1. What techniques do you currently use?
QPS uses various types of ligand-binding assays with colorimetric detection and chromatographic methods with MS/MS or UV detection.

2. What other technologies do you currently use?
Currently, QPS has three platforms of quantitation of oligonucleotides: hybridization-ELISA, LC/MS/MS, and LC/UV.

3. Do you work on multiple-analytes assays?
That depends on the assay requirement. Chromatographic assays have higher selectivity for the parent and the metabolites; however, ligand-binding assays have better sensitivity. In some cases, the parent and the metabolites may co-elute in a chromatographic method, and thus the selectivity is lost. The most common hybridization-ELISA assay will not distinguish the parent from the metabolites, however, it is possible to develop a "parent-only" hybridization-ELISA assay.

On the positive side the N-1, N-2, etc. metabolites usually have the activity as the parent, and so far, the agency has accepted this limitation in current technology.

4. How many GLP methods have you develop and validate in a year?
What is the ratio of method development vs. method transfer?
Between 2003 and 2007, QPS has worked on seven (7) compounds of this class, ranging from LC/MS/MS to LC/UV to hybridization-ELISA. This translated to development and validation of more than 7 methods and 4 methods are on-going. The ratio of method development to method transfer is approximately 2:1.

5. What sample clean-up procedure do you use for chromatographic method?
We try a variety of methods, such as protein precipitation, SPE, LLE, solid support LLE, and column switching as part of the method development procedure. The exact procedure depends on the analyte(s), the internal standard(s) and the matrix.

6. Do you explore sample stability as part of method development?
Compound stability from the collection point to sample work-up is a very important part of method development, and is part of the guidance.

7. What is the scope of your hybridization-ELISA method development for GLP studies?
The hybridization-ELISA assay is based on the capture and detect oligo probes that hybridize specifically to the oligonucleotide compound, coupled with colorimetric ELISA assay. Each hybridization-ELISA assay is oligonucleotides specific and requires method development for optimal assay conditions. This is usually accomplished through method transfer followed by method optimization and full GLP method validation.

The method will have to demonstrate the following before going into GLP method validation:
 ►Minimum required dilution
 ►Precision and accuracy of full assay range
 ►Analyte selectivity test in matrix (minimum 6 lots)
 ►Linearity of dilution (or parallelism if endogenous)
 ►Hybridization Specificity to drug metabolites vs. parent compound
 ►Stabilities of focus

8. What is the scope of your LC/MS/MS or LC/UV method development for GLP studies?
QPS will examine the following parameters as part of method feasibility/method development to optimize for linearity, precision, accuracy, sensitivity, selectivity, extraction recovery, reproducibility, matrix effect, potential cross-talk, and interference peaks. If required, additional studies, such as whole-blood stability, hemolysis effect, endogenous species, interference from co-administered drugs, and enzymatic reaction to check for Phase II metabolites will be conducted.

The method will have to demonstrate the following before going into validation:
 ►Linearity of the calibration curve (1-day calibration curves with minimum 6 concentration levels in duplicate)
 ►Precision and accuracy (1-day quality controls with minimum 4 concentration levels, including LLOQ; n = 3)
 ►Batch size evaluation
 ►Absolute recovery (low and high concentrations, n = 3)
 ►Selectivity test (LLOQ level; n = 6)
 ►Matrix effect test (low and high concentrations; n = 3)
 ►Carry-over test (extracted blank after a high standard)
 ►Room temperature stability (minimum 4-hour; 2 concentration levels; n = 3)

9. What is the process to validate a hybridization-ELISA assay? What are your validation criteria for hybridization-ELISA assay?
Once the method is developed, a validation protocol is written, in which the acceptance criteria is set up for evaluation of all validation parameters. The hybridization-ELISA assays are validated by multiple runs usually involve more than one analyst. A typical validation includes 6 runs for precision and accuracy, room temperature and freeze/thaw stability, matrix selectivity, dilution test, hook effect, and long-term frozen stability.

The scope of the full or partial validation which follows the latest Crystal City Bioanalytical Validation guideline, and the AAPS Journal 2007; 9 (1), E30-E42 article "Workshop/Conference Report - Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and Ligand Binding Assays" is defined below. A validation report will be issued which will be suitable for regulatory filing.

The full validation will demonstrate:
 ►Precision and accuracy (3-day quality controls with minimum 4 concentration levels, including LLOQ)
 ►Dilution test (1 concentration level, three dilution factors; n > 2)
 ►Matrix Selectivity test (LLOQ level; six individual lots)
 ►Stock solution stability (-20oC or below to cover stock storage time; n = 5)

Sample stability:
 ►Long term frozen stability (at about one month, minimum of 2 concentration levels @ -20oC or below; n = 5). The concentrations will lie within the validation assay range.
 ►Room temperature stability (minimum 4-hour cycle; 2 concentration levels; n = 5)
 ►Freeze/thaw stability (one 24-hour cycle, and two 12-hour cycle; 2 concentration levels; n = 5)

10. What is your validation criteria for LC/MS/MS or LC/UV assay?
The scope of the full or partial validation which follows the latest Crystal City Bioanalytical Validation guideline, and the AAPS Journal 2007; 9 (1), E30-E42 article "Workshop/Conference Report - Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and Ligand Binding Assays" is defined below. A validation report will be issued which will be suitable for regulatory filing.

