Leachables and Extractables: What the Container Adds to Your Solution

Every solution takes the shape of its container, and it also takes on a little of the container's chemistry. Plastics contain antioxidants, slip agents, plasticisers and polymerisation residues. Rubber stoppers contain vulcanisation chemicals. Syringes carry silicone lubricant. Glass releases ions. None of this is a defect; these materials exist because they make the container work. But the result is that the liquid in the vial contains things you did not add, and in at least one documented case the amount needed to cause a problem is far smaller than anyone would guess.

Quick answer

Do containers contaminate peptide solutions? They contribute measurable chemistry, and whether it matters depends on the sensitivity of what you are measuring. The clearest published case is bis(2,4-di-tert-butylphenyl)phosphate, a breakdown product of a common polyethylene antioxidant additive, which was shown to be highly detrimental to mammalian cell growth at concentrations well below the parts-per-million range, with a rapid drop in mitochondrial membrane potential. Its migration out of the film is time- and temperature-dependent, and gamma irradiation — the step that sterilises the container — was identified as an important condition for generating it. Extractables are what a material can release under forcing conditions; leachables are what actually migrates during normal contact.

Two words that are not synonyms

Extractables define the worst case and tell you what to look for. Leachables are the subset that genuinely ends up in your solution. A long extractables list is not evidence of a problem; it is a map of what a leachables study should go looking for.

The case worth knowing about

A 2013 investigation into single-use bioprocess containers identified one compound as conspicuously harmful among the many species extractable at low levels from the materials of construction.

The compound is bis(2,4-di-tert-butylphenyl)phosphate, abbreviated bDtBPP. It derives from the breakdown of tris(2,4-di-tert-butylphenyl)phosphite, a common antioxidant additive present in many polyethylene formulations — polyethylene being one of the polymers routinely used as the fluid-contact layer in bioprocess containers.

Three findings from that work are worth carrying:

• The potency. Cell growth experiments across several mammalian cell lines showed harmful effects at concentrations well below the parts-per-million range. The cellular response was rapid and included a significant decrease in mitochondrial membrane potential.
• The migration is conditional. Movement of bDtBPP out of polyethylene-based films was shown to be time- and temperature-dependent. Longer storage and warmer storage both increase what ends up in the liquid.
• Sterilisation generates it. Exposure of oxidised antioxidant to ionising radiation — gamma irradiation, the standard way these containers are pre-sterilised — was identified as an important condition for generating significant amounts of the leachable.

That last point is the uncomfortable one. The processing step that makes a container safe from a microbiological standpoint is the step implicated in producing a chemically active leachable. The two forms of cleanliness are not the same thing and are not obtained by the same means.

The other usual suspects

Silicone oil deserves its own line because it is both ubiquitous and specifically implicated. Work on agitation-induced aggregation found that the presence of silicone oil was a condition under which aggregation and associated effects were markedly worse, and that an appropriate excipient could suppress it. A siliconised surface plus agitation is a combination worth avoiding where the solution matters.

How much contact are you actually giving it?

Leachable transfer scales with contact area, contact time and temperature. For a research peptide the exposures are usually short, which is the main reason this is a smaller problem than it is in pharmaceutical manufacturing. The exceptions are predictable:

• Long-term storage of a working solution in a plastic tube rather than glass.
• Small volumes in large tubes, where the surface-to-volume ratio is high. This is the same geometry that drives adsorption loss, and the two effects run in opposite directions: the container takes peptide out and puts chemistry in.
• Warm storage, since migration is temperature-dependent.
• Organic solvent in the system. DMSO is a far more aggressive extraction medium than water, so a DMSO stock in a plastic tube extracts more than an aqueous one — one more reason to keep DMSO contact in glass. See DMSO in peptide work.

