Sterilization: definition
From a public health perspective — whether in hospitals, food canning, cosmetics or medical device manufacturing — many sectors of ultra-cleanliness, and particularly the pharmaceutical industry, face the challenge of eliminating viable micro-organisms both during manufacturing stages and in terms of finished product quality.
Not only does the elimination of all biological contamination — whether or not it poses an infectious pathogenic risk — appear self-evident, but the pursuit of sterility is also driven by the obligation to control all required interactions and stability conditions. Numerous sterilization methods exist for this purpose.
Since the awareness of the existence of micro-organisms in the infinitely small — initiated by Louis Pasteur — and the first techniques developed by Nicolas Appert to control them in a given environment, the collective consciousness and therefore the authorities have expressed concern, asked questions and harboured doubts in the face of this sometimes poorly understood invisible threat. Given the prolific — indeed extensive — literature available on the subject, our objective is limited to presenting a comprehensive overview of the most widely used concepts and treatment methods today.
Glossary
- Viable micro-organism: a microscopic living unicellular organism, such as bacteria, viruses, fungi, yeasts and protists (algae and protozoa)
- Sterility: an absolute state, probably demonstrable (1), ensuring the absence of viable micro-organisms
- Sterile: probably (1) free from viable micro-organisms (EN 556-1:2001, definition 3.4)
- Sterilization: a validated process aimed at rendering a product free from viable micro-organisms (per ISO 11139:2001, definition 2.42). Sterilization is therefore a treatment whose objective is to achieve the state of sterility — i.e. to guarantee, with a controlled risk, the absence of viable micro-organisms.
- (1) A correctly sterilized product is not "sterile" but "probably sterile", or thermally treated with a Probability of Non-Sterile Unit (PNSU) of less than 10⁻⁶.
Chemical methods
Contact of the micro-organism, under specific conditions, with one or more molecules that generally destroy the metabolism and particularly the nucleus of the micro-organism. These treatments are described as "cold" processes, although they sometimes use temperature as a catalyst to accelerate the reaction. They may operate in continuous or batch mode.
1. Verdunisation / Chlorination
Either by immersion in chlorinated water, or by addition of gaseous or liquid sodium hypochlorite onto surfaces or into the environments to be treated. Strong oxidising potential. Causes corrosion even on austenitic stainless steels.
2. Ethylene oxide
Very rarely used in its pure form, more often as a mixture at low or high pressure with carbon dioxide (CO₂) or nitrous oxide, ethylene oxide — under very strict humidity and temperature conditions — enables sterilization to be achieved according to a combination of gas concentration and exposure time. Its use is becoming increasingly rare in industry due to the stringent environmental controls required given the hazards involved. Desorption is always very lengthy (24 to 48 hours).
3. Propylene oxide
Generally used in the food industry, it provides surface decontamination.
4. Hydrogen peroxide (H₂O₂)
Strong oxidising potential: in liquid form by dissolution or fogging, or by vaporisation (VHP) onto surfaces (non-penetrating). Leaves no residue. Recommended concentration: 1 to 2% (w/v); duration: 2 to 24 hours.
Applications: machine SIP, osmosis systems, isolators, material airlocks, controlled atmosphere zones.
| Temperature (°C) | H₂O₂ concentration (mg/L) | PPM | D-value in minutes |
| 4 | 0,3 to 0,5 | 350 | 8 to 12 |
| 25 | 1 to 2 | 700 à 1500 | 1 to 2 |
| 37 | 3 to 4 | 2000 à 3000 | 0,5 to 1 |
5. Ozone
Produced by electrical discharge in an atmosphere of very dry air (pure O₂), this unstable gas (O₃) is highly reactive at low concentrations but also irritating, toxic and malodorous. A powerful oxidant used as a bleaching agent (waxes, oils, textiles…), its germicidal action is employed to disinfect air and water in the pharmaceutical industry.
Its use requires ultraviolet radiation (253 nm) for residual degradation. As a disinfection and decontamination agent, it generally cannot guarantee sterilization at SAL 10⁻⁶.
6. Peracetic acid vapour
Primarily used for decontamination of isolators, it is easy to eliminate but is limited to a decontamination action (certain spores are resistant). Recommended concentration: 0.2 to 0.8% (w/v); 1 to 2 hours.
7. Hydrogen peroxide gas plasma
Powerful oxidation techniques acting on micro-organisms. This patented batch process (Sterrad) is accepted by the AFSSAPS and validated in the hospital sector as a sterilization technique, but not yet in industry, due to the difficulty of guaranteeing homogeneity of gas plasma concentration — enriched with oxidising species — throughout the entire exposure time, owing to absorption or phase transformation by the load.
Plasma being a gas highly enriched in energy with unstable elements, after their reaction, they regain stability by emitting radiation and recombining as H₂O₂ + O₂.
8. Formaldehyde (formalin)
Despite its powerful biocidal activity, its use generates carcinogenic, mutagenic and reprotoxic (CMR) risks for humans. By decision of the European Commission 2011/391/EU and the French Ministry of Ecology (Official Journal: 14/09/2011), its placing on the market has been prohibited from 01/07/2012 and its use from 01/01/2013.
Radiation methods
Radiation methods provide bombardment (electromagnetic radiation of varying energy levels) that destroys the internal structures of the micro-organism's nucleus. Treatments are carried out cold, in continuous or batch mode, on-site or more often at a specialist service provider.
