Raymond Colton, Laura Okhio, Oscar W. Reif, Maik W. Jornitz and A. Mark Trotter
Introduction
Disposable equipment from polymeric materials is increasingly used in the production of pharmaceutical products. Sterile filters are routinely used in aseptic processing steps, media bags are replacing stainless steel tanks or carboys, elastomeric tubing is used for product transfers and the final product filled into plastic containers via a blow-fill-seal-filler. As with any polymeric material in a pharmaceutical process, its use must be appropriately validated. One aspect of this validation includes proving that the filters, storage vessels, or tubing do not contribute significant amounts of toxic or deleterious substances to the final product.
Potential contaminants from plastics and polymers can be defined as extractables or leachables. For the purpose of this paper, we define extractables as materials that could potentially migrate from a plastic into a formulation. Extractables are usually components of the plastic such as oligomers, antioxidants, mold release agents or plasticizers. An extractable profile is obtained by exposing the material to extreme extraction conditions with regard to solvent, temperature and contact time. Leachables are the subset of extractables that actually do migrate into an actual pharmaceutical formulation. The extraction conditions for the actual product contact are usually relatively mild with respect to solvent, temperature and time. Thus, leachables are a much smaller subset of extractables.
Sterile filtration is one example of a process using a polymeric component for which leachables are a concern. Ideally sterile filtration removes unwanted particles and bacteria while allowing the formulation to remain unadulterated. Although hundreds of liters of pharmaceutical formulation may be filtered through just a few square meters of filtration area, the effect of leachables cannot be underestimated due to intimate interaction of the formulation with the polymer, especially when the filtered component is of corrosive or aggressive nature.
Furthermore, disposable filter/tubing/bag assemblies are commonly gamma sterilized, which in itself can cause degradation of polymeric materials. While the filtration area may only be a few square meters, the surfaces that experience product contact is 1000-times larger due to the
“sponge” pore structure of the polymeric filter material. Leachables may also come from the filter housing membrane support layer, an o-ring, or any other polymeric component of the filter. Identifying and quantifying these contaminants can be an analytical, albeit necessary, challenge. The necessity is more apparent in the final steps of downstream processing, where impurities may have a greater effect on the product’s safety, purity and efficacy.
Filter manufacturers often perform extractables tests with their filters using standard “model” solutions such as water or ethanol to provide an extractable profile. These data can be used as preliminary information for the choice of an appropriate filter. Further examination with regard to the filters interaction with the specific drug product formulation is usually required to satisfy regulatory requirements. This analysis may be performed with the pharmaceutical formulation itself or, under certain circumstances, with a model solution at worst-case operating conditions.

Regulatory Guidance
The regulatory bodies give guidance regarding when and how to test for extractables and leachables. The Federal Food and Drug Administration (FDA) has published Guides to
Inspections that reference the requirement of manufacturers to “establish an appropriate impurities profile for each Bulk Pharmaceutical Chemical” (1). These guidelines specifically state the need for validation procedures, which approximate routine production as closely as possible.
The Center for Drug Evaluation and Research (CDER) has published industry guidances that reference the requirement of process validation in applications for human drug products. “The specific drug product solution filtration processes should be described….Any effects of the filter on the product formulation should be described (e.g. extractables) (2)”. This requirement inclu des the need for scientifically valid methods performed at conditions “fully representative and descriptive of the procedures and conditions proposed for manufacture of the product”(2).
Similarly, the Code of Federal Regulations (CFR) mandates that materials used in the production of pharmaceutical products be compatible with the drug products. CFR Title 21,
Part 211.65 states: “Equipment shall be constructed so that surfaces that contact components shall not be reactive, additive, or absorptive so as to alter the safety, identity strength, quality, or purity of the drug product beyond the official or other established requirements.
” (3)
CDER has published A Guidance for Industry entitled Container Closure Systems for
Packaging Human Drugs and Biologics. This guidance, while not specifically addressing the processing equipment, gives an indication that the FDA considers extractables to be more of a concern for higher risk routes of administration (injectables, inhalants) and in packaging that provides intimate product contact (solutions, aerosols). It states, “When feasible, the preferred solvent (for extractables testing) would be the drug product or a placebo vehicle.
”(4)
In addition, PDA Technical Report No. 26, Section 4.4 (5) specifies
“It is the users responsibility to demonstrate that the product does not contain objectionable levels of extractables from the filter” and “The filter user is responsible for obtaining extractable data for the drug product formulation” and finally “When the product formulation precludes the use of standard analytical methodology, a suitable model may be used to measure the levels of extractables .”
Test Conditions for Extractable and Leachable Analysis
When choosing the conditions and methods for extractables and leachables analysis, the regulatory guidelines recommend those conditions that are as close to the actual processing conditions as possible. Since it is difficult to perform an extraction test during an actual process, simulated conditions that are at least as severe as the actual processing conditions can be substituted. For instance, extracting a filter in only one liter (1 L) of formulation will lead to a higher level of leachables then if the extraction were performed in an entire process batch which may be 100 L or more. This maximizes the ratio of filter surface area to formulation volume and allows for detection of worst-case leachables concentrations.
Other test conditions that should simulate or exceed the actual processing conditions include pH, temperature, contact time, concentration, autoclave cycles and flush procedures. The choice of test conditions should be the actual process conditions or close to the process conditions to accurately depict results expected with the formulation. If the conditions are too

