STERILISASI DENGAN PENYARINGAN

Farmakope Indonesia edisi IV

Sterilisasi larutan yang labil terhadap panas sering dilakukan dengan penyaringan

menggunakan bahan yang dapat menahan mikroba, hingga mikroba yang dikandung dapat

dipisahkan secara fisika. Perangkat penyaring umumnya terdiri dari suatu matriks berpori

bertutup kedap atau dirangkaikan pada wadah yang tidak permeable. Efektivitas suatu

penyaring media atau penyaring substrat tergantung pada ukuran pori bahan dan dapat

tergantung pada daya adsorpsi bakteri pada atau di dalam matriks penyaring atau bergantung

pada mekanisme pengayakan. Ada beberapa bukti yang menyatakan bahwa pengayakan

merupakan komponen yang lebih penting dari mekanisme. Penyaring yang melepas serat,

terutama yang mengandung asbes, harus dihindarkan penggunaannya kecuali tidak ada cara

penyaringan alternatif lain yang mungkin digunakan. Jika penyaring yang melepas serat

memang diperlukan, merupakan keharusan, bahwa proses penyaringan meliputi adanya

penyaring yang tidak melepas serat diletakkan pada arah hilir atau sesudah langkah

penyaringan awal.

Ukuran penyaring Pengukuran porositas membran penyaring dilakukan dengan

pengukuran nominal yang menggambarkan kemampuan membran penyaring untuk menahan

mikroba dari galur tertentu dengan ukuran yang sesuai, bukan dengan penetapan suatu ukuran

rata – rata pori dan pernyataan tentang distribusi ukuran. Membran penyaring untuk sterilisasi

(yang digunakan untuk memisahkan sebagian besar kontaminan mikroba) adalah membran

yang mampu menahan 100% biakan dari 107 mikroba galur Pseudomonas diminuta (ATCC

19146) tiap cm2 permukaan membran pada tekanan tidak kurang dari 30 psi (2,0 bar). Membran

penyaring semacam itu berukuran nominal 0,22 μm atau 0,2 μm, tergantung pada cara

pembuatan produsen. Pengukuran membran penyaring sdapat juga ditentukan untuk pereaksi

atau media yang harus disterilkan dengan cara penyaringan (lihat perlakuan terhadap Isopropil

Miristat pada Salep dan Minyak yang Larut dalam Isoprpoil Miristat yang tertera pada Uji

Sterilitas <71>). Membran penyaring bakteri (juga dikenal sebagai membran penyaring analitik),

yang hanya mampu menahan mikroba berukuran lebih besar, diberi etiket dengan ukuran

nominal 0,45 μm. Tidak ada satupun cara pengukuran penyaring 0,45 μm yang ditetapkan oleh

badan berwenang, dan pengukuran tergantung kepada cara konvensional dari produsen;

penyaring 0,45 μm mampu menahan biakan tertentu, seperti Serratia marcescens (ATCC 14756)

atau Pseudomonas diminuta. Tekanan uji yang digunakan beraneka ragam, mulai dari yang

rendah (5 psi, 0,33 bar untuk Serratia atau 0,5 psi, 0,34 bar untuk Pseudomonas diminuta)

hingga yang tinggi (50 psi, 3,4 bar). Membran ini digunakan untuk uji sterilitas (menurut

Prosedur seperti yang tertera pada Uji Menggunakan Penyaring Membran dalam Uji Sterilitas

<71>), yang tidak memerlukan retensi mikroba yang sempurna. Kecil kemungkinan untuk

melakukan pengujian contoh yang tercemar hanya oleh mikroba berukuran kecil. Membran

penyaring berukuran nominal yang sangat kecil dapat diuji dengan biakan Acholeplasma

laidlawii atau galur lain Mycoplasma pada tekanan 7 psi (0,7 bar) dan akan berukuran nominal

0,1 μm. Pengukuran nominal yang didasarkan pada sifat retensi mikroba berbeda jika

pengukuran dilakukan dengan cara lain, umpamanya dengan pengukuran retensi lingkaran

lateks dengan berbagai diameter. Merupakan tanggung jawab pengguna untuk memilih suatu

penyaring dengan ukuran yang tepat untuk tujuan tertentu, tergantung kepada sifat produk

yang akan disaring. Umumnya tidak layak untuk mengulang uji kapasitas penyaringan di tempat

pengguna. Uji tantang mikroba lebih baik dilakukan pada kondisi produsen terhadap tiap bets

membran penyaring yang diproduksinya.

