Expert Review Stability of Protein Pharmaceuticals: an Update
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Pharmaceutical Research, Vol. 27, No. 4, April 2010 (# 2010) DOI: 10.1007/s11095-009-0045-6 Expert Review Stability of Protein Pharmaceuticals: An Update Mark Cornell Manning,1,4 Danny K. Chou,2 Brian M. Murphy,1 Robert W. Payne,1 and Derrick S. Katayama3 Received October 6, 2009; accepted December 27, 2009; published online February 9, 2010 Abstract. In 1989, Manning, Patel, and Borchardt wrote a review of protein stability (Manning et al., Pharm. Res. 6:903–918, 1989), which has been widely referenced ever since. At the time, recombinant protein therapy was still in its infancy. This review summarizes the advances that have been made since then regarding protein stabilization and formulation. In addition to a discussion of the current understanding of chemical and physical instability, sections are included on stabilization in aqueous solution and the dried state, the use of chemical modification and mutagenesis to improve stability, and the interrelationship between chemical and physical instability. KEY WORDS: formulation; protein stability; protein stabilization. INTRODUCTION the physical state of the protein does change. This includes denaturation, aggregation, precipitation, and adsorption In 1989, Manning, Patel and Borchardt wrote a review (Table I). The term precipitation is used here to denote summarizing what was known at the time about the stability insolubility rather than insoluble aggregate formation. and stabilization of protein pharmaceuticals (1), an article Our knowledge of all protein degradation pathways is that has been referenced almost 500 times. In the late 1980s, markedly greater than it was 20 years ago. Therefore, the there were only three recombinant protein products on the emphasis of this review is on the progress that has been made US market: Orthoclone (OKT-3), human insulin, and tissue since 1989. In addition, there were degradation processes and plasminogen activator. If one included plasma-derived prod- topics that were barely discussed or observed at that time. Those ucts, the number of approved proteins only numbered about are now included as separate sections below. For example, there a dozen. Clearly, recombinant DNA technology has drasti- have been many articles on increasing conformational stability cally changed the pharmaceutical market. Now there are of proteins with various excipients, both in aqueous solution and nearly twenty antibody products and almost 150 approved in the dried state. In addition, a brief overview is provided of protein-based products that are commercially available in the protein stabilization methods, including various drying methods, US alone. In addition, our knowledge regarding protein chemical modification, and site-directed mutagenesis. Finally, a stability and formulation has increased dramatically. The discussion of the interrelationship between chemical and purpose of this review is to provide an update regarding physical instability is provided. what we have learned in the past 20 years. In addition to updating the sections of the original review article, some discussion is provided regarding topics that were not found in CHEMICAL INSTABILITY the literature at the time, such as the interrelationship of chemical and physical instability, instabilities that occur Deamidation during bioprocessing, the impact of lyophilization cycle on protein stability, and the importance of packaging in main- Twenty years ago, it was already appreciated that taining protein stability. deamidation, which involves the hydrolysis of Asn and Gln One can separate protein instabilities into two general side chain amides, was a common degradation pathway for classes: chemical instability and physical instability. Chemical proteins and peptides. It is still regarded as the most common instabilities involve processes that make or break covalent chemical degradation pathway for peptides and proteins. bonds, generating new chemical entities. A list of the more From a regulatory perspective, deamidation generates proc- commonly observed chemical degradation processes is listed ess-related impurities and degradation products. In addition, in Table I. Conversely, there are physical instabilities for it may contribute to increased immunogenicity (2). proteins in which the chemical composition is unaltered, but At the time of the original review article, there were a few examples of deamidation in pharmaceutically relevant 1 Legacy BioDesign LLC, Johnstown, Colorado 80534, USA. proteins, including human growth hormone (hGH) (3,4), γ 2 Genzyme, Framingham, Massachusetts 01701, USA. insulin (5), -globulin (6), and hemoglobin (7). Moreover, the 3 Amylin Pharmaceuticals, San Diego, California 92121, USA. effect of extrinsic