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1674 1229.4 Sterilizing Filtration of Liquids / General Information USP 39

4. Pall D.B., Kirnbauer E.A., Allen B.T. Particulate retention by bacteria retentive membrane filters. Colloids Surfaces.
1980;1(34):235256.
5. Leahy T.J. Validation of Bacterial Retention by Membrane Filtration: A Proposed Approach for Determining Sterility Assurance
[dissertation]. Boston: University of Massachusetts; 1983.
6. Williams R.E., Meltzer T.E. Membrane structure: The bubble point and particle retention: A new theory. Pharm Technol.
1983;7(5):3642.
7. Reti A.R. An assessment of test criteria in evaluating the performance and integrity of sterilizing filters. Bull Parent Drug
Assoc. 1977;31(4):187194.
8. Carter J.R., Levy R.V. Microbial retention testing in the validation of sterilizing filtration. In: Meltzer T.H., Jornitz M.W., eds.
Filtration in the Biopharmaceutical Industry. New York: Marcel Dekker; 1998:599.
9. Tolliver D.L., Schroeder H.G. Particle control in semiconductor process streams. Microcontamination. 1983;1(1):3443.
10. Leahy T.J., Sullivan M.J. Validation of bacterial retention capabilities of membrane filters. Pharm Technol. 1978;2(11):65
75.
11. Sunderam S., Auriemma M., Howard G. Jr., Brandwein H., Leo F. Application of membrane filtration for removal of dimin-
utive bioburden organisms in pharmaceutical products and processes. PDA J Pharm Sci Technol. 1999;53(4):186201.
12. Johnston P.R., Meltzer T.H. Comments on organism challenge levels in sterilizing-filter efficiency testing. Pharm Technol.
1979;3(11):6670,110.
13. PDA. Sterilizing filtration of liquids, Technical Report no. 26. PDA J Pharm Sci Technol. 2008;62(suppl. 5):260.
14. Elford W.J. The principle of ultrafiltration as applied in biological studies. Proc Roy Soc (Lond). 1933;12B:384406.
15. Zierdt C.H., Kagan R.L., MacLawry J.D. Development of a lysis-filtration blood culture technique. J Clin Microbiol.
General Chapters

1977;5(1):4650.
16. Tanny G.B., Meltzer T.H. The dominance of adsorptive effects in the filtrative sterilization of a flu vaccine. J Parenteral Drug
Assoc. 1978;32(6):258267.
17. Zahka J.C., Grant D.C. Predicting the performance efficiency of membrane filters in process liquids based on their pore-
size ratings. Microcontamination. 1992;9(12):2329.

1229.6 LIQUID-PHASE STERILIZATION

INTRODUCTION

Microorganisms are subject to destruction in a variety of ways. Aside from the classical methods of steam, dry heat, and
radiation, destructive sterilization may also occur by immersion in a chemical solution. This is termed liquid-phase sterilization
(1). A number of chemical agents, such as aldehydes, acids, bases, and strong oxidants in solution, under the appropriate con-
ditions, are capable of destroying bacteria and fungi, including both vegetative cells and spores in a quantitative fashion (2,3).
Objects to be sterilized are immersed in the solution of the chemical agent, after which the agent must be removed in a man-
ner that preserves the sterilized object from recontamination. Removal of the chemical sterilant from the exposed surfaces that
have been sterilized must be accomplished in a manner that maintains the sterility of the item postprocessing. Recontamina-
tion falls outside the scope of usual consideration for sterilization processes. However, in liquid chemical sterilization it is cus-
tomary to include the agents removal (whether this is accomplished by physical or chemical means) in the overall process,
together with any needed additional steps to avoid recontamination.
A substantial number of liquids in aqueous solution are capable of sterilizing articles during immersion. Examples include the
following:
Aldehydesglutaraldehyde, formaldehyde
Acidsperacetic acid, nitric acid, sulfuric acid
Basessodium hydroxide, potassium hydroxide
Oxygenating compoundshydrogen peroxide, ozone, chlorine dioxide
Halidessodium hypochlorite, chlorine
As is the case for gas sterilization, the effectiveness of chemical sterilants varies with concentration and temperature. Other
factors that affect antimicrobial activity include pH, extent of mixing (if present), and presence of cellular or other debris. Be-
cause of the limited number of variables, process control for sterilization by liquids is relatively simple.
Because there are no widely accepted biological indicators for sterilization by liquids, the use of a common mesophilic spore-
former such as Bacillus atrophaeus or B. subtilis is commonplace because these are the likely worst-case bioburden isolates.
The agents used for sterilization by liquids vary with respect to sterilant stability, effective pH range, concentration, tempera-
ture, contact time required, and potential interaction with the materials. When selecting the most appropriate sterilant, manu-
facturers must consider its effect on the materials, package components, and equipment, as is the case with all other sterilizing
processes. The variety of agents, process diversity, and potential applications preclude a material-by-material review of these
agents in this chapter. Manufacturers should note that these agents are highly toxic, and appropriate safety measures should
be practiced at all times during cycle development, validation, and routine operation.