Pemakai harus menetapkan parameter penyaringan yang digunakan dalam pembuatan

yang mempengaruhi efisiensi retensi mikroba secara bermakna. Beberapa hal penting yang

harus diperhatikan pada validasi proses penyaringan, meliputi kemampuan kompatibilitas

produk, penyerapan obat, pengawet dan atau zat tambahan lainnya, dan pengeluaran awal

kandungan endotoksin.

Karena efektifitas proses penyaringan juga dipengaruhi oleh beban mikroba larutan

yang akan disaring, penetapan kualitas mikrobiologi larutan sebelum penyaringan, merupakan

aspek penting validasi proses penyaringan sebagai tambahan pada penetapan parameter lain

dari prosedur penyaringan, seperti tekanan, laju alir dan karakteristik unit penyaring. Cara lain

untuk menguraikan kemampuan penahanan penyaring adalah menggunakan log nilai reduksi

(LNR). Umpamanya, penyaring berukuran 0,2 μm yang dapat menahan 107 mikroba galur

tertentu akan memiliki LNR tidak kurang dari 7, pada kondisi yang ditetapkan.

Proses sterilisasi larutan dengan cara penyaringan, pada akhir – akhir ini telah

menghasilkan tingkat kepuasan yang baru, sebagian besar merupakan hasil perkembangan dan

kemajuan tekhnologi penyaring membran. Kelompok media penyaring ini menjurus ke arah

pengendalian pembakuan dan mutu yang lebih efektif dan juga memberi kesempatan yang

lebih luas pada pengguna untuk memastikan karakteristik atau sifat rakitan penyaring sebelum

dan sesudah penggunaan. Kenyataan bahwa penyaring membran adalah lapisan tipis polimer

memberikan banyak keuntungan, tetapi juga memberikan beberapa kerugian jika dibandingkan

dengan penyaring tebal seperti penyaring dari bahan porselen atau bahan masir. Karena

banyak dari permukaan membran adalah suatu ruangan yang kosong atau ruang terbuka, maka

penyaring yang cukup baik dirakit dan disterilkan akan memberikan suatu keuntungan berupa

laju alir yang tinggi. Kerugian karena membran umumnya rapuh, sehingga penting untuk

menetapkan bahwa rakitan sudah cukup baik dan membran tidak akan rusak atau pecah

selama perakitan, sterilisasai atau selama penggunaan. Rakitan wadah dan penyaring yang

digunakan pertama – tama harus divalidasi terhadap kompatibilitas dan integritas oleh

pengguna. Jika terbuka kemungkinan untuk mencampur rakitan dan membran penyaring yang

diproduksi berbagai produsen, maka kompatibilitas dari dari rakitan gabungan ini harus lebih

dahulu divalidasi. Disamping itu, terdapat beberapa uji yang harus dilakukan oleh produsen

penyaring membran yang pada umumnya tidak diulang lagi oleh pengguna, meliputi uji tantang

mikrobiologik. Hasil uji terhadap tiap bets membran penyaring yang diproduksi harus diperoleh

dari produsen, dan didokumentasikan oleh pengguna.

Penyaring untuk tujuan sterilisasi umumnya dilaksanakan menggunakan rakitan yang

memiliki membran dengan porositas nominal 0,2 μm atau kurang, berdasarkan pada

pembanding yang telah divalidasi tidak kurang 107 suspensi Pseudomonas diminuta (ATCC

19146) tiap cm2 dari luas permukaan penyaring. Media membran penyaring yang tersedia saat

ini yaitu selulosa asetat, selulosa nitrat, fluorokarbonat, polimer akrilik, polikarbonat, poliester,

poli vinil klorida, vinil, nilon, politef dan juga membran logam, dan ini dapat diperkuat atau

ditunjang oleh bahan berserat internal. Rakitan penyaring membran harus diuji untuk integritas

awal sebelum digunakan, dengan ketentuan bahwa uji tersebut tidak menggunakan validitas

sistem uji, dan harus diuji sesudah proses penyaringan selesai, untuk menunjukkan bahwa

rakitan penyaring mempertahankan integritas sepanjang prosedur penyaringan berlangsung.

