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Novel Excipients Prevent Aggregation in Manufacturing and Formulation of Protein and Peptide Therapeutics

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... Protein aggregates are often considered ordered if they occur as long, rigid fibrils or filaments. The most characterized aggregation state is the amyloid fibril, associated with neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease [14,15]. Protein aggregation can be problematic during drug manufacturing, especially if it is insoluble and tends to precipitate, and typically reduces drug stability and shelf life [15]. ...
... One approach to reduce aggregation is to work with protein solutions at lower concentrations and correspondingly larger volumes. A number of excipients have been used with varying success to reduce protein aggregation; however, each has its own limitations [14]. Nonionic detergents can be used to reduce aggregation induced by shear and heat. ...
... Another approach to reduce aggregation is to use lyophilized dosage forms [16]. Lyophilization is generally regarded as an effective means to stabilize proteins [13,14]. However, proteins can undergo reversible conformational changes in the lyophilized state, which makes them more susceptible to undesirable side reactions. ...
... It is probably fair to say that there has been insufficient focus on alternative surfactants for aggregation prevention in biotherapeutics. Recently, a class of alkylsaccharide excipients developed initially as highly effective transmucosal absorption enhancers for peptide and protein drugs [40][41][42][43] have been shown to very effectively prevent protein aggregation, reduce or eliminate immunogenicity, and increase the solution stability of a number of high-value protein therapeutics [44][45][46]. Alkylsaccharides are nonionic surfactants comprising a sugar moiety coupled to an alkyl chain. They metabolize quickly and cleanly to the free sugar and the corresponding long-chain fatty acid or alcohol [47]. ...
... Perhaps most importantly, they are not subject to oxidative degradation, thus providing a potential alternative to the use of polyoxyethylenecontaining surfactants in biotherapeutic formulations. Published studies show alkylsaccharide stabilization of insulin, IFN-b1a and IFN-b1b, monoclonal antibodies, parathyroid hormone 1-34 and cyclic1-31, pramlintide, and human growth hormone [44,45], among others. Specific studies on the potential for alkylsaccharides to alter the pharmacokinetics of biotherapeutics have not been undertaken; however, since both alkylsaccharides and polysorbates are nonionic surfactants, and since both alkylsaccharides and polysorbates have been shown to be effective in preventing protein aggregation, one may speculate that the effects, if any, are not likely to be divergent. ...
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Polysorbates and other polyoxyethylene-based surfactants are incorporated into most biotherapeutics to prevent protein aggregation in order to minimize loss of efficacy, induction of unwanted immunogenicity, altered pharmacokinetics and reduced shelf life. While they are effective in initially preventing protein aggregation, they contain ether linkages (within polyoxyethylene moieties) and in the case of polysorbate 80 unsaturated alkyl chains that spontaneously and rapidly auto-oxidize in aqueous solution to protein-damaging peroxides, epoxy acids and reactive aldehydes, including formaldehyde and acetaldehyde. Oxidative damage induces unwanted immunogenicity and in some instances promotes re-aggregation. Immunogenicity of biotherapeutics is a serious and growing concern for the US FDA and European Medicines Agency and will have significant and growing impact on the development and regulatory approval of both biosimilar and new innovator biotherapeutics.
... The biomanufacturing itself, purification and concentration using a variety of methods such as ultrafiltration, chromatography, lyophilization, dialysis and precipitation can cause protein aggregation. Preventing protein aggregation has become a major issue for pharmaceutical production because the tendency towards highly-concentrated solutions increases the likelihood of protein-protein interactions favoring aggregation [65]. ...
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Over the last two decades, developments in nanomedicine have resulted in technical advances with application to clinical science. Both organic and inorganic nanoparticles (NPs) have shown tolerability, pharmacologic specificity and biodegradability. A subclass of NPs, protein NPs, have garnered recent attention due to the inherent biocompatibility of protein substrates. Protein NPs are currently being employed widely in pharmaceuticals development with applications in nasal, pulmonary, intravenous, ocular and oral delivery. Despite the distinct advantages of orally administered pharmaceuticals, the development of oral delivery systems has been comparatively limited. Therefore, this review attempts to discuss the most recent experimental and pre-clinical findings in the development of protein NPs for oral delivery, while envisioning upcoming challenges.
... The introduction of novel excipients may become an important consideration for stabilizing certain protein therapeutics [41]. For example, some recent data suggest that novel alkyl saccharide excipients may prove useful in reducing higher order aggregates in high concentration protein formulations [42]. ...
