What is a lyophilized peptide

Freezing, sublimation and secondary drying

Freeze-drying has three stages: freezing, primary drying and secondary drying. During primary drying, ice passes directly into vapour under reduced pressure; secondary drying removes part of the unfrozen water through desorption. These are distinct phenomena. Product temperature and heat and mass transfer influence the process (Tchessalov et al., 2023; PMID: 37783925).

Secondary drying targets a residual water content appropriate to the formulation. Lyophilized therefore does not mean completely anhydrous. There is also no single cycle suitable for every material: thermal limits and the behaviour of amorphous or partly crystalline formulations require specific consideration. We recommend documenting the final state and its measurements rather than inferring them solely from the word “lyophilized” on a label (Tchessalov et al., 2023; PMID: 37783925).

The structure of the dry material

The structure and morphology established during freezing influence drying; the material can form a porous matrix. That macroscopic geometry should be distinguished from the physical state of its components. Amorphous and crystalline describe solid states, whereas cake or powder describes presentation. We recommend recording these descriptions separately and avoiding interpretation of apparent volume as peptide quantity, which requires its own analytical determination (Tchessalov et al., 2023; PMID: 37783925; McCarthy et al., 2023; PMID: 36949371).

In CSP7 formulations, Sahakijpijarn and colleagues used X-ray diffraction to identify amorphous and crystalline states of excipients and salts. The results depended on composition: the lyophilized solids did not all have the same physical state. This example shows why visual appearance cannot replace instrumental characterization or establish a specific structure for a different peptide, even if two dried preparations look similar (Sahakijpijarn et al., 2019; PMID: 31569515, section 3.3).

Residual water and hygroscopicity

Water remaining after drying should be distinguished from water taken up during subsequent exposure to humidity. Hygroscopicity concerns that uptake from the surroundings. For CSP7, exposing lyophilized formulations to humidity increased absorbed water and promoted physical changes, including aggregation. These observations concern those preparations; they do not provide a universal moisture uptake rate or establish that all peptides respond equally to the same exposure (Hengsawas Surasarang et al., 2018; PMID: 28835128).

Water can be determined by Karl Fischer titration; dynamic vapour sorption examines mass changes as relative humidity varies. Sahakijpijarn used these techniques for different purposes. McCarthy measured moisture in peptide standards and controlled its ingress during analysis. We recommend checking method, units and conditions: a visibly dry solid does not establish a measured water content or replace a reported result for the analysed preparation (Sahakijpijarn et al., 2019; PMID: 31569515, sections 2.7 and 2.9; McCarthy et al., 2023; PMID: 36949371).

Why it is used and its limitations

Lyophilization is used to support stability because molecular mobility and reactivity are reduced in the solid state. However, residual or absorbed moisture can contribute to physical and chemical instability. A comparison with solution requires specifying the measurement and observation period. Preservation is the intended advantage, but it cannot be treated as a guarantee independent of formulation, container and storage conditions (Hengsawas Surasarang et al., 2018; PMID: 28835128).

Different lyophilized CSP7 acetate formulations behaved differently during storage. The study also observed acetate loss depending on processing and composition. We therefore recommend assessing chemical integrity, recovery and salt composition separately. Checking that material remains dry is insufficient: counterion preservation and physical stability are separate analytical questions, and a result for one formulation should not be transferred to another preparation without supporting measurements (Sahakijpijarn et al., 2019; PMID: 31569515, sections 3.4 and 3.5).

Laboratory storage and handling

Hoofnagle and colleagues recommend bringing lyophilized powder to room temperature in a desiccator before handling to limit water absorption. For laboratory records, we suggest documenting openings, environmental conditions and closure problems. These precautions follow from the risk of moisture ingress; they cannot replace storage conditions documented for the particular sequence and preparation, or establish a shelf life for an uncharacterized material (Hoofnagle et al., 2016; PMID: 26719571, solubilization and storage discussion).

After dissolution, different conditions need assessment: solubility, adsorption to surfaces and chemical modifications depend on sequence and medium. We recommend recording solid and solution histories separately and basing any holding period on relevant data. A date associated with the dry vial should not automatically be transferred to a solution prepared for an analytical experiment without evidence for that solution and its storage conditions (Hoofnagle et al., 2016; PMID: 26719571).

What to look for in documentation

We recommend reviewing identity, salt form, declared components, water method and the reporting basis of peptide content. The reference standards study distinguishes bulk material characterization from content assignment to the final vial. Freeze-drying, chromatographic purity and net quantity are different information: a process name cannot replace those results or establish the mass of the target sequence (McCarthy et al., 2023; PMID: 36949371).

Verified references

• Tchessalov S et al. Practical Advice on Scientific Design of Freeze-Drying Process: 2023 Update. Pharmaceutical Research, 2023. PMID: 37783925. DOI: 10.1007/s11095-023-03607-9.
• Hengsawas Surasarang S et al. Formulation for a novel inhaled peptide therapeutic for idiopathic pulmonary fibrosis. Drug Development and Industrial Pharmacy, 2018 (electronic publication, 2017). PMID: 28835128. DOI: 10.1080/03639045.2017.1371736.
• Sahakijpijarn S et al. Formulation Composition and Process Affect Counterion for CSP7 Peptide. Pharmaceutics, 2019. PMID: 31569515. DOI: 10.3390/pharmaceutics11100498.
• Hoofnagle AN et al. Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry-Based Assays. Clinical Chemistry, 2016. PMID: 26719571. DOI: 10.1373/clinchem.2015.250563.
• McCarthy D et al. Reference Standards to Support Quality of Synthetic Peptide Therapeutics. Pharmaceutical Research, 2023. PMID: 36949371. DOI: 10.1007/s11095-023-03493-1.