Sterilization by filtration

Although organic substrates such as carboxylic acids are thermally stable and may be sterilized with the basal media, many others including, for example, carbohydrates, esters, or amides are better prepared as concentrated stock solutions, sterilized by filtration through 0.22 pm filters and added to the sterile basal medium. 

Where sterilization by filtration is used, more information will be required. The maximum acceptable bioburden limit prior to sterilization filtration is to be... 

Typically, the manufacture of a batch of biopharmaceutical product entails filling the production vessel with the appropriate quantity of purified water. Heat-stable nutrients required for producer cell growth are then added and the resultant medium is sterilized in situ. This can be achieved by heat, and many fermenters have inbuilt heating elements or, alternatively, outer jackets through which steam can be passed in order to heat the vessel contents. Heat-labile ingredients can be sterilized by filtration and added to the fermenter after the heat step. Media composition can vary... 

Purification entails use of an immunoaffinity column containing immobilized murine antifactor VII antibody. It is initially produced as an unactivated, single-chain 406 amino acid polypeptide, which is subsequently proteolytically converted into the two-chain active factor Vila complex. After sterilization by filtration, the final product is aseptically filled into its final product containers, and freeze-dried. 

HSA is used therapeutically as an aqueous solution it is available in concentrated form (15-25 per cent protein) or as an isotonic solution (4-5 per cent protein). In both cases, in excess of 95 per cent of the protein present is albumin. It can be prepared by fractionation from normal plasma or serum, or purified from placentas. The source material must first be screened for the presence of indicator pathogens. After purification, a suitable stabilizer (often sodium caprylate) is added, but no preservative. The solution is then sterilized by filtration and aseptically filled into final sterile containers. The relative heat stability of HSA allows a measure of subsequent heat treatment, which further reduces the risk of accidental transmission of viable pathogens (particularly viruses). This treatment normally entails heating the product to 60 °C for 10 h. It is then normally incubated at 30-32 °C for a further 14 days and subsequently examined for any signs of microbial growth. 

All of the LC extracts were dissolved in ethanokhexane (1 1) by nsing 1% Tween 80 solntion at a final concentration of 1,024 pg/ml and sterilized by filtration nsing 0.22 pm Millipore (MA 01730, USA) and nsed as the stock solntions. Standard antibacterial powders of ampicillin (AMP, Fako), ofloxacin (OFX, Hoechst Marion Ronssel), and also standard antifnngal powders of ketoconazole (KET, Bilim) and... 

The LB medium and the test tube were sterilized by autoclaving (121 °C, 20 min), while the glucose and the kanamycin were dissolved separately in water and sterilized by filtration through a 0.2 pm filter. After allowing the LB medium to cool to room temperature, glucose and kanamycin solution were added in the appropriate amounts. 

For the US trace element solution (x 1000) all compounds were subsequently dissolved in 1 L of 1 m hydrochloric acid. The solution was sterilized by filtration through a 0.2 m filter. 1 mL of this solution was added to 1 L of M9 medium prior to usage. 

Magnesium sulfate (1 g), kanamycin (50 mg), thiamine (10 mg) and glucose (5 g) were dissolved in water and adjusted to 100 mL volume. This mixture was sterilized by filtration through a 0.2 /im filter and was added together with 1 mL of US trace element solution (see Procedure 2, Section 12.8.2) to the fermenter. 

The obtained liposome dispersion was made sterile by filtration through 200-nm pore-sized filters and stored in sterile bottles till usage at 4°C. The liposomes were stable in size for at least 120 days. 

Purification entails use of an immunoaffinity column containing immobilized murine antifactor VII antibody. It is initially produced as an unactivated, single chain 406 amino acid polypeptide, which is subsequently proteolytically converted into the two-chain active factor Vila complex. After sterilization by filtration, the final product is aseptically filled into its final product containers and freeze-dried. The excipients present in the product include sodium chloride, calcium chloride, polysorbate 80, mannitol and glycylglycine. When freeze-dried in the presence of these stabilizing substances and stored under refrigerated conditions, the product displays a shelf-life of at least 2 years. It has proved effective in the treatment of serious bleeding events in patients displaying anti-factor VIII or IX antibodies. 

Freeze-drying. For a 7000-fold concentration, 70 L of drinking water was lyophilized in a Virtus Unitrap II. The dried residue was then divided into equal weights and packed into two columns (25 X 1.5 cm) with a sintered glass filter. The organic material was eluted consecutively with acetone, ether, and DMSO. The ether in the ether eluate was removed by rotary evaporation, and the dried residue was dissolved in DMSO. The DMSO concentrates were sterilized by filtration over a 0.2-/xm Teflon filter (Millipore). The acetone and DMSO concentrates were tested in the Ames test. 

Bromo-2-deoxyuridine (BUdR) Dissolve 10 mg/mL in distilled water. Sterilize by filtration with a 0 2-pm filter. Store m aliquots at -20°C... 

Ampicillin Dissolve at 50 mg/mL in distilled water, sterilize by filtration through a sterile 0.22-pm disposable filter, and freeze in aliquots at-20°C. 

Sterilization by filtration is a major unit operation used in aseptic processes. Aseptic processes require the presterilization of all components of the drag product and its container. Then all of the components are brought together in a controlled aseptic environment to create the finished sterile product sealed within its container/closure system. The level of sterility attained by an aseptic procedure is a cumulative function of all the process steps involved in making the product. Therefore, the final level of sterility assurance for such a product cannot be greater than the step providing the lowest probability of sterility. Each step in the aseptic process must be validated to known levels of sterility assurance [43],... 

This section will concentrate on that portion of the aseptic process wherein the drug product is sterilized by filtration. From the earlier discussion, sterile filtration is perhaps a misnomer, since the sterile filtrate is almost always processed further under aseptic conditions, which involves a risk of contamination [44], Therefore, to speak of drug product sterilization by filtration as being as final a processing step as the steam sterilization of a product could possibly lead to erroneous assurances or assumptions. Since a sterile filtrate can be produced by filtration, however, we will continue to refer to the process as product sterilization by filtration. 

Assay methods for monitoring any degradation products may be used to justify the time limits for bulk storage. This time would include the period from when the product is formulated to the end of the filling operation. Because most lyophilized formulations do not contain a biological preservative, microbiological quality before sterilization by filtration must also be monitored. The unfiltered solution bioburden would include microorganisms and endotoxin levels. 

The initial pH of the medium was adjusted to 7.0. All chemicals were of analytical grade. The carbon sources (commercial sugar-sucrose, sugarcane juice and molasses, sugarcane juice alcohol stillage, glycerol, mannitol, soybean oil) were added in order to establish an initial substrate concentration of 20 g/L. In some cases, the medium was also aseptically supplemented with 0.1% (w/v) yeast extract and 0.001% (w/v) Na EDTA previously and separately sterilized by filtration through a Millipore membrane with a pore size of 0.22 pm. 

Urea (Lisher Scientific) is dissolved in DI water at 8 Af, sterilized by filtration through 0.45-pm filter and stored at room temperature. 

---