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USP 39 General Information / 1229.6 Liquid-Phase Sterilization 1675

VALIDATION OF STERILIZATION BY LIQUIDS

Experimental evidence has shown that first-order kinetics is appropriate for microbial destruction, which makes validation a
simple exercise. The validation of sterilization by liquids can be accomplished using either the half-cycle approach or the brack-
eting method.

Half-Cycle Approach

The half-cycle approach described below is a modification of the method described in Gaseous Sterilization 1229.7 (see
Figure 1). It is a method that requires the destruction of a resistant microorganism at defined lethal conditions. In routine oper-
ation, the process dwell period is arbitrarily doubled and supports a theoretical reduction of the biological indicator (and thus
the bioburden) to a probability of a nonsterile unit of at least 106 for a full cycle.

General Chapters
Figure 1. Half-cycle sterilization validation.

The half-cycle method originally was used for ethylene oxide sterilization when the relationship between the microorganisms
and the delivered process parameters was less certain.

Bracketing Approach

In this method (see Figure 2), analysts evaluate conditions of concentration and temperature that bracket the defined proc-
ess condition to support both over- and under-treatment of the materials and bioburden, respectively. Users can establish the
death rate for the microbial population for each of the conditions that bracket the routine process.

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1676 1229.6 Liquid-Phase Sterilization / General Information USP 39
General Chapters

Figure 2. Bracketing method.

Regardless of the approach used, in order to complete the cycle development and validation manufacturers must identify a
rapid neutralization method that inactivates the chemical agent to allow microbial quantification after fractional kill exposure.
The exposure periods may need to be brief because many of these agents have rapid kill rates.

Equipment Qualification

Equipment qualification is a predefined program that examines the equipment to confirm that it has been properly installed
and operates as intended before the sterilization process. This activity for sterilization by liquid chemicals is simple, because the
equipment used is rarely complex. Temperature control and agitation/recirculation rates are the essential considerations.

Component and Load Definition

Sterilization by liquids is a surface phenomenon, and all surfaces of the materials must be immersed in the sterilant. Treat-
ment uniformity can be ensured by recirculation or mixing of the sterilant during the process. Penetration into needle lumens,
closely fitted parts, and porous materials should be confirmed. The use of a maximum load per defined vessel or container
represents the worst case because it provides the maximum surface area to be sterilized.

Biological Indicators

The common indicator organisms for chemical sterilization are B. atrophaeus ATCC 9372 or B. subtilis ATCC 6633. The spore
challenge is inoculated directly onto the items. End users should determine the populations of inoculated items. Manufacturers
should place indicators within loads at locations believed to be hardest for the agent to reach, on the basis of visual examina-
tion.

Process Confirmation/Microbiological Challenge

The core of the validation activity is confirmation of acceptable process parameters and inactivation of the microbial chal-
lenge. The end user should expect a linear death curve for the spore challenge and require total death of the challenge. The
end user can consider adjustment in chemical sterilant concentration, process time, agitation, and other factors. Proof of cycle
efficacy is provided in replicate studies in which the biological indicators are killed, and physical measurements are taken as
documentation.

