How Your Peptide Vials
Are Created

The active ingredient arrives as a raw bulk powder from a chemical or pharmaceutical supplier. Each batch should come with a Certificate of Analysis (CoA) confirming identity, purity, and absence of contaminants. The powder is precisely weighed on an analytical balance to calculate the exact amount needed for the batch based on the intended dose per vial and total vial count — accounting for dead volume losses in filters and tubing.

Glass vials are not sold pre-sterile and must be processed before use. They are baked in a dry heat oven at 250°C (482°F) for 30–60 minutes. This achieves two critical goals simultaneously: sterilization (killing all microorganisms including heat-resistant spores) and depyrogenation — destroying bacterial endotoxins, which are lipopolysaccharide fragments from bacterial cell walls that survive standard steam sterilization and cause fever, inflammation, and septic shock when injected.

After baking, vials are cooled inside the oven with the door closed, then transferred directly into the laminar flow hood in a covered, clean container.

The rubber stoppers are purchased pre-sterilized and pre-siliconized from a pharmaceutical supplier — typically steam autoclaved in sealed, double-bagged packaging. Unlike glass vials, rubber cannot withstand 250°C dry heat without degrading. For stoppers, endotoxin control is handled at the supplier level, which is why sourcing from a reputable pharma-grade supplier is essential.

Stoppers are kept sealed in their original packaging and only opened inside the laminar flow hood immediately before use. Any stopper that contacts a non-sterile surface is discarded.

Every step from this point forward must be performed under a laminar flow hood (LFH) — a workstation with a HEPA-filtered, unidirectional airflow that continuously sweeps particles away from the work surface. This creates an ISO Class 5 (Class 100) environment with fewer than 100 particles ≥0.5µm per cubic foot — compared to tens of thousands in typical room air.

A vertical downflow hood is preferred for aseptic filling — air flows down from above, protecting open containers. The hood should run for 15–30 minutes before use. The operator wears nitrile gloves, a face mask, and a lab coat, and avoids movements that sweep across or upstream of open containers.

The weighed powder is dissolved into Water for Injection (WFI) — highly purified, endotoxin-tested water meeting pharmacopoeial standards for injectable preparations. Regular distilled or RO water is not acceptable; WFI is tested to endotoxin levels below 0.25 EU/mL. The solution is prepared at a concentration such that the fill volume delivers the target dose per vial.

Beyond WFI and the active compound, a properly formulated vial contains several excipient layers. The specific combination depends heavily on the compound being made. Getting this right is the difference between a vial that retains potency for 12–24 months and one that degrades within weeks.

── Layer 1: Bulking Agent / Cryoprotectant ──

Mannitol is the most common. It crystallizes during lyophilization, producing a clean, firm, elegant white cake with excellent structural integrity. Ideal for simple peptides and vitamins.

Trehalose remains amorphous (glassy) during lyophilization. This glassy matrix forms a protective shell directly around each molecule, preserving tertiary structure and conjugation sites. Strongly preferred for complex peptides, proteins, and fatty-acid conjugated compounds (GLP-1 agonists).

Sucrose behaves similarly to trehalose and is used interchangeably in many formulations. Some formulations combine mannitol + trehalose to get both structural integrity and molecular protection.

── Layer 2: Buffer & pH ──

Most compounds have a pH window where they are most stable. Sodium phosphate (pH 6.0–7.5) is the most common general-purpose buffer. Sodium citrate (pH 3.0–6.5) is preferred for acidic-stability compounds like NAD+. Histidine (pH 5.5–7.0) is the preferred buffer for GLP-1 agonists (semaglutide, tirzepatide, retatrutide) — it also has mild antioxidant and metal-chelating properties.

── Layer 3: Surfactant (Aggregation Prevention) ──

Polysorbate 80 (Tween 80) at 0.01–0.05% is the standard choice for GLP-1 agonists — used in the commercial semaglutide formulation. It prevents the peptide from aggregating at surfaces. Polysorbate 20 is used for smaller, less hydrophobic peptides.

── Layer 4: Antioxidant Stabilizers ──

Free methionine amino acid (1–10mM) acts as a sacrificial antioxidant — any oxidizing species in the vial preferentially attacks the free methionine rather than the methionine residue in the peptide backbone. Ascorbic acid is a direct antioxidant scavenger. EDTA at 0.01–0.1mg/mL is a metal chelator that removes trace metal contamination which catalyzes oxidation reactions.

── Compound-Specific Formulation Reference ──

The prepared solution is passed through a 0.22 micron sterile PES membrane filter — a syringe filter for small batches, or an inline capsule filter for larger volumes. This is the final bioburden reduction step, removing all bacteria and fungal spores. The filter is single-use and discarded after the batch. Filtration is performed directly into the sterile fill vessel inside the laminar flow hood.

