Review article
Strategic approaches for overcoming peptide and protein instability within biodegradable nano- and microparticles

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Abstract

This paper reviews the major factors that are closely involved in peptide and protein degradation during the preparation of biodegradable nano- and microparticles. The various means usually employed for overcoming these obstacles are described, in order to bring to the fore the strategies for protein stabilization. Both processing and formulation parameters can be modified and are distinctly considered from a strategic point of view. We describe how partial or full protein stability retention within the carriers and during drug release might be achieved by individual or combined optimized strategies. Additionally, problems commonly encountered during protein quantification, stability determination and release are briefly reviewed. Artefacts that might occur during sampling and analytical procedures and which might hinder critical interpretation of results are discussed.

Introduction

The advent of recombinant DNA technology has made possible the commercial production of proteins for pharmaceutical applications from the early 1980s. The development of pharmaceuticals directly from this ever emerging technology has enabled to provide the market with products of exceptional high purity. In order to maintain such high standards of quality, manufacturers have to make sure that the protein drugs are fully active upon administration and that they contain no toxic by-products. Formulations must be able to release an intact active moiety and actually, the ability to demonstrate protein stability inside pharmaceuticals and during delivery becomes a requisite for reaching the product approval stage [1], [2]. Nevertheless, protein stability certainly still remains one of the most important hurdles for their successful incorporation in biodegradable systems, such as nano- or microparticulate carriers. Indeed, in the field of protein nano- and microencapsulation, ensuring product consistency is a rather tricky matter, insofar as each component of such a formulation has not only to be recognized as safe, but conjugated complexes between drugs and polymeric macromolecules might also be considered as new chemical entities by health authorities [3]. With respect to the safety of the polymers used for encapsulation, it should be underlined that those derived from d,l lactic and glycolic acids (like poly(d,l-lactic-co-glycolic acid), PLGA) have gained some popularity, mainly because of their noteworthy properties in terms of tissue compatibility and biodegradability [4].

According to the FDA, a pharmaceutical product is considered as stable as long as it deteriorates by no more than 10% in 2 years. But as far as proteins are concerned, the term stability needs to be defined with more accuracy. Chemical stability involves typically the integrity of the amino acid sequence (primary structure) and the reactivity of the side chains. Most of the time, the activity of peptides depends on the primary and possibly secondary structures, whereas proteins possess an additional tertiary and sometimes even a quaternary structure, that allows the protein chains to fold and adopt a three-dimensional conformation [5]. The chemical stability of a pharmaceutical protein can be impaired during for instance proteolysis. Such a degradation pathway leads to two or more products of smaller molecular weight, which should be characterized. It is indeed not only necessary to recover high amounts of therapeutically active protein from the pharmaceutical, but even low amounts of degraded protein must also be safe, since degradation products might be therapeutically inactive, or cause unpredictable side effects, such as toxicity or antigenicity. Physical stability is generally defined as the ability of a protein to retain at least its tertiary structure, that is crucial for the biological activity too. Common chemical and physical degradation pathways have been reviewed and discussed, for instance, by Witschi [6] within the framework of the encapsulation of a peptide drug into microparticles. Briefly, chemical degradation includes deamidation, isomerization, hydrolysis, racemization, oxidation, disulfide formation and β-elimination. Physical degradation involves reversible or irreversible denaturation through a loss of tertiary structure and unfolding with further reactions like chemical degradation, aggregation and precipitation. Most of the time, the pharmaceutical properties of therapeutic proteins closely depends on the retention of their biological activity. Consequently, the pharmaceutical efficacy (and hence the three-dimensional integrity) should be assessed by monitoring the biological activity at the end of the formulation process. In the same way, the control of antigenicity is needed to guarantee the efficacy of protein therapeutics like vaccines or to exclude any protein degradation (e.g. aggregation often causes increased antigenicity) [7]. The antigenicity of a protein is dependent on the intactness of its antigenic determinants (well defined sub-units of the molecular structure). It must be emphasized that a protein might undergo a loss of enzymatic activity but preserve its antigenicity, because the active site region responsible for the expression of activity is often different from the epitope that reacts with a polyclonal antibody [8], [9], [10]. Additionally, results obtained from in vitro enzymatic activity measurements and antibody assays should be compared to those obtained during in vivo assessments, since in vitro–in vivo correlation is not always possible [11], [12], [13], [14], [15], [16]. Ideally, these experiments should also be completed by structural analysis in order to make sure that protein stability is totally retained [1].

Nowadays, the assessment of protein stability during manufacture, storage or release is increasingly being integrated into research programs and in the past few years, the number of publications relating to encapsulated protein stability has considerably grown. Additionally, some excellent reviews have addressed many aspects concerning protein stability, such as the factors responsible for instability and the stabilization techniques [17], [18], [19], [20], [21], [22]. For protein degradation to be at least reduced and preferentially avoided, an accurate stabilization rationale is required. However, it is most of the time not possible to adopt a general strategy for protein stabilization. For instance, an additive that efficiently stabilizes a protein might damage another one [23]. Thus, proteins are traditionally formulated by an empirical trial and error approach, each protein being different one from the other. Generally, the need to keep the protein stable within the formulation has right of way over any release considerations. But obviously, the ideal goal would be to achieve satisfactory protein stabilization along with an appropriate drug release by adopting the same strategic approach. Further additional tedious developments of the manufacturing process are thereby avoided [24], [25].

The present article reviews the state of the art of manufacture techniques and discusses parameters that induce perturbations in entrapped proteins, as well as the strategies adopted for peptide or protein stabilization during the encapsulation and release process. The paper focuses on the studies performed with biodegradable nano- and microparticles. Since critical interpretation of results can be considered as being of major importance, sampling procedures and some analytical techniques for protein stability analysis are also discussed. Initial protein quality, properties and preformulation are addressed as they might drastically influence final protein stability in the carriers. Finally, the section concerning the use of additives presents typical examples of successful and failed stabilization trials of different commonly used model and therapeutic proteins. Protein stability issues during freeze-drying procedure and during storage are not addressed.

Section snippets

Sampling and analytical issues

The assessment of stability and the precise quantification of an encapsulated protein still remain difficult tasks and major obstacles are encountered during the sample preparation before analysis and during the analysis itself [5], [15], [16], [26], [27]. Protein integrity evaluation is indeed likely to be affected by artefacts during these operations [28], thereby preventing the scientist from critically ascribing detected protein denaturation to manufacturing conditions. In vitro, there is

Processing parameters affecting protein stability

Among all the possible techniques for protein encapsulation, the most widely used is certainly the water-in-oil-in-water (w1/o/w2) double emulsion method. This is a suitable method for hydrophilic drug incorporation, insofar as the drug is first dissolved in the inner aqueous phase (w1) and then trapped within a polymer resulting in a matrix (sphere) or a reservoir (capsule) system. Briefly, an aqueous solution of the hydrophilic drug is emulsified into an organic solution of the polymer. Ethyl

Formulation parameters affecting protein stability

This section includes the formulation factors that might affect protein stability, i.e. the protein properties, the polymer employed and the excipients added in the formula.

Conclusion

The production of biodegradable nano- and microparticles containing a stable therapeutic peptide or protein still remains a major challenge, mostly in terms of technical obstacles. Beyond the traditional techniques usually used for stabilization of protein in solid state or in solution, some strategies are specific to protein encapsulation such as the development of new manufacturing methods or the choice of a suitable polymer. To enable protein stabilization, the optimization of each step of

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    Present address: Bracco Research SA, Route de la Galaise 31, 1228 Plan-les-Ouates, Geneva, Switzerland.

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