What are system suitability tests (SST) of analytical methods?

Written by Dr. Janet Thode Posted in Method validation

In today’s blog article we will learn about the System Suitability Testing of analytical methods in the pharmaceutical field, its importance in quality control (QC) of drugs and how it differs from the Analytical Instrument Qualification.

The System Suitability Testing (SST) is used to verify that an analytical method was suitable for its intended purpose the day the analysis was done. It is an essential parameter to ensure the quality of the method for correct measurements. An SST is run each time an analysis is performed and each SST is specific to an individual method with pre-defined acceptance criteria for certain parameters e.g. absorbance values being between 0.2 and 1.0 for a photometric content determination method or some resolution factors for chromatographic methods.

 

System Suitability Testing versus Analytical Instrument Qualification

A notable point to mention here is that SSTs must not be confused with analytical instrument qualification (AIQ). A laboratory should absolutely not skip the SST because of having already an AIQ procedure in place. And vice versa, as emphasized in the Aide-mémoire AiM 07123101 of the ZLG: "A system suitability test (which is always method-specific), as required by the pharmacopoeia, does not replace the necessary qualification of an analytical device." This is a big mistake as both the United States Pharmacopoeia (USP) as well as the European Pharmacopoeia (Ph. Eur.) have strong recommendations about SST performance (e.g. for chromatographic methods check USP <621> or Ph. Eur. chapter 2.2.46) and FDA warning letters are issued in case of incorrect behavior as can be seen in this example. In case an SST fails, the method can’t be used for the analysis of the corresponding samples, check also USP chapter <1034>: "If an assay (or a run) fails system suitability, the entire assay (or run) is discarded and no results are reported other than that the assay (or run) failed.". Although different, both ensure the quality of obtained results in a QC lab.

AIQ can be regarded as the basis for all analytical procedures. It proves that the instrument is operating as intended by the manufacturer across the operating ranges defined by the lab. It is done initially and later on in regular intervals. Thus, it is related to the instrument while an SST is method related. An SST is run each time immediately before or in parallel to the analysis of the samples to be investigated. The basis for the SST working reliably is that the instrument has previously been appropriately qualified and the method has been validated. USP chapter <1058> defines SST as “Verify that the system will perform in accordance with the criteria set forth in the procedure. These tests are performed along with the sample analyses to ensure that the system's performance is acceptable at the time of the test.“. This statement actually questions if the method used on the system is working as expected the day the samples are analyzed.

 

System suitability test criteria for chromatographic methods

Let’s now discuss this topic in detail with an example of a chromatographic system.

Obtaining true and precise chromatographic data is the sign of a well behaved chromatographic system, like e.g. a HPLC system. There are multiple factors in a chromatogram that can be assessed as a part of the SST if appropriate. Some examples are as follows:

  1. Precision or injection repeatability: This demonstrates the performance of the system within the defined environment, plumbing conditions and column usage. Unless otherwise stated in a specific monograph, 5 replicates of a standard are used if a relative standard deviation (RSD) of max 2.0% is required and 6 replicates for an RSD >2.0%. A calculation for the maximum permitted RSD is also provided in USP <621>. It should be considered that measured sample values should not differ from the ones of the reference standard for more than the obtained RSD of the reference standard replicate testing described above. In contrast, the Ph. Eur. imposes stricter requirements for repeatability, which is particularly useful for narrow limits of the specification. The requirements are based on a formula that takes into account both the specification upper limit and the number of replicates to be injected (3 to 6). A maximum repeatability of 1.27% is allowed when B = 3.0 (i.e. a specification upper limit of 103.0%) and 6 replicates are injected.
  2. Signal-to-noise ratio (S/N): This parameter can be useful as SST.
  3. Relative retention (r): This is an important tool to have when dealing with impurity determinations. Relative retention measures the relative location of two individual peaks (with one being the compound of interest and the other one the one the corresponding reference).
  4. Resolution (RS): This parameter is essential for quantitation and measures how well the two peaks are separated by considering their retention times and widths at the bases. This parameter comes handy when dealing with potential interference peaks. A clean separation between the peaks is always desired for quantitation.
  5. Capacity factor (also known as retention factor k): It is the relation of the amount (or time) of the substance in the stationary phase against the one in the mobile phase. This essentially means the location of the main peak with respect to the void volume. The peaks must be absolutely free from void.
  6. Tailing factor (also called symmetry factor AS): Peak tailing is a notorious phenomenon and can affect the accuracy estimation of a chromatographic system as peak integration based on where the peak ends could be very challenging. It is essential to determine the location of the upslope and downslope, failing which the accuracy may drop.

 

Establishment of a SST

If not performed earlier (e.g. during method qualification), the SST criteria are established during method validation. For the establishment of the STT, some useful points should be considered:

  • If possible, the sample and the reference standard should be dissolved in mobile phase or in similar amount of organic solvent.
  • The concentration of the sample and the reference standard should be comparable.
  • When filtering samples, it must be taken into account that apart from removing particulates adhesion of the analyte to the filter might also occur, especially at lower analyte concentrations.

 

Examples of SSTs of other methods

For an API manufacturer wishing to recombinantly produce its drug substance in E.coli, it is necessary to test the E.coli strains for the presence of the coding plasmid by checking their antibiotic resistance. In this test, the bacteria are plated on media containing antibiotics. SSTs to be carried along may comprise the simultaneous plating of a positive control as well as of a plasmid-free strain as negative control. In order to prove the viability of the plasmid-free strain, it must be incubated in parallel without selection pressure.

The use of a positive control also makes sense as SST in an E.coli identification test using selective chromogenic media. In case of non-existent growth, it detects a lack of quality of the nutrient medium and on the other hand it helps to distinguish between colored and colorless colonies.

For SDS-PAGE, a clear separation of the bands of a molecular size marker carried along in the gel is a common possibility for a SST. Furthermore, it is possible to define where the bands of a reference standard must be located, since their sizes are known. If a quantification is to be carried out for which different concentrations of the reference standard are applied, the coefficient of determination of the linearity determined therefrom can also be a SST criterion.

For photometric protein determination, e.g. several measurements of a reference standard of known concentration are made and the standard deviation of the measured values is not allowed to fluctuate more than a defined value. In addition, for a successful SST, the mean of the measurements of the reference standard could be in a certain range around the known concentration, e.g. ± 5% of the nominal value.

In case of a ready to use ELISA kit e.g. to determine the host cell protein (HCP) concentration, it may be sufficient as SST to check whether the means of the smallest and highest standard are within the manufacturer's specification.

These examples demonstrate how diverse and method specific SSTs can be.

 

With strong instrument qualification, correct method validation, and strict system suitability criteria, the reliability of the generated data can be ensured. Only with trustworthy validated methods, data that are generated during release and stability testing, are reliable.

 

Update 05.09.2019

Recently, the FDA has also published an answer to the question of the material to be used for SSTs of chromatographic methods. It is expected that high pure primary or secondary reference standards will be used, which were previously qualified against the former reference standard. In addition, they are not allowed to be originated from the same batch as the samples to be tested. Furthermore, written instructions are expected to be complied with and, in terms of data integrity, completeness of the records and their review.