Why Peptides Are Freeze-Dried: Lyophilisation, Mannitol and Cake Appearance

Last reviewed 9 August 2026

A vial arrives holding a firm white plug that sits flat on the base and fills most of the glass. Another vial of the same compound arrives holding a thin scatter of powder against the wall. They look like different products. Very often they are the same product, and the difference between them is a consequence of how much solid was in the fill solution, what was dissolved alongside it, and what happened to the vial between the freeze-dryer and the bench.

What follows describes the process that produces a cake, why an excipient is frequently present when the compound itself amounts to a few milligrams, what the pharmaceutical literature has established about the relationship between how a lyophilisate looks and what it is, and where visual inspection stops being informative. Every question here is a materials-science question. None of it is a statement about what any compound does.

What freeze-drying removes, and in what order

Freezing. As ice nucleates and grows, everything dissolved in the water is excluded from the advancing crystal and pushed into the shrinking liquid between crystals. That residual phase — the freeze concentrate — reaches a solids content far above the starting solution, then either crystallises or becomes so viscous that it stops flowing and vitrifies into a glass. The temperature of that glass transition in the maximally freeze-concentrated phase is written T_g′, distinguishing it from the T_g of a dry amorphous solid. It is a measured property of a particular formulation rather than a constant, usually determined by differential scanning calorimetry and with well-documented difficulties on protein-containing systems (Pansare and Patel, 2016).

Primary drying. Chamber pressure is taken below the equilibrium vapour pressure of ice and the ice sublimes, passing to vapour without melting. The triple point of water, at 0.01 °C and 611.657 Pa, is the ceiling: below that pressure there is no liquid phase for ice to pass through on the way out. The ice leaves as vapour and leaves its shape behind. The pore network of a finished cake is a cast of the ice crystals that used to occupy it. That single fact explains most of what a cake looks like — large, slowly grown crystals leave coarse open channels, fine crystals a dense and resistant matrix.

The constraint here is that the drying front must stay below the temperature at which the freeze concentrate softens and flows. That collapse temperature sits close to T_g′ and is usually slightly above it, the exact difference depending on the formulation. Above it, the matrix relaxes and the structure the ice left behind closes up before the ice has finished leaving.

Secondary drying. Once the ice is gone, water remains sorbed within the amorphous phase and is removed by desorption at a higher shelf temperature. This stage — not the previous one — sets the residual moisture of the finished cake.

Why the material is a solid rather than a solution

Lai and Topp (1999) reviewed the solid-state chemical stability of proteins and peptides and named the reactions that continue after drying: deamidation, peptide bond cleavage, oxidation, the Maillard reaction, β-elimination and dimerisation or aggregation. They identified the governing factors as temperature, moisture content, excipients, and whether the formulation is amorphous or crystalline. Note the direction of that finding. Freeze-drying does not stop chemistry. It slows it, and it changes which routes dominate. Chang and Pikal (2009), reviewing the mechanisms of stabilisation in the solid state, gave the reason for drying at all: proteins show marginal stability when dissolved, and chemical and physical degradation can occur on the time scale of the drying process, distribution and use.

Water does two separate jobs in solution, and removing it withdraws both. It is a reactant — hydrolysis of the peptide backbone and deamidation at asparagine and glutamine residues both consume it. And it is a plasticiser: it lowers the glass transition temperature of the amorphous phase and raises molecular mobility within it. The second effect is what makes residual moisture a specification rather than an afterthought. This page covers the solid; the behaviour of the same material once back in solution is a separate question with separate kinetics.

Drier is not automatically better

Breen and colleagues (2001) examined a lyophilised humanised monoclonal antibody formulated with sucrose, histidine and polysorbate 20, at residual moisture between 1 and 8 per cent, stored six to twelve months at 5 °C to 50 °C. They reported glass transition temperatures varying from 80 °C at 1 per cent moisture to 25 °C at 8 per cent; that high-moisture cakes aggregated faster than drier samples when stored above their T_g; and — the part routinely forgotten — that intermediate-moisture formulations were more stable against aggregation than the driest ones. Their stated conclusion was that increased moisture reduced chemical stability regardless of whether the material was glassy or rubbery, while physical stability was not compromised and could be greater at higher residual moisture provided storage was below T_g. Lo Presti and Frieß (2025) found the same shape in human serum albumin lyophilisates across glucose-based sugars from monosaccharide to tetrasaccharide, reporting that smaller sugars stabilised the protein better than larger ones and that raising residual moisture from 1 to 2 per cent increased stability, particularly for the larger sugars.