harsh, then unrealistically high levels of leachables may be present. If the conditions are too moderate, the reduced tendency to leach will give a false sense of security. The extractions conditions are unique to each application. For this reason, the limited testing performed by filter manufacturers may not be used in place of actual testing and rather be used as a guideline. The vital interaction of the formulation with the filter polymers is not necessarily mimicked with a less complex formulation. The choice of extraction medium is a fundamental decision with regulatory impact.
Test Solutions for Extractable and Leachable Analysis
Based on the regulatory references it is apparent that the first choice of extraction medium and conditions should be the formulation and the respective process condition itself. Under circumstances where interfering substances preclude available analytical methods, then the use of a substitute solution is appropriate. Complex schemes for choosing these substitute solutions have been developed and published (6, 7, 8). As a result of these schemes, extractables analysis may have to be performed with multiple substitute solutions, with each one approximating the effect of one or more of the formulation’s components. The
“equivalency” of the substitute solutions is justified based on their likelihood to extract to a similar degree as the actual solution component.
The use of substitute solutions has been popularized by the perception that interfering substances in the actual formulation almost always preclude analytical methods. This theory is mainly due to the low levels of leachables expected when compared to the concentrations of components in the formulation (6, 8).
Analytical Techniques for Leachables Analysis
When analyzing for leachables, it is important to choose the most appropriate analysis technique for a specific application. The details of techniques for the detection of extractables and leachables are discussed by others (9, 10). The common analytical techniques for the measurement of extractables and leachables fall into two categories, non-specific and specific.
Non-specific analytical techniques such as non-volatile residue (NVR) and total organic carbon
(TOC) measure the total non-volatile weight (in the case of NVR) and total organic carbon.
However, they do not differentiate between the components of a drug formulation and the leachables. A buffer or salt component in a formulation will cause an NVR weight that is much higher than the weight of the potential leachables. Likewise, the presence of an organic drug product in a formulation can lead to a TOC value that is much higher than the potential leachables. In these cases, the level of leachables is likely to fall within the error range of the test method.
Specific analytical techniques include standard analytical techniques such as High
Performance Liquid Chromatography with a UV detector (HPLC-UV), High Performance Liquid
Chromatography with a mass spectroscopy detector (LC-MS) and Gas Chromatography with a mass spectroscopy detector (GC-MS). Unlike non-specific analytical methods, these methods do detect separate individual components. Thus, it is possible to detect a small level of an leachable in the presence of a much higher level of the drug product. In general, no single analytical technique should be relied upon. For instance, GC-MS is very valuable technique but will only detect volatile and semi-volatile compounds. HPLC-UV can detect most compounds but may not detect certain compounds without a significant UV chromophore such

as certain saturated aliphatic oligomers. The experienced analytical chemist will choose the appropriate methods depending on their knowledge of potential extractables from polymer and rubber materials.
Fourier Transform Infrared Spectroscopy (FTIR) is another powerful analytical technique that can be used to identify individual leachables after fractionation with a specific analytical technique such as HPLC-UV. It has been suggested that FTIR can be used in combination with a non-specific analysis technique such as NVR to identify leachables (6)(7). However,
FTIR does not adequately differentiate between identical chemical moieties from different compounds. An example can be seen in an ethanol extraction of a PDVF filter. The FTIR of the NVR (Figure 1) detects siloxanes from the silicone o-ring and Irgonox, a polypropylene stabilizer. A GC-MS of the same extracts show numerous extractables (Figure 2). The identification of these extractables show 12 structures which were not identified in the FTIR including, the stabilizer BHT which was the peak with the largest area. Clearly, reliance on the combination of FTIR and NVR will not reliably categorize an extractable profile.
Figure 1.: FTIR Chromatogram of Non Volatile Residue from Ethanol Extraction of PVDF Filter
Description of the Model Approach
The model stream approach allows the use of a suitable model solution, or solutions, to approximate the leachables that would be present when filtering the actual pharmaceutical formulation. Model solutions are identified as those that have a similar effect on extractables
(8) and/or fall into the same main solvent group as the formulation components (6). For example, a formulation containing monosodium phosphate would be modeled using water, and a formulation containing polyethylene glycol would be modeled using 100% ethanol. Model solvents are used under the actual process conditions to simulate the actual effect on the polymeric equipment used. The rationale for these model solvents is that they have similar interactions with the filter device.

