Solid‐Phase Aggregation of Proteins under Pharmaceutically Relevant Conditions†

Costantino, Henry R.; Langer, Robert; Klibanov, Alexander M.

doi:10.1002/jps.2600831205

Cited by 165 publications

(130 citation statements)

References 41 publications

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“…The number of lysine residues missing in the aggregate compared with the control was greater than for any other amino acid residue. Two well-known reactions involving lysine include transamidation and formation of lysinoalanine cross-links (13). Since the latter did not increase in the aggregate (Fig.…”

Section: Resultsmentioning

confidence: 92%

“…This indicates that the water-sorbing capability is not the only important characteristic of the excipient which influences solid-state aggregation. Sorbitol and other sugars have been reported to preserve the conformation of proteins upon lyo- philization (24)(25)(26)(27) which could improve solid state stability if aggregation requires prior unfolding (13,14).…”

Section: Resultsmentioning

confidence: 99%

“…Previously reported mechanisms for moisture-induced aggregation of proteins include disulfidemediated and noncovalent routes (13). To examine whether these pathways were involved in TT aggregation, vaccine aggregates formed at various humidities (10-day incubation) were reconstituted in reducing and/or denaturing solvents.…”

Section: Resultsmentioning

confidence: 99%

“…We have focused on the rehydration step and subsequent exposure to moisture within the polymeric carrier during release since (i) these conditions take place with any lyophilized microsphere preparation and (ii) moisture induces the aggregation of many solid proteins (11)(12)(13)(14). We report that lyophilized TT becomes insoluble upon exposure to elevated humidity and physiological temperature.…”

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confidence: 99%

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Stabilization of tetanus and diphtheria toxoids against moisture-induced aggregation.

Schwendeman

Costantino

Gupta

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

103

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The progress toward single-dose vaccines has been limited by the poor solid-state stability of vaccine antigens within controlled-release polymers, such as poly(lactide-co-glycolide). For example, herein we report that lyophilized tetanus toxoid aggregates during incubation at 37°C and elevated humidity-i.e., conditions relevant to its release from such systems. The mechanism and extent of this aggregation are dependent on the moisture level in the solid protein, with maximum aggregation observed at intermediate moisture contents. The main aggregation pathway is consistent with formaldehyde-mediated cross-linking, where reactive electrophiles created and stored in the vaccine upon formalinization (exposure to formaldehyde during vaccine preparation) react with nucleophiles of a second vaccine molecule to form intermolecular cross-links. This process is inhibited by the following: (i) succinylating the vaccine to block reactive amino groups; (ii) treating the vaccine with sodium cyanoborohydride, which presumably reduces Schiff bases and some other electrophiles created upon formalinization; and (iii) addition of low-molecular-weight excipients, particularly sorbitol. The moisture-induced aggregation of another formalinized vaccine, diphtheria toxoid, is also retarded by succinylation, suggesting the generality of this mechanism for formalinized vaccines. Hence, mechanistic stability studies of the type described herein may be important for the development of effective single-dose vaccines.

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Section: Resultsmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Stabilization of tetanus and diphtheria toxoids against moisture-induced aggregation.

Schwendeman

Costantino

Gupta

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

103

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“…At the end of release, protein residue in PLGA-Glu millicylinders/microspheres was characterized by a previously reported method (Costantino et al, 1994;Kang and Schwendeman, 2002;. Briefly, polymers were dissolved in acetone and centrifuged.…”

Section: Analysis Of Protein Stability Inside Plga-glu Millicylindersmentioning

confidence: 99%

Stability of proteins encapsulated in injectable and biodegradable poly(lactide-co-glycolide)-glucose millicylinders

Kang

Lambert

Ausborn

et al. 2008

International Journal of Pharmaceutics

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Purpose-To characterize protein stability in poly (lactide-co-glycolide) 50/50-glucose star (PLGA-Glu) injectable millicylinders and to compare results with linear PLGA 50/50.Methods-Bovine serum albumin (BSA), a model protein, was encapsulated in PLGA-Glu and linear PLGA millicylinders by solvent extrusion and incubated under physiological conditions. Important system properties were characterized, including: polymer molecular weight distribution, soluble acidic residues, polymer morphology, polymer water uptake, microclimate pH, protein content and release, and protein aggregation. The polymer microclimate late in the release incubation was simulated and protein recovery was analyzed by UV 280 , size exclusion chromatography, amino acid analysis, and a modified Bradford assay.Results-PLGA-Glu contained higher levels of low molecular weight oligomers, more rapidly biodegraded, and exhibited a lower microclimate pH than the linear 50/50 PLGA, which is the most acidic type in the PLGA family. BSA, when encapsulated in PLGA-Glu millicylinders, underwent extensive noncovalent insoluble aggregation over 2 weeks in vitro release, which was almost completely inhibited upon co-encapsulation of Mg(OH) 2 . However, by 5 weeks release for base-containing formulations, although insoluble aggregation was still suppressed, the soluble fraction of protein in the polymer was unrecoverable by the modified Bradford assay. Polymer microclimate simulations with extensive protein analysis strongly suggested that the low recovery was mostly caused by base-catalyzed hydrolysis of the oligomeric fraction of BSA.Conclusions-In PLGA-Glu, the acidic microclimate was similarly responsible for insoluble aggregation of encapsulated BSA. BSA aggregation was inhibited in millicylinders by coincorporation into the polymer insoluble base, but over a shorter release interval than linear PLGA likely because of a more acidic microclimate in the star polymer.

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Long‐Term Storage of Proteins

2002

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This unit provides a summary of some of the issues that researchers face when attempting to store purified proteins. It briefly explains the stresses that induce protein aggregation the major causes for chemical degradation. It also discusses how to use various storage strategies to increase the long-term stability of proteins. When appropriate it points out critical mistakes to avoid. This unit provides a summary of some of the issues that researchers face when attempting to store purified proteins.

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Solid‐Phase Aggregation of Proteins under Pharmaceutically Relevant Conditions†

Cited by 165 publications

References 41 publications

Stabilization of tetanus and diphtheria toxoids against moisture-induced aggregation.

Stabilization of tetanus and diphtheria toxoids against moisture-induced aggregation.

Stability of proteins encapsulated in injectable and biodegradable poly(lactide-co-glycolide)-glucose millicylinders

Long‐Term Storage of Proteins

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