So the target is an optimum, it is formulation-specific, and it is a figure on a certificate. It is not visible.

Why there is something else in the vial

Start with the arithmetic, because it is the whole reason bulking agents exist. A freeze-dried cake is mostly void: it occupies roughly the volume the frozen solution occupied, while weighing what the dissolved solids weighed. A fill solution carrying 10 mg of peptide per millilitre is a 1 per cent w/v solid. At 2 mg per millilitre it is 0.2 per cent. There is very little material there from which to build a free-standing structure, and below a certain solids content there is not enough to build one at all.

Two excipient jobs that are routinely confused

• A bulking agent is chosen because it crystallises during freezing. The crystals form a rigid scaffold that holds the cake’s shape, and because that scaffold does not depend on an amorphous phase staying below its collapse temperature, it permits primary drying at a higher product temperature. Mannitol and glycine are the common choices.
• A lyoprotectant is chosen because it does not crystallise. Sucrose and trehalose remain amorphous, vitrify around the dissolved solute and stay hydrogen-bonded to it (Chang and Pikal, 2009).

The two requirements pull in opposite directions, which is exactly why they are frequently used together and why the ratio between them is a formulation decision rather than a detail. Johnson, Kirchhoff and Gaud (2002) reported four developmental proteins freeze-dried from 4 per cent mannitol with 1 per cent sucrose, using a lyophilisation process that produced a cake of crystalline mannitol and amorphous sucrose. They reported that the crystalline mannitol allowed primary drying at a product temperature of −10 °C, where the amorphous sucrose alone would have required primary drying below −35 °C to avoid collapse, and that formation of an unstable mannitol hydrate was avoided by conducting secondary drying at 40 °C or higher.

Anko and colleagues (2019) examined mannitol-to-sucrose ratios and monoclonal antibody concentrations by thermal analysis and powder X-ray diffraction, and reported that the ratio between sucrose and mannitol and the antibody concentration had a decisive impact on which physical form the mannitol adopted. The excipient ratio is not a filler decision. It determines what the solid actually is.

Mannitol, identified

Name

D-mannitol; IUPAC name (2R,3R,4R,5R)-hexane-1,2,3,4,5,6-hexol

Molecular formula

C₆H₁₄O₆

Molecular weight

182.17

CAS number

69-65-8 (PubChem CID 6251)

Chemical class

Hexitol — a sugar alcohol, or polyol. Not a sugar

Pharmacopoeial status

The subject of European Pharmacopoeia monograph 0559, harmonised through the Pharmacopoeial Discussion Group, with sorbitol, maltitol and isomalt named as specified impurities A, B and C

Reducing?

No. There is no anomeric carbon and therefore no reducing end

That last row does real work later on. Because mannitol has no free carbonyl, it cannot condense with an amine and cannot initiate a Maillard reaction. Sucrose is also non-reducing — but sucrose hydrolyses to glucose and fructose, and both of those are reducing, which is why Schersch and colleagues (2013) tracked sucrose degradation as a cake property in its own right, alongside glass transition temperature, excipient crystallinity, reconstitution behaviour and residual moisture.

Mannitol is not a well-behaved excipient

Thakral and colleagues (2023) reviewed mannitol as an excipient in freeze-dried formulations and concluded that its physicochemical properties make it desirable and preferred as a bulking agent when compared with glycine, while stating plainly that its use may pose challenges such as vial breakage or its existence as a metastable crystalline hemihydrate in the final cake, both requiring mitigation. Three consequences are worth stating separately.

The hemihydrate

Depending on process conditions mannitol can crystallise as the α, β or δ anhydrous polymorph, as the hemihydrate, or remain amorphous (Seiler et al., 2023; Thakral et al., 2023). The hemihydrate is metastable and carries water into the finished cake that can be released later.

Anko and colleagues (2019) reported that a higher secondary drying temperature had the largest effect on complete dehydration of the hemihydrate; that annealing temperature affected hemihydrate content in the final product, with the higher annealing temperature favouring anhydrous mannitol; and that increasing amounts of mannitol and of the antibody both reduced hemihydrate formation. They also reported that, contrary to what they described as a widespread assumption, they detected no impact of the hemihydrate on the stability of the antibody they studied — a physical form that formulators work hard to avoid, and which in that particular system made no measured difference.

