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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...

Gold nanoparticles (AuNPs) exhibit unique properties that have made them a very attractive material for application in biological assays. Given the potentially interesting interactions between AuNPs and biological macromolecules, we investigated AuNPs-induced protein crystal growth. Differently functionalized AuNPs were tested as additives in cocrystallization studies with model proteins (hen egg white lysozyme (HEWL), ribonuclease A (RNase A), and proteinase K) as well as with case studies where there were problems in obtaining well-diffracting crystals. Trials were performed considering different crystallization drawbacks, from total absence of crystals to improvement of crystal morphology, size, twinning, and number of crystals per drop. Improvement of some of these factors was observed in the cases of HEWL, RNase A, phenylalanine hydroxylase (PAH), myoglobin, native aldehyde oxidase (AOH), and human albumin. In these proteins, the presence of the AuNPs promoted an increase in the size and/or better crystal morphology. From the systematic trials and subsequent observations, it can be concluded that the introduction of AuNPs should definitely be considered in crystal optimization trials to improve previously determined crystallization conditions.