Once the pharmaceutical formulation has been classified and appropriate model solution(s) identified, separate filters are extracted in each model solution at worst-case conditions. A blank analysis is performed by subjecting the model solution(s) to identical test conditions without a filter. NVR levels in the blank and the extracts may then be compared to calculate weight of extractables. TOC may be performed to determine the organic content of the blank and extracts. Specific analytical methods such as FTIR, HPLC, and GC-MS may also be used to identify extractables.
The model approach may fall short of regulatory requirements in some cases since the guidance suggests that the actual formulation should be used, unless analytical interference precludes the use of standard techniques. Without first proving that the actual formulation causes significant interference, the model approach may be viewed as a rush to judgment.
The use of a model solution should not be an automatic reaction to a complex pharmaceutical formulation without first demonstrating the inability to use the actual formulation.
Although the model approach gives only an approximation of what would be observed in actual product (7), there are circumstances when the model approach proves valuable. When the formulation contains biologically active or otherwise hazardous materials, the use of a model solution could be used to reduce safety concerns. When there is unavoidable interference with standard analytical techniques, a model solution is justified according to PDA Technical Report
26 (5). Also, as mentioned previously, filter manufacturers regularly use model solvents to gather preliminary compatibility information for their products. Pharmaceutical manufacturers may use this information to assist in choosing filters for a particular application; however, they should avoid using this information as a replacement to performing their own analysis.
Actual Formulation Approach
When leachables analysis is performed with the actual pharmaceutical formulation, only one solution must be investigated. The individual components of the formulation do not need to be classified and evaluated for their effect on leachables. A justification is not required to defend the choices of model solution(s), and regulatory compliance is achieved.
During extraction, the actual formulation is placed in a container with a filter at worst-case processing conditions. From that point, the methodology mirrors the model approach.
Analytical techniques are employed to detect, identify and sometimes quantify extractables.
As an initial level of detection, RP-HPLC with UV detection and GC-MS can be used. The chromatograms and spectra are compared to find peaks that are in the extract but not the blank. The following chromatograms indicate the expected result when no extractables are present.

40
30
20
10 mAU
70
DA D1 A , Sig=220,4 Ref =450,80 (02025EA A \011-0401.D)
60
50
0
5 10 15
Figure 2. Blank analysis, HPLC-UV
20
DA D1 A , Sig=220,4 Ref =450,80 (02025EA A \013-0601.D) mAU
60
50
40
30
25 30 35 40 45 min
20
10
0
5 10 15 20
Figure 3. Test Extract Analysis, HPLC-UV
25 30 35 40 45 min
In Figures 2 and 3, the expected elution time of leachables is from 5 to 55 minutes. The peaks prior to 5 minutes are generally highly hydrophilic and attributable to the formulation itself.
Thus, there is no interference by the formulation in the region where leachable peaks are expected. Additionally, there are no peaks visible in the extract that are not visible in the blank, indicating a lack of leachables.
If a leachable is detected, HPLC peaks can be collected and subsequently identified using
FTIR or LC-MS. Once identification is complete, an the HPLC method can be validated to determine component concentration. If HPLC interference is a problem, sample preparation techniques may be employed to achieve the required analytical sensitivity. A variety of sample preparation techniques are available in this instance.