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USP 39 General Information / 1229.7 Gaseous Sterilization 1677

Agent Neutralization/Removal

After exposure, the sterilizing agent must be adequately removed from the items or neutralized before further processing.
This segment of the process uses chemical neutralization or physical removal and must be executed in a manner that preserves
the sterility of the items. Aseptic processing with appropriate capability demonstration should be provided. Process simulation
beginning with the completion of sterilization through placement into a sealed sterile container is expected. See Sterilization
and Sterility Assurance of Compendial Articles 1211 for additional information.

Routine Process Control

Liquid-phase sterilization must be subject to controls that maintain the validated state. The practices outlined in Sterilization
of Compendial Articles 1229 address the general requirements for all sterilization systems. Sterilization is accomplished by
means of a number of related practices that are essential for continued use of the process over an extended period of time,
including calibration, chemical and physical measurements, ongoing process control, change control, preventive maintenance,
periodic reassessment, and training.

REFERENCES

1. Agalloco J. Gas, liquid, and vapor sterilization. In: Nema S, Ludwig J, eds. Pharmaceutical Dosage Forms: Parenteral Medica-
tions. 3rd ed. New York: Informa USA; 2010.

General Chapters
2. Favero MS and Bond WW. Chemical disinfection of medical and surgical materials. In: Block SS, ed. Disinfection, Steriliza-
tion, and Preservation. 5th ed. Philadelphia: Lippincott Williams and Wilkins; 2001.
3. Block SS. Peroxygen compounds. In: Block SS, ed. Disinfection, Sterilization, and Preservation. 5th ed. Philadelphia: Lippin-
cott Williams and Wilkins; 2001.

1229.7 GASEOUS STERILIZATION

INTRODUCTION

The use of sterilizing gases for the preparation of materials and equipment is commonly used for items that are susceptible
to damage by heat or radiation processes. Many polymeric materials, especially medical devices, are surface sterilized in this
manner, as is nonpressure-rated process equipment. The sterilization of dry powders using gases is inappropriate due to the
inability of gases to penetrate solid materials. The majority of gas sterilization processes employ ethylene oxide (EO), and pro-
cedures for use with other gases generally are patterned after EO practices. Ozone, mixed oxides of nitrogen, and chlorine
dioxide are some of the other gaseous sterilants used. [Systems that can exist in liquid and gas phase at the operating temper-
atures (e.g., hydrogen peroxide, peracetic acid, and paraformaldehyde) are excluded from consideration in this chapter.] EO's
ability to penetrate through polymers, cellulosics, and other materials allows it to be used for the terminal sterilization of medi-
cal devices in their final packaging. The other sterilizing gases may be suitable for similar applications.
Process control for gas sterilization equipment is accomplished by control of sterilant gas concentration, relative humidity,
temperature, and system pressure. Mixing of the gas in the sterilization chamber may be beneficial. EO sterilization may be
used for parametric release as described in Terminally Sterilized Pharmaceutical ProductsParametric Release 1222.
Gas sterilization differs markedly from processes during which the agent used can condense during the operation. Vapor
sterilization processes will be addressed separately in Vapor-Phase Sterilization 1229.11.
As outlined in Sterilization of Compendial Articles 1229, analysts must take care in ensuring sterility and demonstrating that
the essential quality attributes of the materials are not adversely affected by the process. With respect to gas processes, key
considerations include the immediate effects of sterilizing gas on the materials or equipment being sterilized, residual sterilant,
sterilant byproducts, and potential chemical reactions. The common gas processes differ slightly with respect to process execu-
tion and material concerns and thus are described individually.

ETHYLENE OXIDE

EO is a powerful alkylating agent that destroys microorganisms by chemical reaction, primarily with cell DNA. The destruc-
tive mechanism largely follows first-order kinetics and depends on concentration, humidity, and temperature. The use of EO
for medical devices in their final packaging has, to a large extent, shaped EO sterilization processes (and, to a lesser extent, all
gas sterilization) for other applications (2,3). The usual EO process follows a sequence of prehumidification, air removal, rehu-
midification in the chamber, gas exposure, gas removal from the chamber, and postexposure aeration. The preexposure steps
ensure that adequate moisture is present on and within the items being sterilized. The postexposure steps provide time for the

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