factors, such as pH, temperature, and ionic 4 To whom correspondence should be addressed. (e-mail: manning@ strength, were known as well (8). Since that time, the amount legacybiodesign.com) of information now available on deamidation and related 0724-8741/10/0400-0544/0 # 2010 Springer Science+Business Media, LLC 544 Stability of Protein Pharmaceuticals: An Update 545 Table I. Chemical Instabilities Reported for Proteins of Therapeutic below). However, this mechanism is rarely observed, as the pH Interest must be less than 3. In neutral to basic solution (i.e., above pH 6), the mechanism changes to an intramolecular cyclization Deamidation reaction. The first step involves nucleophilic attack of the n+1 Asp–isoAsp interconversion/isomerization nitrogen of the protein backbone on the carbonyl group of the Racemization Asn side chain (Fig. 1). This step is base catalyzed, since Proteolysis abstraction or partial abstraction of the backbone amide proton Beta-elimination makes the nitrogen more nucleophilic, accelerating the reaction. Oxidation A cyclic imide (also called succinimide or Asu) intermediate is Metal-Catalyzed Oxidation (MCO) formed (Fig. 1) with loss of ammonia. Since ammonia is a gas Photooxidation and is typically not retained in solution, this step is effectively Free radical cascade oxidation irreversible. While the Asu intermediate often can be detected Disulfide exchange as a degradation product in its own right (see below), it is readily DKP formation Condensation reactions hydrolyzed in aqueous solution to form the Asp and isoAsp fi pGlu formation products (Fig. 1). Formation of the Asu ve-membered ring Hinge region hydrolysis intermediate is thought to be the reason that Asn deamidation is Trp hydrolysis more prevalent than Gln deamidation, as five-membered heterocyclic rings are more stable than the six-membered rings associated with Gln deamidation. reactions has increased significantly, as can be found in a Consequently, deamidation generates two degradation number of excellent review articles (9–12) as well as entire products (Asp and isoAsp) at the site of the original Asn books on the subject (13,14). There is even a web site devoted residue. Coupled with the possibility for racemization (15), four to this topic (www.deamidation.org). possible products (L-Asp, D-Asp, L-isoAsp, and D-isoAsp) could be formed. It is now known that racemization does not Asn Deamidation occur to any appreciable extent from the Asu intermediate, as was previously thought. Instead, it appears to be a parallel For those unfamiliar with this reaction, deamidation of Asn degradation pathway (16). Dehart and Anderson have provided residues under acidic conditions takes place by direct hydrolysis a detailed kinetic description of the intramolecular cyclization of the Asn side chain amide to form only Asp. Under these (17). The same observation of a lack of racemization via the conditions, deamidation is subject to acid catalysis. Similarly, cyclic imide intermediate has been made for larger proteins as Gln residues are converted to Glu (as is described in more detail well (18). Fig. 1. General mechanism for deamidation of Asn residues and isomerization of Asp to isoAsp (taken from reference 1). Direct hydrolysis occurs below pH 4 while the cyclic imide pathway predominates at pH 6 and above. 546 Manning et al. Effect of Sequence on Asn Deamidation has been found to play a minimal role in controlling deamidation rates (29). Some work had been done by 1989 on the effects of primary sequence on deamidation, especially recognizing that Asn–Gly Effect of Higher Order Structure on Asn Deamidation were particularly prone to deamidation. Subsequently, the influence of sequence was examined by Robinson and coworkers In 1989, the ability of higher order structure to influence in detail (19,20). Ultimately, their work on sequence effects deamidation rates was just starting to be appreciated. In 1988, resulted in effective predictive schemes (14,21–25). In general, Kossiakoff demonstrated that polypeptide chain flexibility two trends are apparent. First, having amino acids with smaller impacted deamidation rates (30). Other studies have since arrived side chains after the Asn residues leads to faster deamidation, at the same conclusions, examining the relative deamidation rates presumably due to lack of steric hindrance of the initial cyclization for Asn residues dispersed across a given globular protein reaction. Second, succeeding amino acids that have side chains structure (31,32). In addition, a number of studies have shown that can act as hydrogen bond donors tend to accelerate the that placement of the reactive Asn residue within an ordered reaction, likely due to intramolecular hydrogen binding to the secondary structure slows the reaction rate. This has been found carbonyl oxygen