Assay Development Guidelines – Pharmacokinetics
You can navigate through the process of developing assays for pharmacokinetics using the flow diagram (just click on the dark blue fields), or simply scroll down. Click ‘TOP’ to return to the flow diagram.
You can find out more about how Gyrolab® technology has been used to analyze pharmacokinetics in the scientific literature. Learn more >>
There are several assay formats that can be used for PK assays depending on analyte properties, availability of reagents, and sample origin. If a single assay for antibody-based drug molecules is to be used across species, going from pre-clinical studies in animals to clinical studies in humans, a bridging immunoassay is preferred (see below: B or D, depending on access to reagents). If a generic, pre-clinical assay suitable for any type of drug molecule (recombinant antibody) is required, a sandwich immunoassay should be designed based on reagents with low cross-reactivity with host animal proteins.
Assay formats applicable for PK assays. Biotinylated capture reagent is shown in green, analyte in blue and fluorescently-labeled detection reagent in yellow:
- Indirect antibody assay (A) or Bridging immunoassay (B) for studying PK of a therapeutic drug.
- Reversed antibody assay (C).
- Bridging immunoassay for PK of therapeutic antibody using anti-idiotypic antibodies as surrogate molecules for target protein (D).
- Sandwich immunoassay (E) for therapeutic protein not based on an antibody, or for generic preclinical PK assays.
See how a sandwich assay is performed in a Gyrolab Bioaffy CD:
The properties of the analyte and matrix are particularly relevant when planning assay development.
Properties of therapeutic antibodies, fusion proteins*, or protein as analyte:
- pI – this determines the choice of Rexxip® buffer**
- Hydrophobicity – this determines the choice of Rexxip buffer
- Size – Gyroslab technology has been used to analyze analytes in the range 3 kD – 1000 kD. Analytes smaller than 3 kD may require competitive assays.
*e.g the Fc portion of human IgG fused to a recombinant version of the native receptor for a cytokine.
**If pI > 8 then this may lead to carryover problems. In which case, use Rexxip AN or HN.
Gyrolab technology has been used to measure analytes in the following matrices – serum, synovial fluid, plasma, tears, urine, whole blood, sputum, vitreous humor. Determining the Minimum Required Dilution (MRD) in the matrix is a very important initial step in assay development and optimization.
If you already have a plate-based assay then these reagents can be a good starting point for developing a Gyrolab assay.
Indirect antibody assay:
The detection antibody is often a commercially available anti-human IgG (polyclonal or monoclonal).
The target is used as the capture molecule.
The target is used as both capture and detecting reagent.
The anti-idiotypic Ab is used as both capture and detecting reagent. This assay typically uses in-house reagents.
If the analyte is a non-antibody protein then assay performance may be improved by changing the orientation of capture and detection antibodies, and even the antibodies themselves.
Screen all possible combinations, for example when screening two Mabs and one Pab:
Capture-Detection antibodies – the ultimate goal
The overall aim is to identify a good reagent combination that can offer superior analyte affinity, and a lower degree of unspecific reactions, leading to lower background levels and higher sensitivity, with a broad dynamic range (e.g. 1 – 4000 ng/ml).
Additional tips when selecting capture-detection antibody pairs
Freeze-dried (lyophilized) antibody preparations are preferred since these usually are free from substances that may interfere with the labeling reaction (see Labeling). It is also much easier to adjust the antibody concentration when starting from lyophilized material.
Different Mab clones from the same supplier are worth trying in Mab-Mab combinations. You could also try switching roles within the same Mab-Mab pair.
Prioritize among the available antibodies according to the information in product sheets. We recommend that you order at least one Pab, and a number of Mabs for each analyte. If you already have an assay, then test the immunoreagents in all possible combinations.
Finding reagents on the Internet:
- Search for the purified analyte of interest.
- Search for Mabs and Pabs with specificity for the analyte of interest. It is often possible to find antibodies that are pre-conjugated with fluorophore, or pre-biotinylated. You can also contact vendors directly for more information, such as relative affinities and possible epitope overlap, to help with selection of antibodies for screening.
- Read the product sheets carefully. These often include useful (ELISA-based) information that can provide hints on which combinations to try in Gyrolab Bioaffy, e.g. suitability as capture reagent, antibody pairs recommended by the supplier, suitability for different sample types (plasma, serum…) etc.
The majority of assays can be run using Gyrolab Bioaffy 200, which with 112 structures can process the most samples. If sensitivity is critical then Gyrolab Bioaffy 1000 is the CD of choice since it can process 1000 nl of sample.
Changing from Bioaffy 200 to Gyrolab Bioaffy 1000 increases the sample volume 5-fold and boosts the response.
This example illustrates possible consumption in a pre-clinical assay with high sensitivity. Gyrolab Bioaffy 1000 still uses small volumes of sample and buffer, enabling repetitive sampling.
