Sterilization Methods for Biopharmaceutical Consumables

1. Introduction

Sterilization is a process that effectively kills or eliminates microorganisms such as fungi, bacteria, viruses, and spores from the material. Several types of sterilization methods that are commonly used for plastic consumable material sterilization in pharmaceutical manufacturing, including autoclave, gas and chemical sterilization, irradiation (Gamma or E-beam), Cleaning-in-Place (CIP) and Steam-in-Place (SIP). Other methods such as dry heat sterilization and gas plasma (H2O2) sterilization are destructive and not compatible with plastic consumable materials; thus their application is very limited. The choice of sterilization methods and effectiveness depends on the purpose of sterilization, the type and number of microorganisms, and the characteristics of materials that will be sterilized (e.g, size, materials of construction and design).

Sterilization methods

Although gamma irradiation method is the most popular methods for single-use systems that are pre-sterilized for use, other sterilization methods, including autoclaving, CIP/SIP, and gas sterilization are also used for preparing the consumables materials for bioprocessing applications.

Autoclaving  Autoclavingare often used for sterilizing assemblies that are prepared by pharmaceutical users using in-house methods, filters and materials that do not have good irradiation tolerance. Autoclaving is usually conducted at 121oC, 15 psi for 20 min. If the materials are multi-usable, it is good practice to clean and rinse the material with distilled water before autoclaving, because certain chemicals which have no severe effect on resins at room temperature may cause deterioration at autoclaving conditions.

Gas Sterilization Gases for sterilization include Ethylene oxide (EtO), nitrogen dioxide (NO2), and formaldehyde gas. They are low-temperature sterilization methods for heat-sensitive items. EtO and nitrogen dioxide disrupt DNA of microorganisms and prevent them from reproducing, while formaldehyde kills microorganisms by coagulation of protein in cells. The biggest drawback of gas sterilization is that gases may leave residues in the materials, therefore, they should be carefully evaluated before their implementations in bioprocessing materials.

Chemical Sterilization Chemicals used in sterilization purpose may include Benzalkoniium chloride, formalin, ethanol, peracetic acid, Ozone, Bleach, Glutaraldehyde and Formaldehyde, Phthalaldehyde, Hydrogen Peroxide, Peracetic Acid, Ethanol, Silver, etc. Chemical sterilization is typically used for devices that would be sensitive to the high heat used in steam sterilization, and for devices that may be damaged by irradiation. Major concerns of using chemical sterilization is ensuring the material compatibility and control the potential harm to humans exposed to the sterilization chemicals or residuals from the sterilization process.

CIP (Cleaning -in-Place) and SIP (Sterilization-in-Place) Cleaning-In-Place (CIP) and Sterilization-In-Place (SIP) are systems designed for automatic cleaning and disinfecting the interior surfaces of closed systems without disassembly.

The main purpose of Cleaning-In-Place (CIP) is to remove solids and bacteria from tanks, vessels and pipework in the production line. A well designed CIP system will reduce costs in terms of higher plant utilization (e.g., enables clean one part of the plant while other areas continue to operate), minimize downtime in product runs/changeover, also allow significant savings in CIP liquid, water and man-hours. The operators require to enter tanks and vessels to clean them and the cleaning materials do not need to be handled by operators, therefore ensure operation safety. Factors that impact the effectiveness of CIP process include flow velocity, flow rate, temperature, pressure, duration, number and volume of equipment, CIP cycles, and cleaning media (e.g, caustic acid, disinfectant, etc.).

Steaming-in-place (SIP) SIP is a widely adopted method for in-line sterilizing processing equipment, including vessels, valves, process lines, filter assemblies, manifolds, and filling nozzles. The main advantage of SIP relies on manipulation reduction and aseptic connections that might compromise the integrity of the downstream equipment. The critical requirements associated with SIP include proper steam distribution, non-condensable gases removal, and continuous condensate elimination. Good engineering practices, adequate piping design, steam traps, valves, and monitoring instrumentation are essential to ensuring SIP validation. Other key factors that may impact SIP effectiveness include position of gas filter, filter position and limits, temperature, pressure, duration, volume of tanks, SIP cycles, and venting steam.

Irradiation

Gamma and E-beam sterilization are mainly used sterilization methods for single-use and multiple-use consumable materials in pharmaceutical industry, because they are clean methods and do not leave residues. However, because each polymer reacts differently to ionizing radiation, it is important to verify that the maximum dose during the sterilization process will not adversely affect the quality, safety or performance of the product throughout its shelf life.

