The four decisions
Pore size
0.22 µm for sterilization. 0.45 µm for clarification only. USP recognizes 0.22 µm as sterilizing grade.
Membrane material
PVDF preferred (low binding). PES acceptable. Avoid nylon and cellulose acetate for hydrophobic peptides.
Filter size vs volume
13 mm filter for under 10 mL. 25 mm for 10–100 mL. Larger filters lose less peptide to dead volume.
Pre-wet decision
For hydrophobic peptides or valuable material, pre-wet with 0.5 mL of diluent, discard, then filter the main volume to improve recovery.
Filter membrane compared
| Membrane | Peptide recovery | Use when |
|---|---|---|
| PVDF (hydrophilic) | 95–98% | Default choice for aqueous peptide solutions |
| PES | 93–97% | Cost-sensitive workflows, hydrophilic peptides |
| Nylon | 70–90% | Small hydrophilic peptides only |
| Cellulose acetate | 80–92% | Legacy protocols only |
| PTFE (hydrophobic) | n/a | Organic solvent systems, not aqueous |
Step-by-step technique
- Reconstitute the peptide fully. Ensure no undissolved material remains.
- Draw the solution into a clean syringe of appropriate size (10 mL syringe for 5 mL of peptide).
- Attach a 0.22 µm PVDF filter, luer-lock preferred.
- For valuable material, pass 0.5 mL of diluent through first to saturate the membrane; discard.
- Hold the filter outlet inside a sterile receiving vial. Do not touch the vial with the filter tip.
- Depress the plunger with slow, steady pressure. Never force.
- If resistance rises sharply, the filter is clogged. Stop; use a fresh filter for the remainder.
- Cap the receiving vial immediately.
What filtration removes and what it does not
Removes
Bacteria, most fungi, particulates, precipitate, undissolved material, some viruses (larger ones).
Does not remove
Bacterial endotoxin (LPS), mycoplasma, small viruses, dissolved chemical impurities, degradation products.
If endotoxin matters (any injectable use), filter the solution then send an aliquot for LAL endotoxin testing. Filtration is a physical process; endotoxin quantification is chemical.
Related
Filtering Peptides: FAQ
0.22 micron for sterile filtration of aqueous peptide solutions. 0.45 micron is acceptable for clarification only, not sterilization. The pore size must be small enough to retain bacteria (0.22 µm is the sterilizing grade recognized by USP).
PVDF (polyvinylidene fluoride) is preferred for most peptides because of its low protein binding. PES (polyethersulfone) is a close second and often less expensive. Avoid nylon and cellulose acetate for anything hydrophobic or above 10 amino acids; they can bind significant peptide and reduce recovered concentration.
With low-binding PVDF or PES, typical recovery is 92 to 98 percent. With nylon, hydrophobic peptides can lose 20 percent or more. Pre-saturating the filter with a small aliquot before filtering the main volume improves recovery noticeably.
No. Standard 0.22 micron filters remove bacteria but do not remove bacterial endotoxin (LPS), which is a soluble molecule that passes through. Endotoxin removal requires specialized affinity filters, ultrafiltration, or a validated depyrogenation process. If endotoxin is a concern, test the finished solution with a quantitative LAL assay.
For assay and cell culture work, filtration is still valuable to remove particulates that would clog instruments or introduce artifacts. Use the same 0.22 micron PVDF or PES setup. The pressure and technique are the same.
Draw the peptide solution into a syringe, attach the filter, hold the filter outlet over your receiving vial, and depress the plunger with slow steady pressure. Never force a stiff filter; if pressure builds, the filter is clogged and you may be shearing the peptide. Use a fresh filter.
Best practice is yes, before any injection. The bacteriostatic water is sterile at the time of manufacture but the vial has been penetrated and the lyophilized cake was not manufactured under aseptic fill conditions. Terminal filtration is a cheap safety layer.
Filtration does not change chemical stability, only microbial risk. Storage rules from our reconstitution guide still apply: refrigerated 7 to 28 days, frozen aliquots at -80°C for longer. Do not filter and then leave at room temperature.