1. Germicidal UV radiation at 253 nm
Exposure to radiation from ultraviolet lamps under very precise conditions (often not fully observed) achieves a reduction in a large number of micro-organisms but can in no way guarantee sterilization.
2. Beta (β) radiation
Carried out at a specialist centre, off the production site, this bombardment using a beam of accelerated electrons degrades the nucleus structure according to a dose-effect law. Articles packaged in hermetic packaging receive a dose of energy radiation expressed in megarads, controlled by dosimeters that qualify the quantified absorption.
3. Gamma (γ) radiation
Always carried out off-site, the energy is derived from and depends on a radioactive source. The exposure time is therefore several hours and the yield is lower.
4. X-ray radiation
The energy of X-rays, obtained through the deceleration of accelerated electrons, degrades the chromosome structure of the micro-organism's nucleus. Limited industrial application due to its relatively low yield.
Thermal treatments
Thermal treatments are carried out using sterilization autoclaves. Several technologies can be used: dry heat autoclave, moist heat autoclave, air/steam autoclave, saturated steam autoclave, superheated water autoclave, steam autoclave…
1. Dry heat
This is an oxidation by combustion in the presence of energy and oxygen. The integrity of molecular structures is degraded. It may be continuous or batch, with air and steam being the most common heat transfer fluids.
- Continuous: Hot air through a tunnel in the pharmaceutical industry
- Batch: Air — oven/drying cabinet technique with 3 stages of absolute air filtration, heated to 200–225°C by specific resistances. This technique also enables depyrogenation.
2. Moist heat
The sterilizing effect results from a reaction between the micro-organism to be sterilized and the moist heat in contact with it. Saturated steam, superheated water or air-steam mixtures are hydro-energetic complexes that, in the presence of a cooler mass, transfer their heat energy, which in turn degrades the chromosomal structures of the micro-organism's nucleus.
In the presence of dry saturated steam — slightly moist but highly energetic — heating is faster and condensation more intense until the entire load + chamber is thermally homogeneous.
- Continuous:
- Steam in a full sterilization tower in the food industry for products in carton packs, particularly infant nutrition, and occasionally in the pharmaceutical industry when production throughput allows (vials and bags). Providing a pressure of 1 bar during immersion, these towers of 10 to 15 m — horizontal or vertical — manage the various phases using continuous mechanisation passing through hydraulic barriers of the siphon type.
- Ultra-high temperature technique enabling treatment of liquid at high temperature in continuous flow, carried out in plate heat exchangers or tube bundle exchangers fed by steam or infrared heating (Fo +2 Z).
- Batch:
- Subaqual: Consists of total immersion of the load in superheated water
- Superheated water: by cascading pressurised, superheated and sterile water from top to bottom over the entire load, passing through continuous circulation on a heat exchanger fed by steam for heating and by water for cooling
- Air/steam autoclave: A technique enabling mixing of the 2 gases of different density, very well suited in the pharmaceutical industry for products not requiring vacuum technique
- Saturated steam autoclave: Sterilization is independent of steam quality (wet or dry saturated)
- SIP (Sterilization In Place): Sterilization in place of a process device, generally by timed injection of pure steam; the system being at atmospheric pressure (flowing steam disinfection) or under pressure of 1 to 2 bar (dynamic steam sterilization)
Sterilizing micro/nanofiltration
No action on the micro-organism — the "sieving" effect separates and retains micro-organisms upstream of the filters, according to the porosity of the medium.
1. Sterilizing microfiltration (10⁻⁶) on membranes and cartridges
Cartridges with a porosity generally below one micrometre are used: < 0.2 µm
- Very widely used in aseptic filling
- Viruses are not retained
2. Ultrafiltration (10⁻⁷) and nanofiltration (10⁻⁹)
Filtration threshold enabling virus retention. However, these techniques are generally limited to fluids (air and water) as they could also retain pharmaceutical molecules.
3. Single or double reverse osmosis
Different from filtration, applied to water, this solubilisation (high-pressure diffusion) technique — single or repeated twice — purifies water of its ions, microbes and pyrogens by forced passage through special membranes.
Some applications in the food industry, particularly for fruit juices.
Other methods
Numerous other sterilization methods exist. The following is a non-exhaustive list of other sterilization processes.
1. Pulsed light energy
Pure Bright process: Approximately 20,000 times more energetic than sunlight, the repetition (1 to 10) of pulsed light flashes (450 nm) of 0.5 to 2 J/cm³ lasting a few millionths of a second achieves, through accumulation of energy on the targets — with no possibility of dissipation — a photolysis that irreversibly denatures nucleic acids and proteins.
- Liquids and containers must be transparent
- Rapid action and absence of residue
- Process currently being validated in the pharmaceutical industry
2. Pascalisation
A "hyper-bar" treatment ranging from 2,000 to 7,000 bar (< 70°C) for the destruction of bacteria, yeasts and filamentous fungi in the food industry. The mechanism is considered to involve diffusion, expansion, rupture and degradation of these micro-organisms, although it should be noted that bacterial spores can resist pressures of up to 10,000 bar.
3. Microwaves
Based on current scientific knowledge, microwaves are never sterilizing agents as such. However, in air they can produce plasma highly enriched in oxidising elements, or generate steam when rapidly heating water.
Widely used for the treatment of baby bottles and in the food industry, the treatment is classified as decontamination, except in the case of steam production compliant with steam sterilization.