Interactions of formulation excipients with proteins in solution and in the dried state☆
Graphical abstract
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.
Section snippets
Solution
A wide variety of protein stabilizing excipients is used for enhancing the stability of both pharmaceutical and reagent proteins and they are referred to as stabilizing co-solvents [9], [22], [23], [24]. These excipients have been reported to stabilize the structure of native proteins at moderate (0.1 M) to high concentrations (1 M). In fact, these compounds played a critical role at the dawn of classical enzymology and biochemistry of cellular proteins. Many proteins are inactivated when they
Solution
Hydrophilic polymers have often been used to stabilize proteins and enhance protein assembly [152], [153], [154]. Sasahara et al. [155] demonstrated that the stability of a protein against heat treatment was increased through the incorporation of dextran. Manning et al. [156] have studied the effects of polymeric excipients on the thermally-induced aggregation of low molecular weight urokinase, and found hydroxyl ethyl (HETA) starch, PEG4000, and gelatin to all be effective in stabilizing the
Solution
Surfactants are widely used to stabilize proteins, suppress aggregation and assist in protein refolding [217], [218]. Polysorbate 80 (polyoxyethylene sorbitan monooleate) and polysorbate 20 (polyoxyethylene sorbitan monolaurate) are two of the widely incorporated surfactants in marketed protein pharmaceuticals [176], [178], [219], and are typically used in the 0.0003–0.3% range [176]. The effects of surfactants and their interaction mechanism in aqueous solution is described in more detail
Solution
The effects of amino acids in general were described earlier in Section 2. Here, the focus is placed on one specific amino acid, arginine. Arginine is not a protein-stabilizing excipient, but is highly effective in suppressing protein aggregation. Due to this effect and its safety in humans, arginine is frequently used for enhancing the shelf life of proteins. The aggregation-suppressing effect of arginine was accidentally observed by Rudolph and Fischer [256] during their attempt to prevent
Overall discussion on mechanism
The mechanism of each class of excipients for their effects on protein stability, solubility, and aggregation in both liquid and lyophilized formulations has been described above based on their interactions with proteins. In liquid formulations, there are primarily two different modes of interactions present between excipients and proteins, or container surfaces. Those that enhance protein stability demonstrate an unfavorable interaction with the protein. Fig. 9 demonstrates how such an
Conclusion
We have shown here the effects of four classes of co-solvents (excipients), i.e., protein-stabilizers, polymers, surfactants, and arginine on the formulation and stability of proteins in solution and dry state. The efficacy of these excipients in conferring stability to proteins has been approached from the mechanistic point of view, highlighting the various interaction forces present under different protein environmental conditions. Typical protein formulation contains several components, and
References (290)
Conformational stability of globular proteins
Trends Biochem. Sci.
(1990)Instability, stabilization, and formulation of liquid protein pharmaceuticals
Int. J. Pharm.
(1999)Reversible denaturation of ribonuclease in aqueous solutions as influenced by polyhydric alcohols and some other additives
J. Biol. Chem.
(1968)- et al.
The stabilization of proteins by osmolytes
Biophys. J.
(1985) - et al.
Preferential interactions of proteins with solvent components in aqueous amino acid solutions
Arch. Biochem. Biophys.
(1983) - et al.
The mechanism of action of Na glutamate, lysine HCl, and piperazine-N, N′-bis(2-ethanesulfonic acid) in the stabilization of tubulin and microtubule formation
J. Biol. Chem.
(1984) - et al.
On the conformational stability of globular proteins. The effects of various electrolytes and nonelectrolytes on the thermal ribonuclease transition
J. Biol. Chem.
(1965) - et al.
Thermal denaturation of antithrombin III. Stabilization by heparin and lyotropic anions
J. Biol. Chem.
(1981) - et al.
The stabilization of proteins by sucrose
J. Biol. Chem.
(1981) - et al.
Interaction of calf brain tubulin with glycerol
J. Mol. Biol.
(1981)
Strategies to suppress aggregation of recombinant keratinocyte growth factor during liquid formulation development
J. Pharm. Sci.
Protection of bovine serum albumin from aggregation by Tween 80
J. Pharm. Sci.
Stabilizing effects of caprylate and acetyltryptophanate on heat-induced aggregation of bovine serum albumin
Biochim. Biophys. Acta.
The effects of arginine on refolding of aggregated proteins: not facilitate refolding, but suppress aggregation
Biochem. Biophys. Res. Commun.
Suppression of protein interactions by arginine: a proposed mechanism of the arginine effects
Biophys. Chem.