Uji penggunaan khusus adalah uji titik gelembung, uji aliran udara difusif, uji penahan tekanan,

dan uji aliran ke depan. Semua uji harus dikaitkan dengan retensi mikroba.

PROSEDUR UJI STERILITAS MENGGUNAKAN PENYARINGAN MEMBRAN

Jika tekhnik penyaringan membran digunakan untuk bahan cair yang dapat diuji dengan

cara inokulasi langsung ke dalam media uji, uji tidak kurang dari volum dan jumlah seperti yang

tertera pada Pemilihan spesimen uji dan masa inkubasi.

Peralatan Unit penyaring membran yang sesuai terdiri dari satu perangkat yang dapat

memudahkan penanganan bahan uji secara aseptik dan membran yang telah diproses dapat

dipindahkan secara aseptic untuk inokulasi ke dalam media yang sesuai atau satu perangkat

yang dapat ditambahkan media steril ke dalam penyaringnya dan membran di inkubasi in situ.

Membran yang sesuai umumnya mempunyai porositas 0,45 μm, dengan diameter lebih kurang

47 mm, dan kecepatan penyaringan air 55 ml sampai 75 ml per menit pada tekanan 70 cmHg.

Unit keseluruhan dapat dirakit dan disterilkan bersama dengan membran sebelum digunakan,

atau membran dapat disterilkan secara terpisah dengan cara apa saja yang dapat

mempertahankan karakteristik penyaring dan menjamin sterilitas penyaring dan perangkatnya.

Jika bahan uji berupa minyak, membrane dapat disterilkan terpisah, dan setelah melalui

pengeringan, unit dirakit secara aseptik.

FILTRATION

British Pharmacopeia 2005 edisi IV. Publised by The Stationery Office on behalf of the Medicine and Healthcare products Regulatory Agency (MHRA)

Certain active ingredients and products that cannot be terminally sterilized may be subjected to a filtration procedure using a filter of a type that has been demonstrated to be satisfactory by means of a microbial challenge test using a suitable test micro-organism. A suspension of Pseudomonas diminuta (ATCC 19146, NCIMB 11091 or CIP 103020) may be suitable. It is recommended that a challenge of at least 107 CFU per cm2 of active filter surface is used and that the suspension is prepared in tryptone soya broth which, after passage through the filter, is collected aseptically and incubated aerobically at 320. Such products need special precautions. The production process and environment are designed to minimize microbial contamination and are regularly subjected to appropriate sterilization process. It is recommended that the filtration process is carried out as close as possible to the filling point. The operations following filtration are carried out under aseptic conditions.

Solutions are passed through a bacteria-retentive membrane with a nominal pore size of 0,22µm or less or any other type of filter known to have equivalent properties of bacteria retention. Appropriate measures are taken to avoid loss of solute by adsorption on to the filter and to avoid the release of contaminants from the filter. Attention is given to the bioburden prior to filtration, filter capacity, batch size and duration of filtration. The filter is not used for a longer period than has been approved by validation of the combination of the filter and the product in question.

The integrity of an assembled sterilizing filter is verified before use and confirmed after use by carrying out tests appropriate to the type of filter used and the stage of testing, for example bubble-point, pressure hold or diffusion rate tests

Due to the potential additional risks of the filtration method as compared with other sterilization process, a prefiltration through a bacteria-retentive filter may be ensured by other means.

STERILIZATION BY FILTRATION

Diana’M Collett B.Pharm, Phd, MR.Pharms; Michael E.Aulton B.Pharm, Phd, MR.Pharms. Pharmaceutical practice .1990.Singapore.

Sterilization by is a method permited by the BP for solutions or liquids that are not sufficiently stable to withstand the process of heating in an autoclave as described in chapter 20. Passage through a filter of appropriate pore size can remove bacteria and moulds although smaller micro-organisms such as viruses and mycoplasms may not be retained. After filtration the liquid is aseptically distributed into previously sterilized continers which are then sealed.

This method has a number of disadvantages and should be used only for those products where sterilization by alternative means is not available.