Note: 0.22µm filtration does not remove viruses or endotoxins — which is why starting material quality, WFI, and vial depyrogenation all matter independently.

Using a calibrated syringe, repeating pipette, or volumetric fill pump, each vial receives an exact, consistent volume of solution. Dose accuracy is achieved through volume control — not weighing — because the solution concentration is already precisely known. A consistent fill volume guarantees a consistent dose. All filling is performed inside the laminar flow hood with vials never left open longer than necessary.

Vial Sizing & Fill Volume: Fill volume isn't just about dose — it also determines cake geometry and drying performance. As a general rule, the fill should occupy no more than 30–40% of the total vial volume. A 10mL vial, for example, should receive no more than 3–4mL of solution. Fill depth also matters: shallower fills dry faster and more uniformly. Too little fill in an oversized vial produces a thin, fragile disc that customers may mistake for an underdosed or empty vial.

A rubber stopper is placed into the opening of each filled vial but left partially seated — resting at a partially inserted position that allows water vapor to escape during freeze-drying while still protecting the opening from gross contamination. A fully seated stopper would trap water vapor and prevent the product from drying properly. Vials are transferred to the freeze dryer trays immediately after stoppering.

The vials are loaded onto the shelves of the freeze dryer and run through a precisely programmed multi-phase cycle:

1. Freezing — shelf temperature drops to –40°C to –50°C, solidifying the liquid completely. Freezing rate affects ice crystal size, which impacts drying speed and product quality.

2. Primary Drying (Sublimation) — chamber is evacuated to deep vacuum (100–200 mTorr) and shelf temperature rises slightly (–20°C to –10°C). Ice sublimates directly to vapor, which is captured by a cold condenser at –60°C to –80°C. This removes ~90–95% of the water and takes 12–24+ hours depending on fill volume.

3. Secondary Drying (Desorption) — shelf temperature increases further (+20°C to +40°C) to remove residual bound moisture, bringing residual water below 1–2%. Cutting this phase short is a common cause of premature product degradation.

The result is a porous, dry "cake" — a white, solid matrix that reconstitutes quickly when bacteriostatic water is added back.

For compounds susceptible to oxidative degradation — NAD+, peptides with methionine or cysteine residues, and other redox-active molecules — oxygen in the vial headspace will slowly degrade the product over time even in the dry state.

The proper method uses a freeze dryer with a nitrogen inlet port: at the end of secondary drying, instead of venting the chamber with air, the chamber is backfilled with filtered dry nitrogen (or argon) before the stoppers seat. The vial headspace is then nitrogen — no oxygen present.

Smaller operations without this capability can alternatively flow nitrogen gently over the vials immediately before stopper insertion. This is a compromise but meaningfully better than air-filled headspace for sensitive compounds.

When the freeze dryer cycle completes and the chamber is vented, atmospheric pressure outside the vial immediately holds the partially-seated stopper firmly in place — the vacuum inside acts exactly like a canned jar lid held down by atmospheric pressure differential. The stoppers are then fully pressed home and an aluminum crimp cap is applied over the stopper using a hand crimper or benchtop crimping tool.

The crimp cap mechanically locks the stopper and provides tamper evidence — it is not what maintains the vacuum or sterility. The stopper and internal vacuum do the actual sealing work. Labels including compound name, lot number, dose, and reconstitution instructions are applied.

Every finished vial should be individually inspected against a light box or bright lamp against a dark background:

Particulates: visible particles in the cake or on vial walls — indicates contamination or aggregation.

Cake appearance: a good cake is white, uniform, intact, and fills most of the vial. A collapsed cake (sunken, shrunken, sticky residue) means primary drying temperature was too high and the product may be damaged. Browning indicates thermal degradation.

Stopper seating: all stoppers fully seated, none partially out.

Vial integrity: no cracks or chips. Failed vials should be quarantined — they signal a process problem that needs investigation, not just individual rejection.

Lyophilized does not mean indestructible or room-temperature stable. Most lyophilized peptides and supplements should be stored at 2–8°C (refrigerated) for short-to-medium term, or –20°C (frozen) for long-term storage. Light-sensitive compounds should be in amber vials or foil pouches.

An underappreciated problem is freeze-thaw cycling — even though the product is dry, repeated temperature fluctuations from frequently opening a freezer door cause micro-condensation and mechanical stress on the cake matrix. Over time this degrades product quality even without ever reconstituting the vial.

A practical solution is storing vials in an insulated container such as a Hydrapeak or similar vacuum-insulated vessel kept inside the freezer. The thermal mass buffers against the temperature spike every time the freezer is opened. Store vials upright — never on their side.