Protein precipitation and denaturation by dimethyl sulfoxide
Biophys. Chem.
Stabilization of yeast hexokinase A by polyol osmolytes: correlation with the physicochemical properties of aqueous solutions
Biophys. Chem.
Trehalose: current use and future applications
J. Pharm. Sci.
Non-A, non-B hepatitis from intravenous immunoglobulin
Lancet
Heparin protects heparin-binding growth factor-I from proteolytic inactivation in vitro
Biochem. Biophys. Res. Commun.
Stabilization of basic fibroblast growth factor with dextran sulfate
FEBS Lett.
Physical stabilization of acidic fibroblast growth factor by polyanions
Arch. Biochem. Biophys.
Sucralfate and soluble sucrose octasulfate bind and stabilize acidic fibroblast growth factor
Biochim. Biophys. Acta.
Characterization of keratinocyte growth factor binding to heparin and dextran sulfate
Arch. Biochem. Biophys.
Physical factors affecting the storage stability of freeze-dried interleukin-1 receptor antagonist: glass transition and protein conformation
Arch. Biochem. Biophys.
Dehydration-induced conformational transitions in proteins and their inhibition by stabilizers
Biophys. J.
Effects of additives on the stability of recombinant human factor XIII during freeze-drying and storage in the dried solid
Arch. Biochem. Biophys.
Cryoprotection of phosphofructokinase with organic solutes: characterization of enhanced protection in the presence of divalent cations
Arch. Biochem. Biophys.
Stabilization of phosphofructokinase with sugars during freeze-drying: characterization of enhanced protection in the presence of divalent cations
Biochim. Biophys. Acta.
Influence of calcium ions on the structure and stability of recombinant human deoxyribonuclease I in the aqueous and lyophilized states
J. Pharm. Sci.
Fourier-transform infrared spectroscopic investigation of protein stability in the lyophilized form
Biochim. Biophys. Acta.
Control of protein stability and reactions by weakly interacting cosolvents: the simplicity of the complicated
Adv. Protein Chem.
Thermodynamic binding and site occupancy in the light of the Schellman exchange concept
Biophys. Chem.
Thermodynamics of the denaturation of lysozyme by guanidine hydrochloride. II. Dependence on denaturant concentration at 25 degrees
Biochemistry
Studies of the chymotrypsinogen family of proteins. VII. Thermodynamic analysis of transition I of alpha-chymotrypsin
J. Am. Chem. Soc.
Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation
Pharm. Res.
Protein aggregation and bioprocessing
AAPS J.
Effects of protein aggregates: an immunologic perspective
AAPS J.
Defining your product profile and maintaining control over it, Part 4. Product-related impurities: tackling aggregates
Bioprocess Int.
Protein–solvent interactions in pharmaceutical formulations
Pharm. Res.
Increased thermal stability of proteins in the presence of sugars and polyols
Biochemistry
The effects of polyhydric and monohydric alcohols on the heat induced reversible denaturation of chymotrypsinogen A
Eur. J. Biochem.
The effect of ions on the kinetics of formation and the stability of the collagenfold
Biochemistry
Calorimetric study on thermal denaturation of lysozyme in polyol–water mixtures
J. Biochem.
Increased thermal stability of collagen in the presence of sugars and polyols
J. Biochem.
Interaction of calf skin collagen with glycerol: linked function analysis
Biochemistry
Mechanism of protein stabilization by glycerol: preferential hydration in glycerol–water mixtures
Biochemistry
Stabilization of protein structure by sugars
Biochemistry
Stabilization of recombinant human keratinocyte growth factor by osmolytes and salts
J. Pharm. Sci.
Aggregation pathway of recombinant human keratinocyte growth factor and its stabilization
Pharm. Res.
Cited by (297)
Design of a Reciprocal Injection Device for Stability Studies of Parenteral Biological Drug Products
2024, Journal of Pharmaceutical SciencesProcess and isothermal storage stabilities of a live veterinary vaccine formulated with Plectranthus esculentus tuber starch derivatives as stabilizers
2024, International Journal of PharmaceuticsNanocrystal technologies in biomedical science: From the bench to the clinic
2024, Drug Discovery TodayIonic liquids and deep eutectic solvents for the stabilization of biopharmaceuticals: A review
2024, Biotechnology AdvancesRoadmap for Drug Product Development and Manufacturing of Biologics
2024, Journal of Pharmaceutical Sciences
- ☆
This review is part of the Advanced Drug Delivery Reviews theme issue on ”Formulating Biomolecules: Mechanistics Insights in Molecular Interactions”.