The Basics of Bioanalysis: How Do We Develop and Validate Your Bioanalytical Method?

Introduction

Bioanalysis is an essential tool in drug discovery and development for determining the concentration of drugs and their metabolites as well as various pharmacodynamics biomarkers in biological fluids. In these analyses, scientists use developed and validated methods to quantitatively detect analytes and metabolites within biological matrices such as serum, plasma, urine, cerebrospinal fluid (CSF), etc.

Bioanalysis isn’t merely running standard analyses for a biological sample. We must produce results which are quantitative and valid as per FDA guidance as these analyses constitute the foundational block towards drug approval. Due to the significance of these analyses, regulatory authorities generally audit these results for accuracy before approval of the drug.

It’s imperative to have the support and expertise of veterans, such as the team at NorthEast BioLab, when the complexities of bioanalysis get cumbersome. Our scientists help navigate bioanalytical method development and validation, as well as offer insights on the requirements of FDA and other international regulatory authorities. This article alludes how our team can help you with custom assay development that meets all the criteria outlined above.

Table of Content

  1. Sample Preparation
  2. Bioanalytical Method Development
  3. Bioanalytical Method Validation
  4. Sample Analysis
  5. Reporting
  6. Regulatory Guidelines

Sample Preparation

Bioanalysis Sample Preperation

We often go through several steps to clean up and prepare the sample before it is ready for LC/MS/MS analysis. These earlier steps help remove any molecules or substances that interfere with the analyte detection. Ultimately, leading to significant improvement in analyte measurement and instrument performance.

Sample Characterization
Target molecules for bioanalysis, known as analytes in chemistry verbiage, fall into one of two categories - small molecules or large molecules. Small molecules are typically chemical entities (drugs), metabolites, or pharmacodynamic biomarkers. Large molecules include proteins, nucleic acids, lipids, and polysaccharides.

While there are a plethora of different instrumental techniques that can be used for bioanalysis, we primarily use the highly sensitive and sophisticated liquid chromatograph instrument coupled to tandem mass spectrometry (LC-MS/MS) and ELISA, among other technologies. Our expertise in handling biological fluids extends to serum, plasma, urine, cerebrospinal fluid (CSF), etc. Our team can effectively quantify your drugs, and their metabolites as well as pharmacodynamics biomarkers from complex biological matrices.

Protein Removal
Proteins and phospholipids are one of the most common interferents found in biological samples which measuring drugs and metabolites. Proteins can precipitate and clog a chromatographic column if left unremoved from these samples. Proteins can also bind to the small molecules of interest, which may include the analyte, preventing an accurate measurement of analyte concentration. Commonly used techniques for protein removal include protein precipitation (PPT), liquid-liquid extraction (LLE), and solid phase extraction (SPE).

Phospholipid Removal
We know that there are numerous phospholipids in biological samples such as plasma - a common type of biological sample for drug metabolism and pharmacokinetic (DMPK) studies. Phospholipids are organic molecules that are found in cell membranes and are composed of a hydrophilic head group which contains phosphate and choline, plus a hydrophobic fatty acid chain tail. Phospholipids interfere with the reliability of data collected by LC-MS/MS with electrospray ionization (ESI) because, as surfactants, phospholipids aggregate at the surface of the droplets formed during the liquid-to-gas transition of ions. With the phospholipids covering the surface of the droplets, the analyte ions are less able to escape the droplet which suppresses analyte detection. Therefore, to improve analyte detection, the removal of phospholipids before analysis is essential. Commonly used methods for phospholipid removal from samples include LLE and SPE, although some phospholipids can be removed from a sample during PPT as well.

Protein Precipitation
PPT removes proteins from a sample by denaturing the proteins causing them to precipitate. Proteins can be denatured through the addition of organic solvent, an acid, a base, or heat to a sample. Acetonitrile and methanol are the most commonly used organic solvents for PPT. Once the protein has precipitated, it is pelleted by centrifugation, and the supernatant is used for analysis. One downside of this technique is that samples are diluted, and sensitivity may be compromised. To precipitate as much protein as possible, we add two or three equivalents of organic solvent for each equivalent of the sample. PPT can also be followed by solvent evaporation and reconstitution of the analyte in an appropriate solution or buffer and offset dilution of sample.