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The purpose of this study was to characterize the effects of alkylglycosides on the bioavailability of calcitonin following nasal and ocular administration. A salmon calcitonin specific radioimmunoassay kit was used to measure calcitonin levels in anesthetized rats at various times after nasal or ocular administration of calcitonin formulated with saline or with octylmaltoside, a medium chain length alkylglycoside or tetradecylmaltoside, a long chain alkylglycoside. The extent of calcitonin absorption was determined directly from the plasma calcitonin level-time curve and the bioavailability of calcitonin was determined from the area under the plasma calcium level-time curve. The calcium level was determined using a colorimetric method. When the nasal formulation contained calcitonin plus saline or 0.125% octylmaltoside, little or no calcitonin was absorbed. However, plasma calcitonin levels were increased and plasma calcium levels were decreased when the nasal formulation contained calcitonin plus 0.125% or 0.25% tetradecylmaltoside. Maximal calcitonin levels were observed 7.5-10 min after nasal administration of the formulation. Ocular administration of calcitonin formulated with tetradecylmaltoside also resulted in calcitonin absorption, but less calcitonin absorption was found after ocular administration than after nasal administration. The experimental data indicate that tetradecylmaltoside, but not octylmaltoside. can be effectively used to enhance the bioavailability of nasally and ocularly administered calcitonin.
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A major limitation in the successful development of multidose protein formulations is protein aggregation induced by antimicrobial preservatives such as benzyl alcohol, which are included to maintain product sterility. Studies were conducted to evaluate the strategy of developing lyophilized formulations of a therapeutic protein, recombinant human interlukin-1 receptor antagonist (rhIL-1ra), to be reconstituted with a bacteriostatic amount (0.9% w/v) of benzyl alcohol in water. The strategy was based on the following hypotheses. The first was that benzyl alcohol would foster aggregation during reconstitution of the lyophilized sample. The second hypothesis was that the extent of benzyl alcohol-induced protein aggregation would correlate directly with the degree of structural perturbation of rhIL-1ra in the dried solid after lyophilization. Differential structural retention of rhIL-1ra in the dried solid was obtained by using a combination of formulation variables important for lyophilization and included: protein concentration, type of stabilizer, and presence or absence of NaCl. Infrared spectroscopic analysis of the lyophilized samples indicated that high initial solution protein concentration and the stabilizer sucrose minimized structural perturbation of rhIL-1ra during lyophilization. In contrast, NaCl was destabilizing. Reconstitution of the dried solid with 0.9% (w/v) benzyl alcohol caused a greater degree of protein aggregation than reconstitution with water, confirming our first hypothesis. In support of our second hypothesis, the extent of aggregation induced by benzyl alcohol during reconstitution was strongly modulated by the degree of retention of native rhIL-1ra secondary structure during lyophilization. During storage of the reconstituted lyophilized samples at room temperature, benzyl alcohol did not accelerate aggregation of rhIL-1ra. This study demonstrated that for development a multidose lyophilized protein formulation involving reconstitution with a solution of benzyl alcohol, protein structural perturbations during freeze-drying should be minimized with a stabilizing excipient and appropriate choice of protein concentration and tonicity modifier. Furthermore, postreconstitution storage at reduced temperature (e.g., room temperature or 4 degrees C) could minimize the risk of preservative-induced protein aggregation.
2-Trifluoroethanol Toxicity in Aged Rats
,2,2-Trifluoroethanol Toxicity in Aged Rats. J. Tox. Path. 16(1) 1988: 35-45.
Synthetic Long-Chain
  • D Pillion
Pillion D, et al. Synthetic Long-Chain
Alkyl Maltosides and Alkyl Sucrose Esters as Enhancers of Nasal Insulin Absorption
Alkyl Maltosides and Alkyl Sucrose Esters as Enhancers of Nasal Insulin Absorption. J. Pharm. Sci. 91(6) 2002: 1456-1462.
Peptide Pharmaceutical Compositions. US Patent Application No
  • Ruff Pert
Pert, Ruff. Peptide Pharmaceutical Compositions. US Patent Application No. 20070111938A1, 2007.
Nonionic Surfactants. Alkyl Polyglycosides
  • K Kocher
  • H J Wiegand
Kocher K, Wiegand HJ. Nonionic Surfactants. Alkyl Polyglycosides. Balzer D, Lueders H, Eds. Marcel Dekker: New York, 2000: 365-383.