The full validation will demonstrate:
 ►Linearity of the calibration curve (3-day calibration curves with minimum 6 concentration levels in duplicate)
 ►Precision and accuracy (3-day quality controls with minimum 4 concentration levels, including LLOQ)
 ►Absolute recovery (low, middle, and high concentrations; n = 5)
 ►Dilution test (1 concentration level, one dilution factor; n = 5)
 ►Selectivity test (LLOQ level; six individual lots)
 ►Matrix effect test (low, middle, and high concentrations; n = 5)
 ►Carry-over test (extracted blank after a high standard)
 ►Stock solution stability (minimum 6-hour cycle; 1 concentration level @ RT for a minimum of 6 hour and -20oC or below to cover sample storage time; n = 5)
 ►Sample stability:
 ►Long term frozen stability (at about one month, minimum of 2 concentration levels @ -20oC or below; n = 5). The concentrations will lie within the validation assay range.
 ►Room temperature stability (minimum 4-hour cycle; 2 concentration levels; n = 5)
 ►Freeze/thaw stability (one 24-hour cycle, and two 12-hour cycle; 2 concentration levels; n = 5)
 ►Post preparative re-injection reproducibility (minimum of 3 concentration levels in reconstitution solvent in duplicates at room temperature)

The one intraday partial validation will demonstrate:
 ►Linearity of the calibration curve (1-day calibration curves with minimum 6 concentration levels in duplicate)
 ►Precision and accuracy (1-day quality controls with minimum 4 concentration levels, including LLOQ; n = 6)
 ►Absolute recovery (low, middle, and high concentrations; n = 5)
 ►Dilution test (1 concentration level, one dilution factor; n = 5)
 ►Selectivity test (LLOQ level; n = 6)
 ►Matrix effect test (low, middle, and high concentrations; n = 5)
 ►Carry-over test (extracted blank after a high standard)
 ►Sample stability:
 ►Long term frozen stability (at about one month, minimum of 2 concentration levels @ -20oC or below; n = 5). The concentrations will lie within the validation assay range.
 ►Room temperature stability (minimum 4-hour cycle; 2 concentration levels; n = 5)
 ►Freeze/thaw stability (one 24-hour cycle, and two 12-hour cycle; 2 concentration levels; n = 5)
 ►Post preparative re-injection reproducibility (minimum of 3 concentration levels in reconstitution solvent in duplicates at room temperature)

11. Do you run your standard curves and QC with matrix samples?
Yes, this is part of the Bioanalytical Method Validation guidance. Nevertheless, if the matrices are hard to come by, we will look for a scientifically-valid alternative matrix with the sponsor's approval.

QPS also offers "validation test" in patient matrix, if the matrix is available.

12. What are some of the matrices you have worked on?
Probably 90% of QPS GLP studies are plasma and urine. We have validated hybridization-ELISA methods in specific target organ, such as retina and ocular tissues.

13. How many protocols and samples do you run in a year? What is the ratio of clinical to preclinical/nonclinical samples?
Between 2005 and 2007, QPS worked on approximately 10 protocol-driven studies, which equates to approximately 7,500 samples. The ratio of clinical to preclinical/nonclinical samples averages about 10/90 over the years. As many of the preclinical oligonucleotides-based drug candidates are advancing into clinical development, we anticipate a substantial growth in clinical sample analysis in the years ahead.

14. Do you have the capability to run assays with radioactive samples?
QPS can run radioactive samples from mass balance/excretion and metabolism studies.

15. What program do you use to capture the data from your experiments?
We use the Softmax pro 3.1.2 to capture and analyze the ELISA data and to calculate standard curve from our colorimetric assays and the Softmax pro version 4.8 for the fluorescence assays.

In addition we use the Softmax pro for Lmax version 1.1L to analyze the data from the luminescence reader.

16. Do you have a GLP compliant version of software for data management and report writing?
QPS use Watson LIMS v6.4 for all validation and sample analysis projects.

17. How many scientists work in your oligonucleotides quantitation group?
This type of quantitation depends on the technology platform, and the scientists from the LC/MS/MS group and molecular biology group are pulled in on a as-needed basis for a specific programs.

18. What regulatory agencies have audited QPS?
In 2000 EPA audited QPS-US for a series of FIFRA studies performed in 1997-2000. A 483-equilvant was issued with "No Finding".

In 2007 FDA audited QPS-US for a BE study performed in 2002-2004. Three of the four points on the 483 were focused on chromatography. At that time, QPS-US, like the rest of the industry did not have a chromatography SOP. QPS had a draft version of that SOP before the FDA audit. FDA recognizes that although QPS did not have an effective SOP in place, the PI was consistently using the same integration parameters. The final point of the 483 was about documentation, which was addressed before the audit, as the documentation procedure during the 2002-2004 period was different than the current effective procedure.