Liquid-liquid Extraction
LLE is a sample preparation technique that is widely used in the analysis of drugs. LLE begins with the addition of an immiscible organic solvent to a biological sample that's typically aqueous. The analyte is extracted from proteins and lipids into the organic solvent layer while the proteins and lipids remain in the aqueous phase. Neutrally-charged analytes tend to be the most effectively extracted using LLE, but the addition of an inorganic salt to a mixture of a biological sample and a water-miscible organic solvent causes the two solutions to separate and allows hydrophilic analytes to partition into the organic solvent phase. While LLE is quite useful for salt removal from samples, it has some limitations regarding the number of analytes that can be simultaneously extracted based on dispersion between the polarities of each analyte.

Solid Phase Extraction
SPE uses solid sorbent material packed into a column or disc to efficiently and selectively remove interferents from biological samples. When a sample is applied to the packed sorbent material, both analytes and interferents will adsorb to the material. Interferents are washed off the sorbent material using solvents such as acetonitrile or methanol. When acetonitrile is used as a solvent, phospholipids tend to be more readily removed from the sample, whereas the use of methanol tends to lead to the presence of phospholipids in the sample. After interferents are washed from the sorbent material, the analyte(s) are eluted and collected. Typically, either reverse phase or ion exchange SPE cartridges are used in drug bioanalysis.

Solid Phase Extraction

On-line Extraction and Direct Injection
Method sensitivity is increased given lower sample dilution when sample preparation or extraction techniques such as SPE are connected on-line with the analysis instrument. Reproducibility improves as well since the analyte no longer needs to be collected and injected into the instrument.

Sample preparation can be time-consuming and can produce large amounts of waste chemical material, so it can be tempting to inject samples into the instrument for analysis directly. However, a lack of sample cleanup tends to lead to contaminants and physical clogging in the apparatus, so often on-line extraction will be added to the instrument to provide sample cleanup before analysis.

Other Endogenous Material Removal
Other endogenous molecules that are commonly found in biological samples include polysaccharides, salts, nucleic acids, metabolites in urine, and secondary metabolites. Because these molecules can also modify the signal response of the analyte of interest, they are removed via methods such as filtration, centrifugation, or simple dilution. Metabolites are very important as they will have the same daughter ion in MS and will interfere with analysis. Metabolites can be removed during sample cleanup step, but in most cases, it is preferred to collect metabolites in the sample as it is often important to quantitate metabolites as well.

DBS testing
Dried blood spot (DBS) testing involves the collection of a blood sample by pricking either a heel or a finger and blotting blood onto high-quality filter paper. The blood can be eluted from the paper using buffer to dissolve the dried blood before analysis, or the blood can be analyzed directly from the paper using desorption electrospray ionization (DESI) or direct analysis in real time (DART).

Bioanalytical Method Development

Robust assay method development and validation is key to accurate measurements of drugs and biomarkers in biological samples. Several underlying steps are adequately completed during bioanalytical method development. Above all, we must determine and consider the nature and any relevant characteristics of the sample itself. For example, the composition of the sample and the expected concentration of the analyte constitute a couple of noteworthy criteria among several others to be considered.

Step-by-step Protocol
First, we define the goal or purpose as this usually drives the method design. Next, we determine the analyte concentration range and select the reference standard to set the boundaries of the method design space. Consequently, we get ready to create the detailed method steps and various standard operating procedures (SOPs). Figuring out the experimental matrix and sampling plan help to fill in SOP details for testing and analyzing critical study parameters. At this point, the method can be tested and optimized for validation and actual sample analysis.

Selectivity
The selectivity of a method is its ability to differentiate between the analyte, the internal standard, and any other biological matter found in the sample. The selectivity of the method is determined by analyzing at least six blank samples. Blank samples comprise of the biological matrix without any analyte or internal standard.

Carry-over Issues
Carry-over is the presence of the analyte in a blank sample analysis that has been conducted following a high-concentration sample. Analyzing a blank sample after a high-concentration sample or after the upper limit of quantification (ULOQ) calibration standard will show if carry-over is an issue for your analysis or not. Carry-over in a blank sample should not exceed 20% of the lower limit of quantification (LLOQ).

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Lower Limit of Quantification (LLOQ)
The lower limit of quantification (LLOQ) is also the lowest concentration of the calibration standards that is not zero and provides a measure of sensitivity. The LLOQ is the lowest concentration of the analyte that can be quantified reliably. The signal generated by the analyte at the LLOQ should be greater than or equal to five times the signal generated by the zero-calibration standard.

Calibration Curve
A calibration curve is prepared by measuring the response from a set of calibration standards prepared that increase linearly in concentration. Calibration standards are prepared by spiking blank matrix solution with a known concentration of reference standard. The calibration curve should have at least six concentrations of calibration standards plus a blank (a matrix that has been through the sample preparation process but does not contain either an analyte or internal standard) and a zero (blank containing internal standard). Each calibration standard should be measured in three replicates, and the calibration curve should be continuous and reproducible.