FILTER MEDIA

A sterile filter nominal pore size 22 λm less is required ( DHSS 1983, BP 19880. Filters containing asbestos or any other mµedium likely to shed fibres or particles may not be used.

MEMBRANE FILTERS

These are usually the preferred type of filter for sterilization. Membrane filters are made fro cellulose derivatives or other polymers and there are no loose fibres or particles. The retention of particles larger thn the pore size occurs on the filter surface which also makes this type of filter particularly useful for the detection of bacteria.

Advantages of membrane filters include:

1. Rigid structure-unaffected by bubbles or pressure surges.2. High flow rates-80% of filter surface consists of pores.3. Non-fibre shedding.4. Minimal absorption-concentration unaffected5. Minimal wastage-little retention of solution.6. Testable prior to and after filtration.

Although re-useable membrane filter are available, the disposable types are generally preferred.

The use of a pre-filterMembrane filters are generally blocked by particles close in size to the pore size of the

filter. Pre-filtration reduces the risk of blockage of the final filter. Since the filtration method of sterilization carries a potentially greater risk of failure than other methods, a second filtration through a sterilized membrane filter provides an additional safeguard.

Sintered glass filtersSintered glass filters made fromborosilicate glass with anappropriate pore size may be

used to sterilize solutions. These have the disadvantages of slowness of filtration, fragility and difficulty of cleaning.

Filter kaca tersinter

Filter kaca tersinter untuk fromborosilicate gelas/kaca dengan anappropriate ukuran pori-pori dapat digunakan untuk mensterilkan.Filtrasi Ini mempunyai kerugian-kerugian dari lambatnya filtrasi,kerapuhan dan kesulitan pembersihan.

Other filters

Filters media that have been used in the past as bacteria proof filters include asbestos pads, ceramic filters and kieelguhr candles.

Filter lain nya

Media filter mempunyai kegunaan dimasa lalu sebagai filter bakteri seperti abses, filter keramik dan lilin.

Testing of filters

The Bp requires that the integrity of an assembled sterilizing filter be verified before use and confirmed after use by means of a suitable test.

Bacteriological test

The filter may be challenged by the passage of a diluted 24-48 hour broth culture of Serratia marcescens. A sample of the filtrate is collected aseptically and incubated at 25°C for 5 days. This organism ischosen because it has a small cell size ( 0,3-0,4 πm across). It grows vigorously in aerobic conditions and produces a readily detected red pigment.

Bubble pont test

The bubble point of a test filter is the pressure at which the largest pore of a watted filter is able to pass air. The pressure varies with the surface tension of the liquid with which the filter is wetted. Details of bubble pressure testing are given in the relevant British Standard (BS 1752:1963). Sterile membrane filters can be tested before use by a bubble pressure method, usually described in the manufacturer’s literature.

Sterility testing

In-process controls are not generally available for methods of sterilization by filtration. It is therefore advisable to withold the issue of products sterilized by this method until sterility data is available.

FILTRATION STERILIZATION

Kenneth E. Avis; Leon Lachman; Herbert A. Lieberman. 1993. Pharmaceutical DosageForms:Parenteral Medication Volume 3. New York

The validation of 0,2 micron porosity membranes for the removal of viable organisms from liquids and geses differs significantly from other sterilization procedures. In each of the sterilization processes presented earlier in this chapter, the treatment process involved the destruction of the microorganisms by the application of a lethal environment. In sterilization by filtration the organisms are not destroyed, but are separated from the fluid by passage of the fluid through a porous membrane. The nature of filtration processes requires the control of parameters very different from those used in other sterilization procedures in order to reproducibly effect sterilization in this manner. The major parameters of interest are fluid bioburden, filter integrity, and filter pore size. Of less critical significance are parameters such as fluid temperature, pressure, viscosity, filter area solvent type , PH,and other fluid attributes.

Validation program outline

The validation of filter sterilization has been the subject of considerable disagreement within the parental industry. Several attempts to prepare an industry standard for the validation of membrane filtration have failed because of a lack of consensus on the finer points of the integrity testing and microbial challenge methodology. Despite the lack of agreement on some details, there is a degree of acceptance of the general concepts of filter sterilization validation.

