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.