (at a lower temperature); IP2, inflection...

Formulations That Suppress Aggregation During Long-Term Storage of a Bispecific Antibody are Characterized by High Refoldability and Colloidal Stability

“…Both of the latter show reasonable predictions for the ranking of Bis-mAb formulations in order of their effect on protein aggregation during long-term storage. These findings add to our earlier reports with formulations of three IgGs 30,37 , further supporting the hypothesis that the high reversibility of isothermal protein unfolding (induced by denaturants like urea) is a key feature of physically stable formulations of therapeutic proteins. The inset shows the residuals from the fit.…”

“…Formulations containing 10 mM citrate buffer or 70 mM sodium chloride show dramatically lower relative monomer yield (Figure 4b), indicating the high aggregation propensity of the (partially) unfolded Bis-mAb in these conditions. Noteworthy, Bis-mAb has lower much lower RMYs compared to three IgGs that were studied at similar protein concentration in the same formulation conditions, indicating that this bispecific antibody is very prone to aggregation during refolding 30,37 . Whether bispecifics have lower refoldability compared to analogical antibodies with canonical IgG structure is yet to be investigated.…”

“…We previously showed that assessing the aggregation during dilution-or dialysis-refolding from denaturants can be a valuable tool to select monoclonal antibody formulations with higher physical stability and suppressed aggregation during storage 30,37,44 . In an effort to standardize such experiments, we presented a ReFOLD assay that is based on microdialysis in deep multiwell plates 30 .…”

“…We demonstrated that assessing the effect of the formulation on the protein monomer yield (i.e. the fraction of monomer that is not aggregated after unfolding and refolding with urea) from the ReFOLD assay is a valuable approach to qualitatively predict the physical storage stability of three different antibodies in various formulation conditions 30,37 . In the study presented here, we applied this concept to a different molecule -the bispecific antibody Bis-mAb which is a human IgG1κ-scFv protein.…”

Role of NMR in High Ordered Structure Characterization of Monoclonal Antibodies

“…It should be noted that 1 H-detected 1D NMR experiments are also used to analyze the interaction between formulation components and biologics [38]. Finding formulations to provide sufficient stability during long-term storage is important in developing a therapeutic mAb.…”