1. Filter/Fluid Compatibility

The first step in the validation of a new filtration system is the establishment of compatibility between the filter and the fluid (product). This is generally a task shared by the filter manufacturer and the parenteral manufacturer. The filter manufacturer will provide information regarding the likely effect of the fluid on the filter, as well as identifying the components of the filtration system that will come into contact with the fluid. The parenteral firm will closely examine the process fluid for the presence of filter components and any deleterious effect on its process materials. For existing systems, where the filter has been utilized for some years, this step is largely ignored because of the availability of historical data. However, significant change in filter or process fluid should prompt a re-evaluation of compatibility. The downside risk associated with a change in materials is such that many firms will continue to utilized older filter medias to preclude compatibility evaluations required to employ newer filtration systems.

Fluid evaluation. Evaluating the effect of the filter on the process fluid will usually entail some form of stability testing with careful assessment of key product attributes. Samples for this type of testing are prepared by having the filter immersed in the process fluid for an extended period of time at conditions (temperature, pH) approximating those of use. Information and test procedures from the filter manufacturer can be used to determine

whether any components of the filter can be detected in the products. When conducting these type of studies. It is important to remember that in many filtration systems there are additional materials in the cartridge and housings that are not part of the membrane proper. The potential for interaction of these materials with the product must be also evaluated.

In addition to confirming that filter materials have not been introduced into the product at unacceptable levels, careful analysis of the fluid product must be conducted. There are numerous references to filters selectively removing individual components of a formulation [46]. The potential to change in the fluid, as a consequence, of contact with materials in the filter and its support systems, is assessed through the completion of accelerated and long-term stability studies that will confirm the compatibility of the chosen system. Assistance from the filter manufacturer in the identification and quantification of filtration system materials and advice in the selection of the most appropriate filtration medium for a particular fluid, is essential to the success of any compatibility determination effort.

Filter evaluation. Inparellel with the evaluation of likely effects on the process materials as a consequence of contact with the filtration system, a careful review of the component of the filtration system can be beneficial. Changes in the appearance and properties of the filter system compenerits may be easily detected and lead to a more rapid determination of potential incompatibilities. The filter suppilier should be able to provide a comprehensive set of test criteria that can be utilized to confirm materiam suitability. Common method for this evaluation include change in weight , chang in integrity test result, change in appearance, and so on . while the absence of a significant change in the filtration system is no aclear indication of compatibility with the prosess fluid, the detection of measurable changes in the filter system materials should be a warning of potential problems.

2. Filter Integrity

The confirmation of filter integrity is required for every sterilizing filtration. Parenteral firms will test their sterilizing filtration systems before (in many cases) and after use to confirm the filter’s integrity. Testing before filtration is sometimes omitted for smaller systems where the costs associated with refiltration, in the case of integrity failure post-filtration, is acceptable low. The confirmation of filter integrity after completion of the sterilizing filtration is universal. For the most part, in-plat integrity test utilize physical methods that have been correlated to the microbial retention capabilities of the filter. Common integrity test include the bubble-point test, diffusive flow test, and pressure hold test, each of which rely on the physical measurements taken in the parenteral facility. The physical test utilized must be supported by filter manufacturer’s data which establishes microbial retention for filters exhibiting similar physical test results.

Bubble-Point Test. The most commonly utilized membrane integrity test is the bubble-point test. In this case, the filter is fully wetted with the test fluid (WFI and various alcohols are the standard fluids), the supply of liquid is stopped, a low-pressure gas stream is applied to the upstream side of the filter membrane, and the pressure is slowly increased. The pressure at which the largest pore in the filter is opened to the passage of the gas is the bubble point. The determination of the bubble point is somewhat operator dependent. Several filter supplier have introduced automated test aparatur that can provide greater reliability in the test result, but their use is not widespread because of the high cost of the automated unit. The bubble point is dependent upon the size of the largest pore in the filter and the viscosity and surface tension of the fluid. Filter users must establish the appropriate bubble point for their process fluids; such values may be either higher or lower than those utilized by the filter manufacturers to control their production.

