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Ligand binding assays

29 important questions on Ligand binding assays

What is a biopharmaceutical?

It is a biological medical product manufactured in extracted from or synthesized in biological sources.

What kind of large molecules do we have?

- proteins/peptides (natural, recombinant, antibodies)
- carbohydrates
- nucleic acids/ oligonucleotides (DNA, RNA, siRNA)

What is the aim of analysis of large molecules?

determination of
- PK
- imunogenicity
- PD

What are the techniques that can be used for the analysis of large molecules?

- ELISA: Enzyme(-linked) Immuno (Sorbent) Assay
- ECLIA: Electrochemiluminescence immuno assay (MSD)
- ELLA: Simple plex
- Singlulex: single molecule counting
- FACS: Flow cytometry

What is the basis structure of an immunoassay?

The analyte is captured between 2 antibodies. One is connected to a solid matrix (96-wells plate for instance) and the other is labelled so that a signal can be caught.

What is the secondary antibody?

In case an antibody serves as an antigen a goat anti-mouse IgG is used as a capture tool.

What kind of enzyme conjugates are there?

-horseradish peroxidase (HRP)
- Alkaline phosphatase (AP)
- Beta-galactosidase

What is the difference between monoclonal antibodies and polyclonal antibodies?

monoclonal antibodies are monospecific antibodies made by identical immune cells which are clones of an unique parent cell. They bind to exactly the same epitope. The advantage of using these is the high affinity antibodies with availability of large quantities that can be continuous produced.

polyclonal antibodies (antiserum): are obtained from the serum of animals that were immunized with a specific antigen. The antibody pool obtained from serum is the result of many b-cell clones each secreting one specific antibody.
from the serum antibodies can be extracted that can be used in heterogeneous immunoassays.

What are the sandwich immunoassays?

1) the first antibody extracts the analyte from the sample.
2) second antibody (with a chemical label) identifies the presence of the analyte
3) The antigen is sandwiched between 2 antibodies
4) This type of assay measures the amount of analyte in the sample by looking at the amount of labeled antibody that binds to the analyte on the solid support.

How is the quantification carried out?

-based on a calibration curve.

What are the competitive binding immunoassays?

This is a quantitative method based on competition between analyte in sample and a fixed amount of labeled analyte for a limited number of antibody binding sites (equilibrium method)
- This indirectly measures the amount of analyte in the sample by looking at the amount of labeled analyte that it displaces from the antibody.

Here the calibration curve functions the other way around. (It starts high and ends up low)

Why PK analysis?

It helps to understand and predict clinical efficacy.

What is a study of efficacy?

The rate at which a drug action begins and the duration of the effect (formulation of optimum dosing)

What kind of PK assays are there available for biologics?

- immunoassays: structural epitopes
- LC-MS/MS assays: limited part of unique primary sequence
- receptor/target binding assays: PK on location
- bioactivity/potency assays: activity-based PK profile

What are the most common methods for PK?

- immunoassays (ELISA, ECLIA)
- chromatography assays (HPLC, LC-MS/MS)

There is more shift from immunoassays towards LC-MS/MS
LC-MS/MS for biologics may be more precise but it is not always sensitive.

What are the plusses and negatives of ligand binding assays?

Plus:
- sensitive
- specific for structural conformation
- combination of epitope recognition
- idiotype specific (antibody drugs)
- specific assays for total, target-bound or free analyte

negative:
- relative variability/imprecision (no internal standard)
- dependent on availability (and changes) of critical reagents
- sensitive to structural changes
- influence of anti-drug antibodies
- time consuming assay optimization

What are the strengths and limitations of LC-MS/MS?

plus:
- quantitative assays
- good precision (use of internal standards)
- relatively fast assay optimization

negative:
- sensitivity
- focused on primary structure and signature peptides digestion
- limited association between concentration and activity
- sensitive to biotransformation of the parent drug
- analysis of free drug only
- production of internal standard.

How an immunoassay is developed? (required information)

First you need the required information.
- from preclinical studies you know a certain cmax. Then you could ask yourself if a dilution step is necessary or not.
- Determine the LLOQ next to the ULOQ to make sure that also the samples from the lowest dose cohort can be measured.
- You need to ask yourself do you need to transfer from a method or development? e.g. species transfer or a matrix transfer.

How an immunoassay is developed? (acquire reagents)

- It takes months to produce monoclonal antibodies.
- you need to get all the other materials.

How an immunoassay is developed? (Assay "proof of concept" experiments)

Here the proof is given, but it is not optimized and validated yet.

What do you need to take into account if you want to optimize your assay?

conditions like:
- type of ELISA plate
- coating concentration
- blocking buffer
- composition of assay matrix
- incubation time
- sample diluent
- wash buffer
- concentration of detection antibody
- incubation time
- define the expected assay range
- linearity of dilution
- matrix issues (this occurs in patients and not in healthy patients usually)
- perform a pre-validation (5 different dilutions, 6 times in triplicates, 10 individual matrices, stock stability)

How the assay is validated?

It is essential to know if the assay fits for the purpose, therefore issues to consider are:
- purpose (it's the purpose which defines the choice of technique, degree of validation claim of GLP etc)
- degree of validation (a limited validation is needed in case of exploratory biomarkers)
- regulatory requirement for validation
- GLP

What is the difference between partial/limited validation and complete validation?

partial/limited validation: precision and accuracy: 3 runs in 3-fold at 5 levels. Next to this selectivity needs to be validated.

complete validation: precision and accuracy: 6 runs in 3-fold at 5 levels. Next to this also the dilutional linearity, prozone, selectivity, and stability needs to be validated.

How do you validate precision and accuracy?

5 different concentrations which you test in triplicates 6 times. Then you calculate back with your calibration curve if you can find back what you put into it. (does it correspond with the calibration curve?)

How do you validate the dilutional linearity and the prozone?

You have several dilutions which you will test and back calculate.

How do you validate selectivity?

You have 10 individuals. (10 different matrices) with a known amount of the compound. See if the signal will be different for lipemic, healthy, diseased and hemolytic samples.

How do you validate the stability?

By multiple freezing and thawing. Or by bench-top stability.
long term frozen storage stability (usually until 1 year)

Where are the regulatory requirements coming from?

FDA guidance for industry: bioanalytical method validation
- EMA guideline on bionanalytical method validation

What are the GLP objectives?

- promote the quality and validity of generated test data
- protect human health and the environment
- facilitate data recognition (animal welfare, harmonization, time and resource efficiency, avoidance of duplicative testing)
GLP is no guideline for scientific quality!

The question on the page originate from the summary of the following study material:

introduction

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