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Lyophilized vs solution stability

Lyophilized versus solution stability is the comparison between how a peptide or protein changes over time when it is kept as a freeze-dried solid and when it is kept dissolved in water. A review by Wang states that protein pharmaceuticals usually have to be stored under cold conditions or freeze-dried to achieve an acceptable shelf life.[1]

Which degradation pathways does the literature describe?

Manning, Patel and Borchardt summarized chemical and physical decomposition pathways for proteins. They list chemical instability as including proteolysis, deamidation, oxidation, racemization and beta-elimination, and physical instability as including aggregation, precipitation, denaturation and adsorption to surfaces.[2]

A 2010 update by Manning and colleagues discusses chemical and physical instability, stabilization in aqueous solution and in the dried state, and the interrelationship between chemical and physical instability.[3]

ICH Q5C, the stability guidance for biotechnological and biological products, refers to degradation changes that may result from deamidation, oxidation, sulfoxidation, aggregation or fragmentation during storage, and names electrophoresis, high-resolution chromatography and peptide mapping among the methods that may detect them.[4]

How quickly can a peptide deamidate in solution?

Geiger and Clarke studied deamidation, isomerization and racemization at asparaginyl and aspartyl residues in synthetic peptides. They reported that the hexapeptide L-Val-L-Tyr-L-Pro-L-Asn-Gly-L-Ala deamidated with a half-life of only 1.4 days at 37 °C and pH 7.4, forming an aspartyl succinimide.[5]

In that study the succinimide went on to react by hydrolysis and by racemization, leaving a mixture of L- and D-aspartyl and isoaspartyl hexapeptides. Replacing the glycyl residue next to asparagine with a bulky leucyl or prolyl residue slowed degradation 33- to 50-fold.[5]

The review of peptide impurities by D'Hondt and colleagues classes succinimide formation as a degradation route for synthetic peptides, alongside diketopiperazine and pyroglutamate formation.[6]

What role does water play?

ICH Q1A(R2) asks stress testing of a drug substance to evaluate its susceptibility to hydrolysis across a wide range of pH values when it is in solution or suspension.[7]

Water still matters after drying. Bell, Hageman and Muraoka reported that the denaturation temperature of lyophilized bovine somatotropin and of lysozyme, measured by differential scanning calorimetry, fell as moisture increased, irrespective of the excipient.[8]

Lai and Topp note that peptide and protein drugs are often formulated in the solid state to provide stabilization during storage, but that reactions still occur in the solid state. Their review covers deamidation, peptide bond cleavage, oxidation, the Maillard reaction, beta-elimination and dimerization or aggregation, and names temperature, moisture content, excipients and the amorphous or crystalline state of the solid as influencing factors.[9]

Why is freeze-drying used to make a solid form?

Wang writes that, to overcome the instability barrier, proteins often have to be made into solid forms to achieve an acceptable shelf life as pharmaceutical products, and that lyophilization is the most commonly used method of preparing them.[10]

An FDA inspection guide on lyophilization lists among the advantages of the process enhanced stability of a dry powder, removal of water without excessive heating of the product, and enhanced product stability in a dry state.[11]

Does a freeze-dried solid stop degradation?

Wang's review states that the lyophilization process itself generates freezing and drying stresses that can denature proteins to various degrees, and that even after successful lyophilization with a stabilizer, proteins in the solid state may still have limited long-term storage stability.[10]

The FDA inspection guide describes meltback, a form of cake collapse caused by incomplete sublimation in the vial, and states that the associated change in the physical form of the drug substance or a pocket of moisture may result in greater instability and increased product degradation.[11]

The same guide calls the amount of moisture present in vials an obvious concern for a lyophilized product, and states that the expiration date and moisture limit should be established from worst-case data showing adequate stability at the moisture specification.[11]

Limitations

Much of the literature cited here concerns proteins or model peptides. The rates reported by Geiger and Clarke apply to the hexapeptides they studied at 37 °C and pH 7.4, and their own substitution experiments show that neighbouring residues change those rates substantially.[5],[2]

General descriptions of degradation pathways are not stability data. ICH Q1A(R2) describes stability testing as providing evidence of how the quality of a particular drug substance or product varies with time, which a review of mechanisms cannot supply.[7]

This page describes degradation chemistry in general terms. It contains no handling or preparation instructions and does not describe any PepGenex material.

This page describes what the cited documents say. It is not a statement that any PepGenex material has been manufactured, tested or released under any of them.

Compound profiles

Research peptide profiles in this library. What this page describes applies to peptide lots in general; none of these profiles reports a result of it for any lot.

References

  1. Wang W. Instability, stabilization, and formulation of liquid protein pharmaceuticals. Int J Pharm. 1999;185(2):129-188. PMID 10460913 · DOI 10.1016/s0378-5173(99)00152-0
  2. Manning MC, Patel K, Borchardt RT. Stability of protein pharmaceuticals. Pharm Res. 1989;6(11):903-918. PMID 2687836 · DOI 10.1023/a:1015929109894
  3. Manning MC, Chou DK, Murphy BM, Payne RW, Katayama DS. Stability of protein pharmaceuticals: an update. Pharm Res. 2010;27(4):544-575. PMID 20143256 · DOI 10.1007/s11095-009-0045-6
  4. ICH Q5C Quality of Biotechnological Products: Stability Testing of Biotechnological/Biological Products. U.S. FDA guidance for industry, July 1996. FDA guidance, July 1996 · 61 FR 36466
  5. Geiger T, Clarke S. Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation. J Biol Chem. 1987;262(2):785-794. PMID 3805008 · DOI 10.1016/S0021-9258(19)75855-4
  6. D'Hondt M, Bracke N, Taevernier L, et al. Related impurities in peptide medicines. J Pharm Biomed Anal. 2014;101:2-30. PMID 25044089 · DOI 10.1016/j.jpba.2014.06.012
  7. ICH Q1A(R2) Stability Testing of New Drug Substances and Products. U.S. FDA guidance for industry, November 2003. FDA docket FDA-2002-D-0222
  8. Bell LN, Hageman MJ, Muraoka LM. Thermally induced denaturation of lyophilized bovine somatotropin and lysozyme as impacted by moisture and excipients. J Pharm Sci. 1995;84(6):707-712. PMID 7562408 · DOI 10.1002/jps.2600840608
  9. Lai MC, Topp EM. Solid-state chemical stability of proteins and peptides. J Pharm Sci. 1999;88(5):489-500. PMID 10229638 · DOI 10.1021/js980374e
  10. Wang W. Lyophilization and development of solid protein pharmaceuticals. Int J Pharm. 2000;203(1-2):1-60. PMID 10967427 · DOI 10.1016/s0378-5173(00)00423-3
  11. U.S. FDA. Guide to Inspections: Lyophilization of Parenteral (7/93). FDA Inspection Guide, July 1993