In Bioprocess Technology Consultants (www.bioprocessconsultants.com) third annual survey of biopharmaceutical manufacturing capacity and production, biopharmaceutical manufacturers and CMOs said the primary reason they werent expanding the use of single-use disposables was concern over how to validate single-use systems in relation to leachables and extractables.
When using single-use systems for products that will come in contact with the human body (IV fluids, for example), two extremely important questions must be asked: Is the polymer safe? and Is it compatible with the solution it is in contact with?
Guidance documents from the FDA and EMEA help to define the level of validation and qualification necessary for the safety of the single-use systems. An FDA guidance document issued in May 1999, Container-closure Systems for Packaging Human Drugs and Biologics, suggests a general approach for both extractable testing and toxicological evaluation.
The EMEA recently issued Guidelines on Plastic Primary Packaging Materials, which explains how to submit information on plastic primary packaging materials used for storage of active substances and medicinal products. Depending on the type of product affected by the validation, decision trees list interaction studies to be performed.
Beyond the classic biocompatibility and physicochemical tests performed on plastic materials and described by European and U.S. Pharmacopoeias, Stedim (www.sted im.com) provides the industry with further information on extractable and leachable interaction studies, protein adsorption studies, and chemical resistance studies.
Extractables and Leachables
Model solvent extraction tests were conducted to generate extractables/leachables from containers using low, neutral, and high pH solutions, as well as pure ethanol. Extracts were identified and quantified using a purge-and-trap method coupled with gas chromatography to identify volatile compounds. A solvent extraction method was coupled with GC/MS for the identification of semivolatile compounds. The nonvolatile compounds were identified by liquid chromatography.
Mass spectra were interpreted by comparison with a NIST mass spectral library. Internal standard methods were used to quantify the chemical compounds extracted from the plastic material containers. Finally, metal analysis was performed with a solution of low pH in contact with the bag system.
Conditions of test. 500-mL bag samples were assembled with standard components (tubing and connectors), all previously sterilized by gamma radiation at 50 kGys.
Each 500-mL bag was filled with 200 or 250 mL of extract. This represents the worst-case scenario in comparison with the larger volume bags because as users move to larger bag volumes, the relative contact surface area per milliliter of solution decreases. The effect of the film and potential extractables on the stored solution decreases as the ratio of contact surface per mL of solution decreases.
The bag samples were stored for four months at room temperature (25C 2C) for each analysis except for the metal analysis, which was done using the inductively coupled plasma method. Those samples were stored for two months at 40C 2C. The targeted analyses per extract are outlined in Table 1.
Conclusions. A small quantity of extractables were identified. For a polyethylene-based container, the cumulative results of volatile, semivolatile, and nonvolatile leachables tests showed a maximum level of 350 g/L in a neutral aqueous extract after a four-month contact time (Figure 1).
For a 50-L container, the total quantity of leachable in a neutral water-for-injection extract was 24 ppb after four months. For a larger capacity, such as 2,350 L, the total level of leachable under the same conditions was less than 0.1 ppb.
Protein Adsorption Study
Single-use systems are increasingly used in downstream processing, final formulation, and filling to process fluids of a critical nature, including protein-containing solutions. These systems gained acceptance for storage and processing at manufacturing scale of recombinant proteins or monoclonal antibodies in liquid or frozen forms. Container-protein interactions may include potential adsorption of the protein onto the container surfaces.
In this study, traditional glassware used in analytical and bioprocess applications was also tested.
The quantities of adsorbed protein were low, thus RP-HPLC was selected for its sensitive and quantitative method of amino acid analysis.
The major driving forces influencing the adsorption of proteins onto solid surfaces are hydrophobic and electrostatic interactions. These interactions are responsible for nonspecific protein binding on a variety of surfaces. Interaction factors between the surface and protein are modulated by the physical state of the surface, the composition and pH of the solution, the storage conditions (temperature, contact time) and the concentration and structural and conformational properties of the protein.
Test conditions. Protein adsorption levels are dependent on a variety of different parameters, chosen according to standards used in the field. The flexible single-use containers were made of ethylene vinyl acetate copolymer (Evam and Stedim 71) or low-density polyethylene (Stedim 40). The study was performed under worst-case conditions in respect to contact surface-to-volume ratio.
Two model proteins commonly used in biotechnology were chosen: bovine serum albumin (BSA) and bovine polyclonal Immunoglobulin G (IgG). 10 mg/mL BSA and 1 mg/mL bovine IgG solutions were prepared in a phosphate-buffered saline at pH 7.2.
The bags and glass containers were filled with the protein solutions and stored at 5C and 37C. Sample containers were removed from storage and analyzed at regular time intervals (4 hours, 1 day, 3 days, 1 week and 1 month).
Conclusions. Low binding levels of the model proteins were measured on polymeric surfaces and on the borosilicate glass surface. The maximum amount of protein adsorbed (g/cm2) over the storage period for each container is summarized in Table 2. The adsorption level of the two proteins to tested plastic films was low.
The maximum level of protein potentially adsorbed onto Stedim bags filled at nominal capacity at 5C is summarized in Table 3 and illustrated for Flexboy bags in Figure 2.
These values were obtained for the bags with the greatest surface-to-volume ratio for each bag range, which represent a worst-case compared to large volume bags.
The extrapolation of the results obtained with the Stedim product range gives a maximum level of adsorption of 0.14% of the initial protein content, which is low and not significant according to the ICH Harmonized Tripartite Guideline Q1A titled Stability Testing of New Drug Substances and Products.
Chemical Resistance Study
A chemical resistance study was conducted to evaluate the resistance of Stedim containers to chemical reagents. This test was inspired by the standard test, Method for Resistance of Plastic to Chemical reagents D543-95, and by Stedim internal protocol.
For each time parameter evaluated, three bags per batch were tested. One bag filled with water was used as a reference for each parameter. The following tests were performed:
Drop test/Tightness test
Tensile strength on the film
Infrared analysis of the film
Thickness of the film
The bag systems showed excellent compatibility with buffers, alkaline and acidic solutions, and amino-acid solutions. Table 4 summarizes the chemical resistance study. In general, interaction with a high concentration of organic solvents is not recommended and should be tested on a case-by-case basis.