Vial breakage

Williams and Dean (1991) examined frozen mannitol solutions by differential scanning calorimetry and reported an exotherm appearing on warming at −25 ± 1 °C that correlated with the temperature at which frozen mannitol solutions broke vials. They reported that additives including lactose, glycine, potassium chloride and sodium chloride, even at under 1 per cent of the mannitol concentration, substantially reduced breakage or stopped it occurring; that the stereoisomers sorbitol and dulcitol produced neither the exotherm nor breakage; and that different vial types showed different susceptibility. Cracked glass in a delivered box is far more likely to be transit damage than this, but the failure mode is real and it is a property of the excipient rather than of the compound.

Reconstitution behaviour

Kulkarni and colleagues (2018) investigated the mechanism by which crystalline mannitol shortens the reconstitution time of high-concentration lyophilised protein formulations. They reported that phase separation of crystalline mannitol from the protein-rich amorphous matrix increased the cake’s wettability and the penetration of liquid into it, and that crystalline mannitol created weak points allowing the cake to disintegrate more readily. They also reported no evident correlation between the degree of crystallinity and the reconstitution time. The corollary is worth keeping: a cake that goes into solution quickly is not thereby evidence of anything about its crystallinity, and a cake that goes slowly is not evidence of degradation. The arithmetic of concentration is unaffected by any of it.

What an acceptable cake looks like, and what “acceptable” is a statement about

Patel and colleagues (2017), in a commentary co-authored from industry and academia, set out a harmonised nomenclature for this. The commentary does two things. First, it supplies that nomenclature together with a set of representative images for describing variation from the ideal of a uniform, elegant cake, so that two people looking at the same vial use the same word for it.

Second, and more useful to anyone holding one vial: it states that cake appearance is an important attribute of freeze-dried products which may or may not be critical with respect to product quality — that is, to safety and efficacy. Non-ideal appearance sometimes has no bearing on product quality at all, and can be inherent to the product given its formulation, presentation and processing. Their recommendation is a science-based and risk-based approach to setting acceptance criteria, rather than holding cosmetic elegance to be a requirement in its own right. That is a stronger position than it first reads as.

The appearances people actually notice

Collapse is the counter-intuitive one

Schersch and colleagues ran three studies on exactly this question, and they are the most directly relevant work in the literature to anyone looking at a slumped cake. They induced collapse deliberately — by adjusting the mannitol-to-sucrose ratio and by running an aggressively fast freeze-drying process — and compared collapsed against non-collapsed lyophilisates of identical formulation.

• Part I (2010) examined a monoclonal IgG₁ antibody, a second protein designated PA01, and L-lactate dehydrogenase, the last included for its documented sensitivity to freeze-drying stresses. They reported that collapsed cakes had comparable residual moisture to non-collapsed lyophilisates, and that protein stability was not relevantly different between the two.
• Part 2 (2012) ran the same comparison over six months at 2–8 °C, 25 °C, 40 °C and 50 °C, on a monoclonal IgG antibody, tissue-type plasminogen activator and L-lactate dehydrogenase, monitoring soluble and insoluble aggregates, biological activity and conformation. They reported full protein stability in the collapsed cakes, and on some stability-indicating parameters reported greater stability in the collapsed cakes than in the non-collapsed ones.
• Part III (2013) compared cakes that collapsed during freeze-drying against cakes of the same formulation and comparable moisture that collapsed later, during three months of storage at 40 °C and 50 °C. They reported that the incorporated protein was significantly better stabilised in the cakes that collapsed during the freeze-drying process, and related the difference to the onset of crystallisation, hydrolysis of the stabiliser and non-enzymatic browning.

Two things follow. Collapse during manufacture is a process deviation with a cosmetic signature, not a self-evident stability failure. Collapse that develops afterwards is a different observation entirely: it says the material has spent time above its glass transition temperature since it was made, and the temperature history is the finding — not the shape.

Fogging is a filling artefact, not a leak

Abdul-Fattah and colleagues (2013) investigated fogging in lyophilised products and reported that drug product creeps up the inner vial wall during the filling process and then dries in position, and that the main factor controlling it is the hydrophilicity or hydrophobicity of the inner vial surface. They reported contributions from glass quality, vial processing history and formulation surfactants, and that depyrogenation of the glass was of limited effectiveness while hydrophobic containers reduced the defect. The haze is therefore material that went up the glass as a liquid before drying started. It is not condensation, and it is not evidence that the closure has been breached.