Accuracy vs. Precision
Accuracy is defined by the closeness of the measured analyte concentration to the actual strength of the analyte. Precision is the closeness of repeated measurements of analyte concentration to each other. Both accuracy and precision are measured using Quality control samples. Accuracy is expressed as a percentage and is acceptable if at least five QC standard replicates at three concentration levels are within 15% of the nominal concentrations. Precision is expressed as the coefficient of variation (CV; also known as relative standard deviation, RSD) and is acceptable if the CV is within 15%.

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Dilution Integrity
If a sample needs to be diluted to fit within the detection range of the instrument, it’s vital to ensure that the act of diluting the sample will not affect the accuracy and precision. To test whether dilution affects accuracy and/or precision (the dilution integrity), a sample of blank matrix should be spiked with analyte at a concentration above the upper limit of quantification (ULOQ) and then diluted with blank matrix to within the range of the study dilutions and then tested for accuracy and precision values.

Reference Standards
For any bioanalysis, a well-characterized solution of an analyte, known as a reference standard, is used to prepare calibration standards and quality control (QC) standards in the biological matrix. Calibration standards are used to generate the calibration curve, and quality control samples are used to measure the performance of the method. Calibration and QC standards can be prepared either by diluting reference standard in organic solvent and then spiking that into the biological matrix or by spiking the reference standard into biological matrix and further diluting the standards with more matrix.

The reference standard should be obtained from a reliable source and should be received with a certificate of analysis (CoA) that demonstrates identity and purity, as well as information such as the batch/lot number, storage conditions, and expiration date.

A structurally similar or an analog of the analyte is generally used as Internal Standard, but a stable labeled analyte of the molecule is an ideal internal standard if available. Most importantly an ideal internal standard must not interfere with the analyte and stable labeled internal standard must not contain over 1-2% of unlabeled analyte as that can interfere with quantitation.

Matrix Effects

Matrix Effects Representation

Matrix effects occur when any molecule (for example, metabolites or decomposition products) other than the analyte that is found in the sample changes the way the analyte responds during detection. It’s possible for matrix effects to cause either matrix suppression or enhancement of the analyte signal, both of which skew the results and reduce the accuracy of the analysis. A value called the matrix factor (MF) can be calculated by determining the peak area of a sample of the blank matrix that has undergone sample preparation and then been spiked with an analyte and determining the peak area of a sample of a pure analyte. Simply put, the ratio of these two values gives the MF. To normalize the MF to the internal standard, divide the MF calculated for the analyte by the MF calculated for the internal standard.

Stability Testing
Analyte stability is determined to ensure that sample handling and storage don’t affect the analyte concentration and therefore the analysis results in a specific sample matrix. Quality control samples are analyzed under varying sample handling conditions including storage at room temperature, in autosampler, during freeze-thaw cycle, and over long-term storage at specific temperatures vs. when freshly prepared, and the results are compared. The values determined should be within 15% of the nominal concentration.

At NorthEast BioLab, we commonly test stability at room temperature for bench-top stability, as well as at -20 °C and at -70 °C for freeze-thaw stability.

Bioanalytical Method Validation

Analytical chemist and molecular biologists working on method development and transfer.

Full Validation
A full validation study should be performed for any custom assay method that is new, or that has been developed based on the literature. A fully validated method shows that it can be used to determine the concentrations of analytes such as a drug and its metabolite(s) realiably in a specific biological sample matrix from a particular species. The key characteristics that need to be determined for the performance of a method to be considered acceptable are selectivity, carry-over, the lower limit of quantitation (LLOQ), the calibration curve, accuracy and precision, dilution integrity, matrix effects, and analyte stability.

Partial Validation
There are times when a full method validation need not be performed. For example, when a method is based on a previously validated method where only minor changes have been made. Partial validation must be run when an analysis is performed in another laboratory or using different equipment, or when the sample origin or storage conditions have changed. A partial validation may be as straightforward as determining the accuracy and precision of the modified method.

Cross-Validation
A cross-validation study must be performed if data has been put together using different methods in a study or if data has been collected in different laboratories. Essentially, cross-validation study allows the data to be reliably compared across disparate sources.

Sample Analysis

Analytical Chemist and Molecular Biologist performing Sample Analysis.

Analysis of samples is performed after method validation once the method has been appropriately verified.