Forward Flow Test. The forward flow test is utilized for all sizes of filtration systems but is most useful in larger systems (those which utilize cartridge filters) where the large volume of the system may make the accurate determination of the bubble point more difficult. In the forward flow test, a fixed pressure and volume of gas is applied to the upstream surface of a wetted filter. The volume of gas that diffuses through the filter in a given period of time is proportional to the size of the pores in the membrane. As the pore size increases, the amount of gas flow will increase. When conducted at a pressure approximately 80% of the bubble point, the forward flow test can confirm filter integrity and differentiate between filters of different pore sizes. The specific values obtained for this test are related to the test fluid utilized. Filter manufacturers will have available data on WFI and other common solvents, but filter users must establish acceptable values unique to their process fluids.

Pressure Hold Test. The pressure hold test is closely related to the forward flow test and relies on a similar concept. As stated earlier, the diffusive flow across a wetted filter is proportional to the size of the pores in the membrane surface. In this test, the supply of gas to the system is stopped and the drop in upstream pressure caused by diffusive flow across the membrane can be related to the pore size of filter. As with each of the advantage of not requiring a down-stream connection to the system making it most suitable for post-sterilization integrity testing, where maintenance of sterility is a major consideration.

3. Microbial Challenge Testing

The performance of a microbial challenge to a filtration system is definitive proof of the filter’s ability to eliminate microorganism in the process fluid. Filter manufacturers utilize specialized test apparatus and conditions to confirm the microbial retention capabilities of their filters in a variety of challenge conditions. The result of the microbial challenge studies are closely related to the physical parameters associated with integrity testing, thereby allowing

filter users to employ the physical methods to establish the microbial retention of their filtration system.

Laboratory Challenge. A number of filter users place their confidence in the completion of laboratory challenges (generally performed solely by the filter supplier), in conjuction with physical integrity tests performed on plant filtration systems, to establish the acceptability of their filtration systems. These firms believe that the correlation between microbial retention in the laboratory and physical methods in the parenteral plant is sufficient to established the sterility of their effluent materials.

Process System Challenge. Other firms believe that the unique aspects of the production environment preclude the use of physical measurements alone to establish the retention capabilities of the filtration system. These firm adapt the laboratory challenge methods of the filter manufacturer and employ microbial challenges in a production size filtration system with appropriate modifications to facilitate sampling.

Process Fluid in Laboratory. A third approach is the hybridization of the previously described methods. In this procedure, the process fluid is microbially challenged in a laboratory setting under conditions that closely approximate those in use in the operating area. In this instance, the use of the process fluid rather than the saline system commonly utilized by the filter manufacturer is intended to stimulate the production situation more closely.

Each of these techniques has advantages and disadvantages relative to the other methods. The inability of the industry to establish a single approach to filter sterilization validation has to a large extent failed because of the very strong opinios of proponents of one method or an other.

Microbial Challenge Procedure. The confirmation of the filter integrity via microbial challenge is a task of some complexity. Details of the test procedure vary according to the pore size of the membrane (0,45; 0,2; or 0,1 micrometer) and the test organism (S. marcesans, P. diminuta, and A.Laidawii, respectively)utilized. In general terms, the organism. The differences in test procedure from one filter manufacturer to another are subtle but significant. Various adaptations have been made by different investigators to challenge filters or different sizes, in gas phase applications, over extended time periods, etc. While integrity tests appear to be definitive proof of the filter’s ability to retain organisms, the specialized circumstances of the test methods are such that direct confirmation of filter integrity in the parenteral plant appears impossible. Essentially, one place faith in one’s supplier that the filters being supplied will yield a sterile effluent as confirmed by the integrity test with the process fluid.

4. Filter Sterilization

A Satisfactory means for sterilization of the filter medium and its housing must be identified and validated. For larger fluid systems, this will likely entail sterilization-in-place as described earlier in this chapter, for smaller filtration systems, sterilization in a steam autoclave is the preferred method. In some instances, the filtration medium or its supportive materials cannot withstand steam sterilization and the filter must be sterilized using an alternative procedure. Regardless of the type of sterilization procedure utilized , confirmation of the filter’s integrity after sterilization must be performed. This is usually accomplished through the performance of an integrity test. In applications such as tank or sterilizer vents, it is recommended that filter-life studies be conducted to establish the maximum number of cycles to which the filter can be subjected without risk of failure.