Material on the closure is the exception

Mehta, Roy and Yang (2018) reported a lyophilised monoclonal antibody batch, filled by peristaltic pump, in which roughly 40 per cent of vials showed residual product around the stopper; the authors attribute the defect to handling errors when loading filled vials onto the freeze-dryer. Comparing those against the 60 per cent from the same batch with no such defect, they reported no significant difference in protein concentration, residual moisture or aggregation — but reported that container closure integrity, tested by blue dye ingress, was compromised in the affected vials. Their stated conclusion was that a seemingly cosmetic defect may impact product quality.

That is the honest counterweight to the collapse literature. “Appearance does not determine quality” is a rule with a clear exception, and the exception is the class of defects involving the seal rather than the solid — which is also where the specification of research-grade material is decided.

Colour, and the assumption that white is the reference

Browning in a lyophilisate is conventionally read as Maillard chemistry, which requires a reducing carbonyl and a free amine (Lai and Topp, 1999). Mannitol supplies no carbonyl at all. Where sucrose is present the precursor can be generated in place by hydrolysis, which is why Schersch and colleagues (2013), comparing collapsed against non-collapsed cakes in storage, related the stability difference they measured partly to non-enzymatic browning.

The more common error, though, runs the other way: assuming that white is the reference appearance. It is not universally. GHK-Cu is the copper(II) complex of the tripeptide, and is a deep blue to blue-violet solid, the colour arising from copper(II) d–d absorption in the visible region. For that material a colourless cake is the anomaly. Read the expected appearance for the specific compound in its library entry before reading the vial, and do not compare against a photograph of something else — cake density, pore structure and colour are all formulation-specific.

What appearance cannot establish

The list is short, and it is close to total. A cake is a bulk physical observation of a mostly empty solid, and it records process history rather than composition.

• Identity. Established by comparing a measured mass against the sequence, not by looking at the glass.
• Purity. Chromatographic area percent, which is a different number from net peptide content and answers a different question.
• How much peptide is in the vial. Net peptide content, after counterion and residual water are accounted for. A larger, denser-looking cake may simply carry more bulking agent.
• Sterility and endotoxin. Specification questions, settled by test method.
• Residual moisture. Karl Fischer titration. The single physical property most likely to matter, and entirely invisible.

Inspecting a vial on arrival

This is the one section addressed to the person handling the material, and it is about evidence rather than reassurance.

1. Inspect the sealed vial before breaking any seal — against a white background, then against a light source. Once diluent goes in, the evidence is gone and cannot be recovered.
2. Photograph it in focus first. Verbal descriptions of cake appearance are unreliable enough that Patel et al. (2017) set out a harmonised nomenclature with representative images to standardise them. A photograph is worth more than any description of it.
3. Assess the closure and the crimp separately from the cake, and first. On the published evidence that is the observation most likely to correspond to something real.
4. Record where the material is — base, wall, closure, or distributed — and whether the vial appears to have been inverted or shaken in transit. Loose powder in a low-solids vial is a mechanical observation, not a chemical one.
5. Record the condition the parcel arrived in, including its apparent temperature. A cake that has slumped since manufacture is a temperature-history question, and the answer is not in the glass.
6. Note which diluent is intended before opening. The choice between a preserved and an unpreserved vehicle is a compatibility question, and it is decided before the cake is disturbed, not after.

What a photograph cannot settle

Everything a cake’s appearance raises resolves into a documentary question: what the material is, how pure it is, how much peptide the vial holds, and what the certificate for that specific material records. None of those is answered by looking, and reading a certificate is a different skill from reading a vial. NovoVita’s published position on purity is stated as a floor rather than a point value: Third-party tested at greater than 99% purity. What is and is not published behind that figure is set out separately. A question about a specific vial is better asked than searched for, and the contact page is where it belongs.

The arithmetic that follows reconstitution is indifferent to all of it. Concentration is the mass of solid divided by the volume of diluent added, and the reconstitution calculator resolves that relation and nothing beyond it. A cake’s appearance changes what the vial looks like. It does not change what is in it, and it does not change the arithmetic.

References

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18. European Pharmacopoeia. Mannitol, monograph 0559. Harmonised through the Pharmacopoeial Discussion Group; see the United States Pharmacopeia PDG notice for the harmonised text and the specified impurities. Regulatory document.
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