Sample Preparation Techniques
HPLC interference can result from excessive protein concentration, emulsions, or any UVactive component in the formulation. The goal is to remove the interfering component while preserving the leachables at a level comparable to their original level. To evaluate the sample preparation techniques on this basis, a positive spiked control is analyzed alongside the extracts. This establishes the efficiency of the sample preparation technique to preserve leachables.
Simple chemical separations are employed to isolate the leachables from the interferences.
Example #1
A formulation with significant UV-interference was extracted with hexane to recover leachables into the organic layer. The sample was evaporated to original volume to preserve leachable concentration and analyzed on HPLC.
DA D1 A , Sig=220,4 Ref =450,80 (03181EA D\006-0901.D) mAU
60
50
40
30
20
10
0
0 5 10 15 20 25 30
Figure 4. Interference in HPLC before Sample Preparation
35 40 45 min

mAU
DAD1 A, Sig=220,4 Ref=450,80 (03181EAD\009-1201.D)
7.693
60
Spiked Typical Extractables
50
27.851
40
30
25.037
20
10
5.074
0
0 5 10 15 20 25 30 35 40 45 min
Figure 5. Spiked Control after Sample Preparation
In Figure 5, the sample was spiked with three typical leachables prior to the sample preparation. After sample preparation, the typical leachables are clearly visible at 7.7 minutes, 25.0 minutes and 27.9 minutes while nearly all of the interference has been removed.
Example #2
A product containing 9 mg/mL of protein indicated significant HPLC-UV interference. To remove the interference, the sample was passed through a solid phase extraction column of
C18 -treated silica gel. The protein passed through the column while the leachables were bound to the gel. The column was lyophilized to remove excess moisture, followed by the elution of the leachables with methanol and methylene chloride. The eluate was evaporated to original volume to preserve extractable concentration and analyzed on HPLC.

Spiked Typical Extractables
Figure 6. HPLC before Sample Preparation Figure 7. HPLC after Sample Preparation
In Figure 6, the HPLC interference obscures the extractables peaks. After sample preparation techniques were applied, the spiked extractable peaks (6, 23 and 35 minutes) are visible and interference is removed as shown in Figure 7.
Conclusions
Regardless of the method used to determine the presence of extractables and leachables detected from polymeric materials it remains the user’s responsibility to determine if extractables will have any effect on the final product with regard to toxicity or efficacy of the drug product. Extractables and leachables can be addressed by either modeling or by testing with the actual pharmaceutical formulation. Testing with the actual formulation gives a direct measurement of extractables and leachables and may more closely comply with the regulatory requirements. Even though extractables and leachables are usually only present in trace quantities, it is possible, using standard analytical techniques to determine their presence in actual formulations. The sample preparation techniques applied to these solutions are methods generally practiced in most analytical chemistry laboratories. Most pharmaceutical formulations do not interfere with HPLC methods. Of those that do, many of them can be treated to remove the interference and preserve extractables. In a minority of cases, no sample preparation technique exists to remove the interference and the model approach is the best option. However, a diligent effort to evaluate the materials when exposed to the actual product formulation clearly leads to more reliable and meaningful results.
References
1.
FDA Publications; Guide to Inspections of Bulk Pharmaceutical Chemicals, Guide to
Inspections of Sterile Drug Substance Manufacturers.
2.
“Equipment Construction,” Code of Federal Regulations, Food and Drugs Title 1, Part
1``.65 (U.S. Government Printing Office, Washington, DC, revised 1 April, 2003

3.
CDER Publication; Guidance for Industry for the Submission Documentation for
Sterilization Process Validation in Applications for Human and Veterinary Drug Products,
November 1994.
4.
CDER Publication: Guidance for Industry for Container Closure Systems for Packaging
Human Drugs and Biologics, May 1999
5.
PDA Journal of Pharmaceutical Science and Technology, Technical Report No. 26,
“Sterilizing Filtration of Liquids”, 1998, Supplement, Volume 52, Number S1.
6.
C.J. Weitzmann, “The Use of Model Solvents for Evaluating Extractables from Filters Used to Process Pharmaceutical Products, Part 1 – Practical Considerations,” Pharm. Tech.,
April 1997, p 44-60.
7.
C.J. Weitzmann, “The Use of Model Solvents for Evaluating Extractables from Filters Used to Process Pharmaceutical Products, Part 2 – Assigning a Model Solvent,” Pharm. Tech.,
May 1997, p 72-77.
8.
T.E. Stone, “Methodology for Analysis if Filter Extractables: A Model Stream Approach,”
Pharm. Tech., October 1994, p 116-130
9.
O.W. Reif, P. Soelkner and J. Rupp, “Analysis and Evaluation of Filter Cartridge
Extractables for Validation in Pharmaceutical Downstream Processing,” PDA J.
Pharm.Sci.Technol., 50, 399-410 (1996).
10.
Reif, O.W. "Extractables and Compatibilities of Filters," Chapter 8: "Filtration in the
Biopharmaceutical Industry ",: Eds., Meltzer, T.H. and Jornitz, M.W.; Marcel Dekker, New
York (1998).