A further consideration in the sterilization of filters is the performance of the initial compatibility studies using filtration media sterilized in accord with normal practices. Conducting the compatibility studies in this manner eliminates the potential for differences in compatibility brought about by the stresses crated during the sterilization procedure.

5. Bioburden Determination

A further confirmation of the suitability of its membrane filtration procedures, the filter user will often instate a bioburden monitoring program for its process fluids. This program will entail the samling of the fluid prior to passage through the final filter. Maximum benefit are gained from a bioburden sampling plan if the plan addresses seasonal variations, alternative suppliers, multiple lots, time period from manufacturing, etc, to accommodate the range of conditions likely to impact the microbial content. The testing plan should include count and identification of the organism(s) found. Additional benefits can be gained if additional data on the process is gathered at the same time as the sample is taken. Information regarding the batch size and filtration area can be utilized in conjunction with the microbial count to determine the maximum number of organisms presented to the filter and allow the determination of a theoretical SAL.

6. Air and vent filter applications

Membrane filters in the 0,2 micrometer range are widely used for the filtration of compressed gases and as vent filters. The validation of filters utilized in these applications requires some adjustment in the methods employed.

Issues with regard to filter-gas compatibility are virtually nonexistent for most common gases. The filter manufacturer should be able to provide information on more exotic gases. Filter manufacturers have also adapted the microbial challenge test to gas filtration systems, using an aerosol challenge. The particulars of this test have been well defined by the filter manufacturers and virtually all users rely on the manufacturer’s data in validating their systems.

Confirmation of filter integrity for gas phase filtration systems introduces alevel of complexity to the user. All of the common filter integrity test methods require a wetted filter surface, which must be dried before the filter can be utilized for filtration of the gas. As the vast majority of air and vent filters are hydrophobic, a suitable solvent must be utilized that will enter the pores of the membrane. The addition of upstream and downstream connection points to the system to allow for testing and solvent removal is required.

Sterilization of membrane filters for gas phase application is performed using methods similar to those employed for liquid-phase filters. The earlier section of this chapter addressing sterilization-in-place addresses the major issues fully.

7. Filter manufacturer Vs. Filter user Responsibilities

Clearly the filter supplier. Plays a far greater role in the validation for filtration than does any sterilizer manufacturer. The pharmaceutical firm is in essential partnership with its filter suppliers for the maintenance of its sterility assurance. The control followed by the filter manufacturer in the conduct of its business are of critical importance to the validation of the filtration sterilization process. For this reason, filter manufacturers utilize many of the GMP concepts and validation method evidenced in the pharmaceutical industry. Open communication between the filter manufacturer and filter user is essential to maintenance of validation.

STERILIZATION BY FILTRATION

USP 30 volume I

Filtration through microbial retentive materials is frequently employed for the

sterilization of heat labile solutions by physical removal of the contained microorganisms. A

filter assembly generally consist of a porous matrix sealed or clamped into an impermeable

housing. The effectiveness of a filter medium or substrate depends upon the pore size of the

porous material and may depend upon adsorption of bacteria on or in the filter matrix or upon

a sieving mechanism. There is some evidence to indicate that sieving is the more important

component of the mechanism. Fiber-shedding filters, particularly those containing asbestos, are

to be avoided unless no alternative filtration procedures are possible. Where a fiber-shedding

filter is required, it is obligatory that the process include a non fiber-shedding filter introduced

downstream or subsequent to the initial filtration step.

Filter rating- the pore sizes of filter membranes are rated by a nominal rating that

reflects the capability of the filter membrane to retain microorganisms of size represented but

specified strains, nor by determination of an average pore size and statement of distribution of

sizes. Sterilizing filter membranes (those used for removing a majority of contaminating

microorganisms) are membranes capable of retaining 100% of a culture of 107 microorganisms

of a strain of pseudomonas diminuta (ATCC 19146) per square centimeter of membrane surface

under a pressure of not less than 30 psi (2.0 bar). Such filter membranes are nominally rated

0,22 µm or 0,2 µm, depending on the manufacturer’s practice. This rating of filter membranes

is also specified for reagents or media that have to be sterilized by filtration (see treatment of

isopropyl Myristate under oils and oily solutions or ointments and creams in the chapter

sterility tests (71)). Bacterial filter membranes (also known as analytical filter membranes),

which are capable of retaining only larger microorganisms, are labeled with a nominal rating