You can download the IFU ‘Bioaffy CD for Gyrolab’ here: Download
Rexxip buffers help you to save time in assay development and get the best performance from your immunoassay. The buffers preserve the integrity of the sample while it flows through the CD, enhance binding between analyte and reagent, and minimize non-specific binding in the capture column. The short contact times in Gyrolab assays minimize matrix effects. In PK assays the Minimum Required Dilution (MRD) may be as low as 1:2.
To get best assay performance, you should dilute reagents, samples, standards and quality control samples in Rexxip buffer before analysis.
Animal samples are diluted with Rexxip A, while human samples are diluted with Rexxip H, which includes blockers to remove the heterophilic antibodies present in these samples. The robustness of Gyros technology to matrix effects enables low dilution levels, which often means that the ‘–max’ versions of these buffers can be used.
All sample and standard dilutions should be:
- comparable with respect to Rexxip buffer properties
- optimized during assay development, with the aim of diluting as little as possible (low MRD) to maximize sensitivity
Choice of Rexxip buffer for PK assays
SS= Rexxip for standard and sample dilution
When the capture reagent is the target molecule then Rexxip A, AN or Rexxip F may be preferable to PBST.
IAA = Indirect Antibody Assay
SIA = Sandwich ImmunoAssay
BIA = Bridging ImmunoAssay
Ab = Antibody
TM = Target Molecule
AIA = Anti-Idiotypic Antibody
Note that fusion proteins, such as IgG plus an interleukin or receptor, can clearly have a wide range of properties. Rexxip A may be the best choice, although the fusion element affects properties so much that another Rexxip buffer may be more suitable.
The Gyrolab method determines how Gyrolab systems process the samples and reagents.
Gyrolab Bioaffy 200 CD
- If the analyte has a pI that is approximately neutral (7) and is not particularly hydrophobic then it may not demand a method with additional washing steps. You can begin with 200-3W-001-A.
- If the analyte has a pI > 8, use the 2-wash method 200-3W-002-A.
- Combine this method with a suitable Rexxip buffer (AN or HN)
- We also recommend that you use Gyrolab Wash Buffer pH 11: Download
Gyrolab Bioaffy 1000 CD
- If the analyte has a pI that is approximately neutral (7) and is not particularly hydrophobic then it may not demand a method with additional washing steps. You can begin with 1000-3W-001-A.
- If the analyte has a pI > 8, use the 2-wash method 1000-3W-006-A.
- Combine this method with a suitable Rexxip buffer (AN or HN)
- We also recommend that you use Gyrolab Wash Buffer pH 11: Download
You can find the methods in the Methods Database. This includes standard methods (marked *) and also a range of additional methods that may help in method optimization for specific applications.
Labeling is most effective if the reagents are at a concentration of at least 1 mg/ml and in a buffer free of stabilizing proteins (for example BSA), ammonium ions, primary amines, and sodium azide.
Please note that microscale labeling kits are available, e.g. Alexa Fluor® 647 Microscale Protein Labeling Kit (A30009) from Molecular Probes. You can find out more about their products here.
Biotin labeling can be scaled according to the manual, maintaining a relationship of between 1:12 (our recommendation) and 1:20 (kit recommendation) of target:biotinylation reagent.
You can find full details and protocols in Section B1: ‘Labelling of capture and detection reagents’ in the Gyrolab User Guide: Download Section B1
Screening may be very limited due to the nature of the assay, which may be based on predetermined reagents. Capture-detection antibody pairs are tested to determine which combination gives the best linearity, dynamic range, signal/background ratio and precision, especially at low analyte concentrations.
- The blanks should be low and this may require experimentation with dilutions to check for matrix effects.
- The Coefficient of Variation (CV) for the concentration should be according to regulatory recommendations.
- Poor precision requires troubleshooting, which may include reassessing the choice of Rexxip buffer, and possibly an alternative Gyrolab Method.
- If you suspect that there is a problem with carryover then you can run a Gyrolab Carryover Check: Download Instruction
- Look for good precision, high response levels, and low blanks.
- Compare the standard curves of different candidates regarding signal level, single/background (S/B), and the curve shape at low concentrations.
Gyrolab Viewer helps you to analyze the results of screening reagents and to identify outliers.
TM: Target molecule
Click on the image to enlarge.
An assay was designed to measure a humanized monoclonal IgG1 antibody using capture and detection reagents selected from Target Molecule, anti-hIgG1 and anti-drug antibodies. Column profiles were compared for three reagent combinations, which all generated distinct peaks at 125 ng/ml. However, anti-drug / Target Molecule was the only combination that did not generate signal in the blank, and was chosen for further studies.
The first ’samples’ should be QC samples – matrix spiked with known amounts of analyte at low, medium and high levels. These spiked samples are checked against the standard curve that is prepared using the same matrix. Any bias (relative error) could be due to matrix interference, sample pretreatment and/or choice of Rexxip buffer and insufficient sample dilution. The assay can then be tested on samples.
