What is Sterile filtration?

Sterile filtration removes bacteria from a solution by forcing it through a membrane whose pores are too small for them to pass. Peptides cannot be autoclaved without degrading, so filtration is how a peptide solution destined for cell culture is made microbiologically clean. In the lab this usually means a syringe filter rated 0.2 or 0.22 µm, fitted to a sterile syringe and discharging into a sterile tube.

What the rating means

"Sterilising grade" is a performance claim, not a pore measurement: such filters are qualified by their ability to retain Brevundimonas diminuta at a challenge of 10⁷ colony-forming units per cm² ^[1]. The rating is not absolute. When eight 0.2/0.22 µm filter types from four manufacturers (nylon, PVDF, PES and cellulose acetate) were challenged with bacteria from a natural water source, all 25 cartridges tested showed consistent penetration at 10¹–10⁴ CFU/cm² ^[1]. In another study, Ralstonia pickettii held in a drug solution shrank from a mean length of 1.25 to 0.84 µm over 24 hours and passed 0.2/0.22 µm membranes, whereas 0.1 µm filters qualified against both B. diminuta and the mycoplasma Acholeplasma laidlawii gave sterile filtrate ^[2]. Filtration lowers bioburden reliably; it is not a substitute for clean handling upstream.

What it does not remove

A filter retains organisms, not the pyrogens they have already released. Endotoxin is shed from Gram-negative cell walls as molecules and aggregates far smaller than a bacterium, and its removal relies on other processes, such as adsorption, aqueous two-phase partitioning, ultrafiltration and chromatography ^[4]. A solution can therefore be sterile and still contain enough endotoxin to activate immune cells in an assay.

Worked example: where the peptide goes

Filtration can cost material in two ways that are easy to confuse. Hold-up volume (liquid left in the filter housing) reduces the volume recovered but not its concentration. Adsorption to the membrane reduces concentration, and because a membrane has a roughly fixed binding capacity, dilute solutions lose proportionally more. With an illustrative capacity of 20 µg:

Real losses depend on the peptide and the device. With a model protein, syringe filters carrying a glass-microfibre prefilter lost 20–80% of the protein to adsorption, membrane-only filters 0–20%, and particles shed from some filters accelerated aggregation when the filtrate was later agitated ^[3]. Filtering at stock concentration, before dilution, and checking the filtrate by UV or HPLC keeps the arithmetic honest; the reconstitution calculator and dilution calculator assume no loss.

Common misunderstandings

• "Lyophilised peptide is sterile." Not unless its documentation says it was filled aseptically; a dry cake can still carry bioburden into a reconstituted stock.
• "Filtered once, sterile forever." Every later entry into the tube is a new chance of contamination. Filtering and then dividing into single-use aliquots protects the result.
• "Any 0.22 µm filter will do." Membrane material and prefilters change recovery and particle shedding ^[3], and filter-shed particles feed aggregation.

The sterile technique guide places filtration within a full aseptic workflow, and sterile water covers the diluent side.