Gamma irradiation kills microorganism by breaking down its DNA. Complete penetration can be achieved depending on the thickness of the materials. The sterilization dose can be defined as the absorbed energy per unit mass [J/kg] = Gy. Dose of sterilization should be chosen according to the initial bioburden, sterility assurance level (SAL) and the radio sensitivity of micro-organisms. It is important to consider the product tolerance when use gamma irradiation because the irradiation can cause ionization and excitation of polymer molecules, generate free radicals and sometimes cause molecular bond reactions like chain breaks, cross-linking or photo-oxidation reactions that can induce polymer structure changes, ultimately affect the polymer material physical or chemical properties. All polymers are affected to some degree, although some polymers have higher resistance to irradiation induced changes. The degree of change may depend both on the basic polymer structure and any additives used. For examples, PTFE can only tolerate 5 KGy, polyacetals, polymethylpentene, polyacrylonitrile, polyacrylate, homopolymer, PP-PE can only go up to 20-25 KGy. In addition, dose rate, dose distribution, and radiation quality are physical parameters that can affect the outcome of irradiation. The most important physiological and environmental parameters are temperature, moisture content and oxygen concentration. It is also important to keep in mind that the effect of radiation is cumulative and repeated irradiation of materials should be avoided.

Table 1. Toleration of polymers to gamma irradiation.

Polymer Name Radiation Effect Tolerance (KGy)
PVC Polyvinyl chloride chain scission and cross-linking 100
PMMA Polymethylmethacrylate radiolytic degradation, yellowness 100
PC Polycarbonate radiation-induced main chain scissions, yellowness 1000
PU Polyurethanes Degradation, wide formulation various 100-1000
Polyolefin Polyolefin oxidation degradation, crosslink to gain strength, loses some elongation 1000
Si Silicon rubber molecular architecture change, increase in Mw and decrease elasticity, crosslink density increase 50-100
Polyacetals Delrin, Celcon avoid use due to embrittlement 5
PTFE Tetrafluroethylene liberate fluorine gas, disintegrates to powder, avoid use 5
FEP Fluorinated Ethylene Propylene -Avoid use but can go up to 50 KGy 50

* More material tolerance to gamma irradiation can be found Gamma Compatible Materials .

E-beam sterilization is a relatively new process with many advantages such as safety, having no emission, and high speed processing. Microorganisms are killed by DNA chain cleavage, the absorbed dose is the amount of interaction between E-beam and product which will be sterilized. E-beam is not generally used for microbial control or sterilization of single-use systems because it’s limited penetration ability. Table 2 compared key differences of Gamma and E-beam irradiation methods.

Table 2. Comparison of Gamma and E-beam sterilization methods

Items Gamma irradiation E-beam
Mechanism Deliver a certain dose from 60Co or 137Cs, electromagnetic quantum waves Deliver a certain dose, source of electricity producing high charge of electrons
Time Minutes to hours depend on the thickness and volume of the product In few seconds, but can only give to small products.
Penetration More penetrating than E-beam Less penetrating
Availability Commonly available Needs an electron accelerator that is very rare
Degradation effect Depend on the polymer and additives Less depend on the shorter exposure time connecting with the dose rate.
Temperature Cold method, increase in temp. is low Cold method, increase in temp. is low
Dose rate Lower than electron beams. not dose flexibility Higher dose rate, well controlled dose range can be achieved

For some materials and products that are sensitive to oxidative effects, such as low molecular weight PP, PTFE and polyacetals, radiation tolerance levels for electron beam (e-beam) exposure may be slightly higher than for gamma exposure. This is due to the higher dose rates and shorter exposure times of e-beam irradiation which have been shown to reduce the degradative effects of oxygen. However, a comparison of radiation’s effect of e-beam versus gamma is not easily accomplished unless product specific characteristics-including part thickness, volume of product, molecular weight, scission to crosslink ratio, oxygen sensitivity, use of anti-oxidants and aging effects-are known to allow the evaluation.

3. Conclusion

There is no single sterilization method fit for all the pharmaceutical materials; because every method has some advantages and disadvantages. In all cases, sterilization methods and process should be selected according to the chemical and physical properties of the consumable materials. Gamma irradiation is by far the most commonly used sterilization method for single-use systems due to its effectiveness, cleanness and availability. Autoclaving is also commonly used as alternative sterilization method when the assemblies are prepared by biopharmaceutical end-user in house, or the materials are not compatible with irradiation methods.   

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