Dosage Validation for Biological
In Vitro Assays

The Problem: How do you relate your results from in vitro receptor or cell based assays, and in particular, toxicology and safety assays to the actual compound dose?

When you report negative results (no binding) – could you be actually reporting an assay without a compound in solution?

What is the consequence of:

• reporting a false negative in a safety assay?
• unknowingly reporting the wrong IC50?
• dropping off a compound for assumed lack of activity – even though it is highly active but insoluble in the assay buffer?
• approving a compound for development that has “passed” ion channel safety assays – even though there was no soluble compound in the assay buffer?

One Approach:

1. Assume the stock concentration is correct
2. Develop a method for measuring each compound concentration
3. Prepare calibration curves for each compound based on dilution factors used
4. Quantify each dose prepared for each assay

Time consuming, expensive, and impractical in discovery

 Analiza’s Solution:

1. Accurate determination of the actual DMSO stock concentration
2. Rapid determination of anticipated solubility limit in the assay buffer, with the proper amount of DMSO, to serve as an upper limit for actual assay dosage
3. Direct determination of the final dose solution

One or more of these options could be selected, depending on the particular assay, and all rely on a patented technique using direct measurement by chemiluminescent nitrogen detection.

The Method: All compounds containing nitrogen can be assayed using a universal calibration curve, conserving precious compound and sample preparation time.

• No assumptions
• No “crashing” of compounds
• No false negatives in safety screens
• No underestimates of potency due to solubility problems

Illustrative Example: The following chart shows that many compounds fall out of solution when diluted 300-fold with assay buffer (typical for biological assays), as compared with DMSO. Initial stock concentrations of 10 to 30mM.

Nominal Ion Channel In Vitro Assay Dosage vs. Actual

Note: early discovery compounds are even more prone to solubility limitation than shown below with commercial compounds.

Solubility Quick Facts

Assay: Aqueous Solubility

Method: Automated, miniaturized gold standard shake-flask method, incubation per client protocol, filtration; with total equimolar chemiluminescent nitrogen detection

Experience: Developer of Automated Phsychem technologies since 1999 with tens of thousands of data points for clients ranging from large pharma to small biotechs

LOD: 0.1µg/mL min, max limited by amount of available materials (nominal, actual compound-specific limits are determined by compound amount, molecular weight, and number of nitrogen atoms in each compound)

Accuracy: Linear fit between automated manual shake-flask and literature data demonstrated fit with r² = 0.9993

Precision: Typically CV < 3%

Failure Rate: 0.3-0.7% based on over 12,000 data points

Source: Dry Powder or DMSO dissolved compounds in 96 well plates, typically 50µL (less when ordered with additional assays), 30mM concentration

Turnaround time: As little as 24 hours, typically within 5 business days

Results: Provided electronically. Tabulated data in Excel per customer instructions. All results are analyst-checked; internal standards are included on each plate.