Instability, stabilization, and formulation of liquid protein pharmaceuticals

Abstract

One of the most challenging tasks in the development of protein pharmaceuticals is to deal with physical and chemical instabilities of proteins. Protein instability is one of the major reasons why protein pharmaceuticals are administered traditionally through injection rather than taken orally like most small chemical drugs. Protein pharmaceuticals usually have to be stored under cold conditions or freeze-dried to achieve an acceptable shelf life. To understand and maximize the stability of protein pharmaceuticals or any other usable proteins such as catalytic enzymes, many studies have been conducted, especially in the past two decades. These studies have covered many areas such as protein folding and unfolding/denaturation, mechanisms of chemical and physical instabilities of proteins, and various means of stabilizing proteins in aqueous or solid state and under various processing conditions such as freeze-thawing and drying. This article reviews these investigations and achievements in recent years and discusses the basic behavior of proteins, their instabilities, and stabilization in aqueous state in relation to the development of liquid protein pharmaceuticals.

Introduction

The advent of recombinant DNA technology has led to a worldwide zeal to develop protein pharmaceuticals in the past two decades. These protein pharmaceuticals or pharmaceutical candidates include functional regulators and supplements, enzyme activators and inhibitors, poly- and monoclonal antibodies, and various vaccines. In comparison with small chemical drugs, protein pharmaceuticals have high specificity and activity at relatively low concentrations. These features have made protein pharmaceuticals indispensable in combating human diseases.

Due to advances in analytical separation technology, recombinant proteins can now be purified to an unprecedented level (Bond et al., 1998). Highly purified protein pharmaceuticals significantly reduce the known and unknown potential side or even toxic effects. However, one of the most challenging tasks remains in the development of protein pharmaceuticals: dealing with physical and chemical instabilities of proteins. Protein instability is one of the two major reasons why protein pharmaceuticals are administered traditionally through injection rather than taken orally like most small chemical drugs (Wang, 1996). Protein pharmaceuticals usually have to be stored under cold conditions or even freeze-dried to a solid form to achieve an acceptable shelf life.

In search for ways of stabilizing proteins, scientists turned their attention to nature for an answer. It is well known that certain natural organisms can grow well at extreme temperatures. Hyperthermophilic organisms (hyperthermophiles) such as anaerobic, methanogenic, or sulfate-reducing archaebacteria grow at temperatures near or above 100°C (Huber et al., 1989, Adams, 1993, Adams, 1994). Proteins in these organisms function normally at high temperatures. For example, enolase and α-glucosidase in hyperthermophilic Pyrococcus furiousus have optimum activity, at >90 and >105°C, respectively (Costantino et al., 1990, Peak et al., 1994). The most thermostable proteins found so far have half-lives in excess of 10 min at 130°C (Daniel et al., 1996). The mechanisms responsible for the high molecular stability of thermophilic proteins include increased hydrophobic interactions, greater molecular packing, more H-bonds, more salt-bridging, loss of surface loops, more helix-forming amino acids, restricted N-terminus mobility, etc. (Vieille and Zeikus, 1996, Cowan, 1997, Vogt and Argos, 1997). Extrinsic factors (not primary structure-related) have also contributed to protein stabilization. One of these is the high cellular content of sugars, salts, or other organic solutes/osmolytes, such as α-glutamate, di-myo-inositol-phosphate and its isomer, β-mannosylglycerate, and di-glycerol-phosphate (Huber et al., 1989, Rupley and Careri, 1991, Martins and Santos, 1995, Martins et al., 1997, Ramakrishnan et al., 1997).

The identification of intrinsic and extrinsic factors that contribute to the stabilization of thermophilic proteins has provided valuable information for stabilizing protein pharmaceuticals and for designing more stable mutant proteins. Yet the structural differences among different proteins are so significant that generalization of universal stabilization strategies has not been successful. Very often, proteins have to be evaluated individually and stabilized on a trial-and-error basis.

To understand and maximize the stability of protein pharmaceuticals or any other usable proteins such as various catalytic enzymes, many studies have been conducted in the past few decades. These studies have been reviewed with emphasis on general protein stability (Jaenicke, 1991, Kristjánsson and Kinsella, 1991), mechanisms of chemical and physical instabilities of proteins (Manning et al., 1989), mechanisms and prevention of major protein degradation pathways (Cleland et al., 1993), and various means of stabilizing proteins in aqueous or solid state and under various processing conditions such as freeze-thawing or drying (Gianfreda and Scarfi, 1991, Arakawa et al., 1993, Timasheff, 1993, Manning et al., 1995, Wong and Parascrampuria, 1997). This article reviews these investigations and achievements in recent years and discusses the basic behavior of proteins, their instabilities, and stabilization in aqueous state in relation to the development of liquid protein pharmaceuticals.