What Does Lyophilized Mean? Freeze-Dried Peptides

What does lyophilized mean?

Lyophilized means freeze-dried. Lyophilization removes water from a frozen sample by sublimation and desorption, in three steps: freezing, primary drying and secondary drying (Roy and Gupta, 2004). A lyophilized peptide is a peptide supplied as the dry cake or powder the process leaves behind. Lyophilized and lyophilise are the British spellings; to lyophilize is the verb, and lyophilizing describes the process under way. Freeze-drying is used for proteins, peptides, antibiotics, vaccines and liposomes because it significantly improves the storage stability of materials that are only marginally stable in water (Izutsu, 2018).

How lyophilization works

Freezing. The solution is cooled until most of the water turns to ice and the dissolved material is concentrated between the ice crystals. This step shapes everything after it: it sets the cake's structure and physical state, its residual moisture and how quickly it redissolves (Kasper and Friess, 2011). Low temperature, freeze-concentration and ice formation are the main stresses at this stage, and freeze-concentration can speed reactions and crystallize buffer components (Bhatnagar et al., 2007).

Primary drying. Under vacuum, controlled shelf heating makes the ice sublime, passing straight from solid to vapor and leaving a porous solid behind. The product must stay below its collapse temperature, which is linked to the glass transition of the freeze-concentrate (Tg'), so shelf temperature and chamber pressure are chosen to hold a target product temperature (Tang and Pikal, 2004).

Secondary drying. Once the ice is gone, the temperature is raised to desorb water still bound in the solid, lowering the residual moisture before the vials are closed (Roy and Gupta, 2004; Tang and Pikal, 2004).

Why peptides are supplied lyophilized

In water, peptides degrade by several chemical routes. An asparagine-glycine model hexapeptide deamidated with a half-life of only 1.4 days at 37°C and pH 7.4, through a succinimide that went on to hydrolyze and racemize (Geiger and Clarke, 1987), and deamidation rates depend on pH, temperature and buffer composition (Patel and Borchardt, 1990).

At acidic pH, an aspartyl hexapeptide underwent cleavage of its Asp-Gly peptide bond (Oliyai and Borchardt, 1993), and with oxygen, iron and an electron donor present, methionine residues in small peptides oxidized to methionine sulfoxide (Li et al., 1995). Proteins often have to be made into solids to reach an acceptable shelf life, and lyophilization is the most common way to do it (Wang, 2000).

A dry peptide is not inert. In the solid state, deamidation, peptide bond cleavage, oxidation, the Maillard reaction, beta-elimination and aggregation still occur, at rates set by temperature, moisture content, excipients and whether the solid is amorphous or crystalline (Lai and Topp, 1999). That is why the powder is still stored cold and dry.

What lyophilized peptide powder looks like

Freeze-dried peptide powder is usually white or off-white. With enough material the solid forms a cake, a porous plug that keeps the shape of the frozen fill; a small fill may leave only a thin film, flakes or loose powder at the bottom of the vial, and a colored compound gives a colored cake. Departures from a uniform cake are described in a harmonized nomenclature, and a non-ideal cake may have no impact on quality, because appearance also reflects the formulation, the vial and the process (Patel et al., 2017).

Excipients and bulking agents

Pharmaceutical lyophilized products usually contain excipients: cryoprotectants protect a protein during freezing and lyoprotectants during drying (Roy and Gupta, 2004). Disaccharides such as sucrose and trehalose, and some amino acids, substitute for the molecular interactions water provides and embed the active ingredient in a glassy solid of low molecular mobility, which reduces chemical reactivity (Izutsu, 2018). Infrared spectra of lysozyme freeze-dried with trehalose or lactose showed the sugars interacting with the dried protein in a way that mimics water (Carpenter and Crowe, 1989).

Bulking agents such as mannitol give the cake its structure, and whether mannitol crystallizes depends on its concentration, the freezing rate and the other solutes present (Kim et al., 1998). The choice matters for peptides: in lyophilized formulations of a model hexapeptide, the type of bulking agent, amorphous or crystalline, had a significant effect on solid-state degradation (Oliyai et al., 1994).

Residual moisture

Secondary drying never removes all the water, and what remains matters. In a lyophilized antibody formulation prepared at 1 to 8% residual moisture, the glass transition temperature fell from about 80°C at 1% moisture to 25°C at 8%, and higher moisture lowered chemical stability whether or not the solid was glassy (Breen et al., 2001).