0,45 µm. no single authoritative method for rating 0,45 µm filters has been specified, and this

rating are depends on conventional practice among manufacturers; 0,45 µm filters are capable

of retaining particular cultures of serratia marcescens (ATCC 14756) or ps. Diminuta. Test

pressures used vary from low (5 psi, 0.33 bar for serratia, or 0,5 psi, o.34 bar for ps. diminuta)

to high (50 psi, 3.4 bar). They are specified for sterility testing (see membrane filtration in the

section test for sterility of the product to be examined under sterility tests) where less

exhaustive microbial retention is required. There is a small microorganisms). Filter membranes

with a very low nominal rating may be tested with a culture of Acholeplasma laidlawii or other

strain of mycoplasma, at a pressure of 7 psi (0,7 bar) and be nominally rated 0,1 µm. the

nominal ratings based on microbial retention properties differ when rating is done by other

means, e.g., by retention of latex spheres of various diameters. It is the user’s responsibility to

select a filter of correct rating for the particular purpose, depending on the nature of the

product to be filtered. It is generally not feasible to repeat the test of filtration capacity in the

user’s establishment. Microbial challenge tests are preferably performed under a

manufacturer’s conditions on each lot of manufactured filter membranes.

The user must determine whether filtration parameters employed in manufacturing

will significantly influence microbial retention efficiency. Some of the other important concerns

in the validation of the filtration process include product compatibility, sorption of drug,

preservative or other additives, and initial effluent endotoxin content.

Since the effectiveness of the filtration process is also influenced by the microbial

burden of the solution to be filtered, determining the microbiological quality of solutions prior

to filtration is an important aspect of the validation of the filtration process, in addition to

establishing the other parameters of the filtration procedure, such as pressures, flow rates, and

filter unit characteristics. Hence, another method of describing filter-retaining capability is the

use of the log reduction value (LRV). For instance, a 0,2 µm filter that can retain 10-7

microorganisms of a specified strain will have an LRV of nit less than 7 under the stated

conditions.

The process of sterilization of solutions by filtration has recently achieved new levels of

proficiency, largely as a result of the development and proliferation of membrane filter

technology. This class of filter media lends itself to more effective standardization and quality

control and also gives the user greater opportunity to confirm the characteristics or properties

of the filter assembly before and after use. The fact that membrane filters are thin polymeric

films offer many advantage s but also some disadvantages when compared to depth filters such

as porcelain or sintered material. Since much of the membrane surface is a void or open space.,

the properly assembled and sterilized filter offers the advantage of a high flow rate. A

disadvantage is that since the membrane is usually fragile, it is essential to determine that the

assembly was properly made and that the membrane was not ruptured during assembly,

sterilization, or use. The housings and filter assemblies that are chosen should first be validated

for compatibility and integrity by the user. While it may be possible to mix assemblies and filter

membranes produced by different manufacturer, the compatibility of these hybrid assemblies

should first be validated. Additionally, there are other tests to be made by manufacturer of the

membrane filter, which are not usually repeated by the user. These include microbiological

challenge tests. Results of these test on each lot of manufactured filter membranes should be

obtained from the manufacturer by users for their records.

Filtration for sterilization purposes is usually carried out with assemblies having

membranes of nominal pore size rating of 0,2µm or less, based on the validated challenge of

not less than 107 Pseudomonas diminuta (ATCC No. 19146) suspension per square centimeter

of filter surface area. Membrane filter media now available include cellulose acetate, cellulose

nitrate, fluorocarbonate, acrylic polymers, polycarbonate, polyester, polyvinyl chloride, vynil,

nulon, polytef, and even metal membranes, and they may be reinforced or supported by an

internal fabric. A membrane filter assembly should be tested for initial integrity prior to use,

provided that such test does not impair the validity of the system, and should be tested after

the filtration process is completed to demonstrated that the filter assembly maintained its

integrity throughout the entire filtration procedure. Typical use tests are the bubble point test,

the diffusive airflow test, the pressure hold test, and the forward flow test. These tests should

be correlated with microorganism retention.