Aliquoting
Aliquoting is the portioning of a sample into precisely known amounts (such as volume or mass) to be used in further analyses. Knowledge of the exact quantity of the aliquot is important for any quantifications made during sample analysis. It is vital to maintain a standard procedure for aliquoting samples to ensure consistency and accuracy.

Analytical Run
An analytical run is the analysis of single batch of the sample. A batch of samples is a complete set of samples along with calibration standards and QC samples that have been processed at the same time in the order in which they will be analyzed. The analysis run-time varies widely as an analytical run may take 24+ hours to complete, or multiple analytical runs may be completed within a single day.

Acceptance Criteria of an Analytical Run
The acceptance criteria of an analytical run should be defined in the method development SOP. One standard criterion is a calibration standard accuracy level where at least 75% of calibration standards should be within 15% of the nominal concentration of the standard determined by back-calculation (using the calibration curve to find out the concentration of the calibration standards). Another standard criterion is a QC standard accuracy level where at least 67% of QC standards should be within 15% of their nominal concentrations.

Calibration Range
The calibration range is determined early in method development and may be re-evaluated later during sample analysis due to a narrower range of analyte concentrations in samples than expected, or due to analyte concentrations of samples being outside of the calibration range. If the calibration curve range is changed, the method should be partially revalidated to ensure accuracy and precision.

Reanalysis of Study Samples
Samples may be reanalyzed for any number of reasons, including the rejection of the analytical run or malfunction of the equipment. Reasons for the reanalysis of study samples should be outlined in the SOP during method development, and the number of samples that were reanalyzed should be found in the study report.

Incurred Sample Reanalysis (ISR)
An incurred sample is one taken from a subject that has been dosed, and incurred sample reanalysis supports the reliability and reproducibility of the reported data as the accuracy and precision of real samples adds to the data collected from QC standards.

Integration
The parameters used for signal integration should be outlined in the SOP and should be maintained in laboratory documentation. Any deviations should be discussed in the bioanalytical report.

Reporting

Documentation of Method Development and Validation in Comprehensive Report.

 

Records must be produced and securely stored to ensure proper method validation. Based on the validation and bioanalytical reports, a study should be able to be repeated as reported. 


Validation Report
The validation report should contain all the necessary information concerning the validation that was performed. The SOPs created during method development should be referred to in the validation report for any needed method details. All the source data should be available upon request in the original format. Any deviations from the validation protocol should be reported.

Bioanalytical Report
Reference should be made to the validation report in the bioanalytical report, and otherwise, the bioanalytical report deals with details of the method and samples, as well as the actual data generated.

Regulatory Guidelines

Federal Drug Administration (FDA) regulates Drug Research and Development in the USA.

With our team of dedicated scientists, we develop and optimize bioanalysis methods on which we then perform assay validation as per FDA and EMA guidelines using Good Laboratory Practice (GLP).

FDA and EMA Guidance on Method Validation
Both the Food and Drug Administration (FDA) and the European Medicines Agency (EMA), provide guidelines for bioanalytical method validation based on GLP principles. By following these regulatory guidelines, a fully validated bioanalytical method is guaranteed to produce data that is accurate and reliable.

Assay development performed at NorthEast BioLab is always validated following the most recently published FDA (2018) and EMA (2011) guidelines.

Good Laboratory Practices (GLP)
Good Laboratory Practice (GLP) is a regulatory concept that was introduced in the 1970s in the USA due to concerns over the validity of data provided in new drug applications. As a quality control system, GLP principles offer a tool to ensure that laboratory studies are planned, conducted, and documented to a minimum standard for data quality and validity. GLP principles also define the responsibilities of facilities and personnel operating in a GLP-compliant bioanalytical lab.

The Organization for Economic Co-operation and Development (OECD) has harmonized GLP principles for international use to allow for the acceptance of previously generated data during the trade of chemicals.

Conclusion

As you can see, bioanalytical testing is a complicated endeavor that provides incredibly valuable information about the safety and efficacy of drugs in a trustworthy manner.

Common applications of bioanalysis performed at NorthEast BioLab include testing the pharmacology, bioavailability, bioequivalence, pharmacokinetic, and toxicology in various studies conducted during preclinical (animal) and clinical (human volunteers) phases of drug development. Similarly, Immunogenicity tests are also considered bioanalytical methods as their targeted analytes are antibodies in serum or plasma.

For expert support in developing and validating bioanalytical methods for your drug development and research, contact us at NorthEast BioLab about our bioanalytical laboratory services. We seek contentment in serving our clients towards their noble mission of bettering the current standards of treatments.