Once you have identified the best assay format and a suitable set of reagents it is important to determine the following:
Titrate reagents concentration:
- Column saturated with capture reagent
- Titration of detection reagent concentration
- It is very important that you run a Gyrolab Carryover Check to avoid the risk of problems later in assay development: Download the Instructions
- Rexxip buffers for dilution
- Curves in different matrix concentrations and in buffer as control (MRD)
Optimization also involves investigating the following in preparation for validation:
- Precision – systemic errors
- Accuracy – random errors
- Range of quantitation – Lower Limit Of Quantitation (LLOQ) and Upper Limit Of Quantitation (ULOQ)
- Dilutional Linearity – multiple dilutions of spiked samples
- Robustness – consistency when assay conditions are changed
- Ruggedness – consistency when routines are changed
Factors that improve assay performance
You can read more about these factors in the sections below.
Changing LIF detector settings can improve dynamic range
In this example, the PMT-settings 5 and 25 % result in a plateau at the high end of the standard curve, which indicates saturation of the detector. A PMT-setting of 1% is in this case sufficient to cover the desired dynamic range.
Titrating detection antibody improves precision and signal/background at low analyte concentrations
Reducing the concentration of the detection antibody can greatly increase performance by reducing background and increasing precision at low analyte concentrations.
An indirect assay was developed for the detection of a therapeutic monoclonal antibody, based on drug-target as capture reagent, drug as analyte, and anti-drug antibody as detection reagent. Titrating the detecting reagent indicated that 1.56 nM gave lower background and lower detection limit than the higher concentrations but was sufficiently high to allow measurements at the high end of the standard curve.
Diluting the sample (MRD) can improve linearity of the standard curve and spike recovery
It is very important that you determine the MRD early in the development and optimization of the assay since MRD has a major influence on assay performance and the success of final validation.
An indirect antibody assay was developed for an approved therapeutic monoclonal antibody drug molecule of the IgG1 subclass. The capture reagent was biotinylated recombinant target molecule that bound the drug molecule, which was detected using a mouse monoclonal antibody directed against human IgG.
To determine the optimal sample dilution factor, standard curves were prepared in different concentrations of pooled serum (40% to 1%) and diluted in Rexxip A. The optimal sample concentration was determined to be 5%.
Titrating the capture reagent
In most cases the capture column is saturated with reagent, which corresponds to a concentration of 700 nM. However, in bridging assays, for example, where target is used as both capture and detecting reagent, saturation may cause binding of both arms of the drug antibody to the captured reagent. In these cases, it is important that you titrate the capture antibody for best performance.
Bridging assay formats – titrating capture reagents with BSA
The performance of some assays can be improved by adding in biotinylated BSA to the biotinylated target molecule to reduce the density of the capture reagent on the solid-phase. This avoids false negatives due to binding of both arms of the drug antibody to the captured reagent.
Note that simply diluting the biotinylated capture reagent alone will not affect the local density of capture reagent – the capture reagent will still enrich at the top of the capture column, leaving the biotin-binding sites in the lower parts of the column unsaturated.
The example above shows the effect of titration on a bridging immunoassay for studying the PK of a therapeutic antibody drug. The capture reagent (drug target) was titrated using different proportions of biotinylated capture reagent and biotinylated BSA. Pure (100%) biotinylated capture reagent gave almost no signal, since the antibody analyte bound to capture reagent molecules with both arms. The assay was improved marginally with a mixture of 75% capture reagent and 25% BSA. Increasing to 95% BSA gave good dynamic range, and 99% BSA/1% capture reagent increased the sensitivity of the assay, whilst maintaining dynamic range.
Choosing the correct Rexxip buffer can improve precision
During assay development and optimization, it is critical that you choose the Rexxip buffer that gives the best assay performance.
Click on the image to enlarge
In this case, changing the Rexxip buffer greatly improved the precision of the assay.
Reversing the reagents may improve performance
Capture and detection reagents were reversed in a sandwich immunoassay for the detection of a therapeutic protein. The version where Antibody A and Antibody B were used as Capture and Detection reagents, respectively, gave the best linearity at low analyte concentrations.
Once the assay has been optimized, you should prepare an Assay Description that defines exactly how the assay should be performed.
You can download examples of assay descriptions here:
- Assay description for rat IgG in mouse serum: Download
- Assay description for human IgG in Cyno serum: Download
Performance parameters to be studied in pre-validation and validation should include at least:
- Standard curve form
- Precision and accuracy
- Range of quantification (dynamic range) – LLOQ & ULOQ
- Specificity and selectivity
- Dilutional linearity
- Robustness – inter-CD and inter-run precision
- Run acceptance criteria
Detailed descriptions of validation can be found in the following documents:
- Recommendations for the bioanalytical method validation of ligand-binding assays to support pharmacokinetic assessments of macromolecules. DeSilva B et al. Pharm Res. 2003 Nov;20(11):1885-900.: Pubmed link
- Fit-for-purpose method development and validation for successful biomarker measurement. Lee JW et al Pharm Res. 2006 Feb;23(2):312-28. Epub 2006 Jan 12.: Pubmed link
- Poster: Validation of a ligand binding assay. Eckersten A. et al : Download
ou can find out more about how Gyrolab® technology has been used to analyze pharmacokinetics in the scientific literature. Learn more >>