What this means in practice

• Glass for stocks and references. Glass is the lowest-leachable common material. Where a solution has to sit, or has to serve as a reference, glass is the default.
• Plastic for short contact. Transfers, brief dilutions and working aliquots in plastic are a reasonable trade for the convenience and the lower breakage risk.
• Never store organic solvent stocks in plastic. Extraction efficiency is the whole issue.
• Keep a consistent consumable. Changing tube brand mid-study changes the leachable profile. That is a real variable, and an unrecorded one — the same argument as in lot-to-lot variability, applied to plastics instead of peptide.
• Record the container in the method. Material, brand and format. If a result stops reproducing and nothing about the peptide changed, consumables are a legitimate suspect and only a written record lets you check.
• Include the container in the control. A vehicle control should sit in the same type of container, for the same time, at the same temperature, as the test condition.

What to expect on a certificate

Nothing, and that is normal. Extractables and leachables assessment is a pharmaceutical container-closure activity carried out by the people who manufacture the container system, not by a peptide supplier, and a research certificate will not carry it. Published programmes of this kind cover prefilled syringe systems across glass and plastic and reference the elemental impurities framework for the metal side of the question.

What is reasonable is to know the container your material arrived in, to keep it in that container until you use it, and to make your own container choices deliberately rather than by whatever tube was nearest.

Key takeaways

• Extractables are what a material can be forced to release; leachables are what actually migrates under real conditions.
• A documented plastic leachable, bDtBPP from a polyethylene antioxidant additive, harmed mammalian cell growth well below one part per million and rapidly reduced mitochondrial membrane potential.
• Its migration is time- and temperature-dependent, and gamma irradiation of the oxidised additive was identified as an important condition for generating it.
• Sterility and chemical cleanliness are different properties obtained by different means, and one process can trade against the other.
• Silicone oil from syringes and stoppers is specifically implicated in agitation-induced aggregation.
• Transfer scales with area, time and temperature, and organic solvents extract far more than water.
• Glass for stocks and references; plastic for short contact; never organic solvent stocks in plastic.
• Record the container in the method, and put the control in the same container.

Frequently asked questions

Should I be worried about the vial my peptide arrived in?

Not particularly. Peptide vials are typically glass with a rubber closure, which is the low-leachable configuration, and the material is dry for most of its life — migration needs a liquid phase. The exposures worth thinking about start after you reconstitute and transfer.

Is plastic always worse than glass?

For leachables, generally yes. For adsorptive loss, not necessarily — some low-bind plastics retain less peptide than untreated glass. The two properties trade off, which is why the answer depends on whether you are protecting the concentration or protecting the chemistry.

Does filtering remove leachables?

No. Leachables are dissolved small molecules and pass straight through a sterilising membrane, which is sized to retain bacteria. The filter also contributes its own extractables, so filtration adds to this problem rather than solving it.

Would a leachable show up in my analysis?

Usually not. A reversed-phase method tuned for a peptide and monitored at a peptide wavelength is not looking where these compounds elute or absorb. Their effect appears as an unexplained biological result, not as a peak.

Does this apply to short experiments?

Much less. Migration is time-dependent, so minutes of contact transfer very little. The risk concentrates in long storage, warm storage, high surface-to-volume ratios and organic solvents.

Sources

• Hammond M, Nunn H, Rogers G, Lee H, Marghitoiu AL, Perez L, Nashed-Samuel Y, Anderson C, Vandiver M, Kline S. Identification of a leachable compound detrimental to cell growth in single-use bioprocess containers. PDA J Pharm Sci Technol. 2013;67(2):123–34. DOI
• Ronk M, Liu J, Gallegos A, Luo Y, Fujimori K, Li K, Lee H, Nashed-Samuel Y. Holistic extractables and leachables program: evaluations of prefilled syringe systems for biotechnology products. PDA J Pharm Sci Technol. 2020;74(6):627–643. DOI
• Kim NA, Noh GY, Hada S, Na KJ, Yoon HJ, Park KW, Park YM, Jeong SH. Enhanced protein aggregation suppressor activity of N-acetyl-l-arginine for agitation-induced aggregation with silicone oil. Int J Biol Macromol. 2022;216:42–51. DOI

Citations retrieved via PubMed. These studies concern bioprocess containers and therapeutic proteins; the mechanisms transfer, the specific compounds and concentrations belong to those systems.

This article describes materials chemistry and laboratory practice only. It contains no usage instructions, no dosing information and no protocols of any kind.

For laboratory and in-vitro research use only. Not for human or veterinary use.