In the model hexapeptide study, residual moisture and storage temperature also affected solid-state reactivity (Oliyai et al., 1994). Residual moisture is commonly measured by Karl Fischer titration (USP <921>, Water Determination). A dry cake readily takes up water, so a cold vial should reach room temperature before it is opened.

Storing lyophilized and reconstituted peptides

Lyophilized: keep vials sealed, cold, dry and dark until use. Temperatures are compound-specific and listed on each product page; the L-Glutathione page, for example, lists -20°C for the powder as shipped, protected from light.
Let the vial reach room temperature before opening so moisture does not condense on the powder.
Reconstitute in a sterile solvent or buffer suited to the assay, adding it gently down the vial wall and letting the powder dissolve rather than shaking it.
Reconstituted: store at 2 to 8°C, protected from light, as product pages list, and prepare working solutions fresh for long incubations.
Split stock into single-use aliquots. Freezing a solution brings back the freezing stresses, including freeze-concentration and ice formation (Bhatnagar et al., 2007), so avoid repeated freeze/thaw cycles.
Record the lot number with every solution and take concentrations from the measured amount on the lot's certificate.

What does non-lyophilized mean?

Non-lyophilized material has not been freeze-dried: it is supplied as a ready-made solution or as a solid made another way. A solution skips reconstitution but keeps the compound in water, where the solution-phase degradation routes above apply, so its storage instructions differ.

The 200 mg/mL L-Glutathione solution, for example, is listed for 15 to 25°C before opening, not to be frozen, and refrigerated once opened, while the lyophilized L-Glutathione powder is listed for -20°C. Lyophilization is also not a sterilization step: freeze-drying is the preferred method for storing culture collections of live microorganisms (Morgan et al., 2006), so sterility, where it matters, is a separate test on the certificate.

Key sources

Roy I, Gupta MN. "Freeze-drying of proteins: some emerging concerns." Biotechnol Appl Biochem. 2004. PMID 15032737. DOI 10.1042/BA20030133. Review: the three steps and protective excipients.
Tang X, Pikal MJ. "Design of freeze-drying processes for pharmaceuticals: practical advice." Pharm Res. 2004. PMID 15032301. DOI 10.1023/b:pham.0000016234.73023.75. Review: collapse temperature, primary and secondary drying.
Kasper JC, Friess W. "The freezing step in lyophilization: physico-chemical fundamentals, freezing methods and consequences on process performance and quality attributes of biopharmaceuticals." Eur J Pharm Biopharm. 2011. PMID 21426937. DOI 10.1016/j.ejpb.2011.03.010. Review of the freezing step.
Izutsu KI. "Applications of Freezing and Freeze-Drying in Pharmaceutical Formulations." Adv Exp Med Biol. 2018. PMID 30288720. DOI 10.1007/978-981-13-1244-1_20. Review: stability and excipients.
Lai MC, Topp EM. "Solid-state chemical stability of proteins and peptides." J Pharm Sci. 1999. PMID 10229638. DOI 10.1021/js980374e. Review: degradation in the dry state.
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. PMID 3805008. Model peptides in solution.
Oliyai C, et al. "Chemical pathways of peptide degradation. VII. Solid state chemical instability of an aspartyl residue in a model hexapeptide." Pharm Res. 1994. PMID 7937533. DOI 10.1023/a:1018998312503. Lyophilized model peptide.
Patel SM, et al. "Lyophilized Drug Product Cake Appearance: What Is Acceptable?" J Pharm Sci. 2017. PMID 28341598. DOI 10.1016/j.xphs.2017.03.014. Commentary on cake appearance.

Frequently asked questions

Is lyophilized the same as freeze-dried?

Yes. Lyophilization and freeze-drying are the same process, and lyophilized is the British spelling.

Why are peptides sold as lyophilized powder?

In water, peptides deamidate, hydrolyze and oxidize; freeze-drying improves storage stability, although reactions continue slowly in the dry state (Lai and Topp, 1999).

Is a shrunken or cracked cake a problem?

Not necessarily. Non-ideal cake appearance can be an inherent result of formulation, vial and process with no impact on quality (Patel et al., 2017). The certificate for the lot is the record of what the vial contains.

Does lyophilized mean sterile?

No. Freeze-drying removes water; it is even used to preserve live microorganisms (Morgan et al., 2006). Sterility is a separate test.

How should lyophilized peptides be stored?

Sealed, cold, dry and dark, at the temperature on the product page. After reconstitution, at 2 to 8°C in single-use aliquots.

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