Interactions of formulation excipients with proteins in solution and in the dried state

Abstract

A variety of excipients are used to stabilize proteins, suppress protein aggregation, reduce surface adsorption, or to simply provide physiological osmolality. The stabilizers encompass a wide variety of molecules including sugars, salts, polymers, surfactants, and amino acids, in particular arginine. The effects of these excipients on protein stability in solution are mainly caused by their interaction with the protein and the container surface, and most importantly with water. Some excipients stabilize proteins in solution by direct binding, while others use a number of fundamentally different mechanisms that involve indirect interactions. In the dry state, any effects that the excipients confer to proteins through their interactions with water are irrelevant, as water is no longer present. Rather, the excipients stabilize proteins through direct binding and their effects on the physical properties of the dried powder. This review will describe a number of mechanisms by which the excipients interact with proteins in solution and with various interfaces, and their effects on the physical properties of the dried protein structure, and explain how the various interaction forces are related to their observed effects on protein stability.

Introduction

Many proteins are structurally unstable in solution, and are susceptible to conformational changes due to various stresses encountered during purification, processing, and storage [1], [2], [3], [4], [5], [6], [7], [8]. These stresses include elevated temperature, exposure to extreme pH, shear strain, and surface adsorption, to name a few [5], [6]. Thus, protein-based pharmaceuticals have the potential to undergo physical degradation (e.g., unfolding, aggregation, and insoluble particulate formation) by a number of mechanisms, which can negatively impact both the efficacy and safety of the therapeutic product [7], [8]. The solvent environment of the protein plays a major role in determining its stability [9]. Numerous solvent additives, the so-called “osmolytes”, have been shown to enhance the stability of proteins and, as a consequence, reduce the aggregation of marginally stable proteins [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28]. In this case, protein unfolding precedes aggregation, and the structure-stabilizing co-solvents reduce aggregation by stabilizing the native structure. The lack of affinity for, or repulsive interaction with, the protein surface is the reason why these co-solvents stabilize the protein structure. Conversely, excipients such as arginine, surfactants, proteins, and polymers are often used to suppress protein aggregation without enhancing its stability [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38]. These additives exert their effects by weakly binding to the protein surface, or by competitively binding to the surface/interface that have the potential to destabilize the protein structure. Some of these excipients are also used to stabilize proteins in the dry state. However, in the absence of water, fundamentally different mechanisms are in effect, as any mechanism that involves excipient-water interactions will not play its part. This chapter summarizes the effects of additives that are used to mitigate protein aggregation and will discuss the mechanistic basis of their effects both in solution and in the dried state. In addition, the effects of additives on protein stability during freezing will also be discussed, as freezing is an intermediate processing step involved in lyophilization. It should be noted that as water is still present, yet is gradually removed during ice crystallization, the freezing process involves an interesting physical state that is described mainly by interaction forces that are present in solution.