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Summary - introduction

  • 1 Introduction

  • What is the purpose of this course?
    To review the role of bioanalysis in the development and marketing of new drugs. Both technological aspects and regulatory aspects are covered.
  • What is the definition of analysis?
    The process of breaking a complex topic or substance into smaller parts to gain a better understanding of it. This can happen in all kind of academic fields (e.g. philosophy, mathematics, computer science, business, psychotherapy, statistics and chemistry)
  • What is chemical analysis?
    It is the process of identifying the (proportions) of components in a complex material. (e.g. in environmental analysis, food analysis, material analysis or in bioanalysis)
  • What is bioanalysis?
    It is the measurement of components of biological materials. This is done in clinical chemistry, doping control, forensic science, toxicology or drug research.
  • What is clinical chemistry?
    This is the science of analysing body fluids to diagnose a disease (laboratorium medicine)
    - it is typically performed by hospital laboratories,
    - it is highly standardized and there is a high level of automation

    e.g. creatinine, bilirubin, cholesterol, glucose, potassium, sodium etc.
  • What is therapeutic drug monitoring?
    It is the science of determining medication levels in blood to help establishing proper dosing (drugs with a narrow therapeutic range (e.g. gentamicin, valproic acid, cyclosporin, phenytoin etc.)
    This is typically performed by hospital laboratories as well.
    - It is also highly standardized and has a high level of automation as well.
  • What comprises bioanalysis in drug development?
    It is the science of analysing biological samples to support the development of new drugs.

    - This is typically performed by commercial laboratories.
    - It is not standardized and has a low level of automation.

    (e.g. any new drug in development)
  • What is qualitative bioanalysis?
    The question within this analysis is, what is present in the sample not the amount. The outcome will always be a specific name for example. So, it is the assessment of the identity of one or more of the components of a biological sample.
  • What is quantitative bioanalysis?
    Here the question is the amount of a specific compound in a biological sample. So the answer will always be a specific amount of concentration.
    This means that it is the assessment of the concentrations of one or more of the components of a biological sample.
  • What is regulated bioanalysis?
    This is bioanalysis that is performed according to scientific standards that are set by drug registration authorities. It is meant to ensure the reliability of the results.
    - It is necessary if you want to sell your drug in other countries for example.

    Key words: validation, quality control, precision, accuracy, selectivity and stability
  • What is the difference between regulated bioanalysis and good laboratory practice (GLP)?
    GLP is a set of procedural guidelines issued by governments that are meant to ensure the integrity of results. This means that it is more focused on documentation, traceability, organization etc.

    - It is required for studies with animals but typically not for studies with humans.
  • What is validation?
    It is a thorough demonstration of the reliability of a bioanalytical method for its intended purpose.

    questions here are:
    - Can you find back what you initial put into it. (the same concentration e.g.)

    - Validation takes place before application of the method.
  • What is quality control?
    It takes place during the application of the method. Its purpose is to control the reliability of the bioanalytical method.
  • What is an analyte?
    It's the compound that needs to be determined (either its concentration or its identity).
  • Into which 2 classes can we divide analytes?
    - biotic or endogeneous analytes: those analytes are naturally occuring in the sample, such as a biomarker. (not easy for a blank)
    - Xenobiotic or exogeneous analytes: are not naturally occurring in the sample (e.g. drugs)
  • What is a matrix?
    This is the type of biological sample that needs to be analysed. So, the material the analyte is in.
  • What kind of matrixes can we have?
    fluid: blood, plasma, serum, urine, saliva, CSF
    Tissue: brain, heart, liver, kidney, skin, muscle
  • What are the most important matrices?
    - plasma: blood without blood cells.
    - Serum: blood without blood cells and without clotting factors.
  • What is a blank matrix?
    This is an aliquot of the relevant matrix which does not contain the analytes of interest.
  • Which 2 types of matrices do we have?
    - authentic matrix: this is the actual matrix itself. (e.g. for xenobiotics obtained from undosed subjects)
    - proxy or surrogate matrix: This is an alternative matrix, preferably with the identical properties.  (there are 2 options for a blank matrix for endogenous analytes, remove the endogenous compound from the matrix or prepare the blank yourself)
  • What is pharmacokinetics?
    It is the fate of an administered drug in the body.
    So what the body does to the drug.
  • What is pharmacodynamics?
    It is what the drug does to the body. e.g. a change in blood pressure, cognitive test or hormone levels.

    - not necessarily concentrations of molecules, but if so then typically endogenous compounds.
  • What are small molecules?
    - molecules with a molecular mass below ca. 2000 Da
    - can usally be chemically produced.
    - can be administered orally
    - majority of registered drugs
    e.g. ibuprofen
  • What are large molecules?
    - molecules with a molecular mass above ca. 10000 Da.
    - typically proteins.
    - cannot be chemically produced.
    - cannot be administered orally, -> broken down by enzymes.
    - Prepared by microorganisms
    - There is a rapidly increasing interest in these molecules.
    e.g. trastuzumab
  • What are the intermediate molecules?
    - mass between 2000 and 10000 Da.
    - typically peptides
    - can be chemically synthetized but not be administered orally.
    e.g. calcitonin
  • What is a callibration curve?
    With this, the unknown concentration of an analyte can be calculated.
    - There is a set of calibrators (samples with ascending known concentrations of the analyte in the matrix.
    - It is a plot of how the analytical response changes with the concentration of the analyte.
    - it is necessary to correct for changes in the instruments that are used.
  • What are typical callibration curves?
    - for LC-MS for example: a linear line.
    - for binding assays or ELISA: a sigmoidal curve.
  • What is a reference standard?
    This is a standard taken into the assay that is not produced in your laboratory. This means that it ensures a high consistency.
    - They produce honest results.

    In fact it is the compound that is used  to prepare the stock solution for a callibration curve.
  • How are calibrators prepared?
    They are prepared (spiked) by adding small volumes of a standard solution of the analyte to an anliquot of blank matrix.

    - a stock solution is prepared first by dissolving the reference standard in a suitable (water- miscible) solvent (often methanol or DMSO)
  • The proportion of non-matrix in the spiked calibrators should not exceed 5%.
  • What is an internal standard?
    - many responses are often normalized by the use of an internal standard. This is necessary to correct for fluctuations in temperature etc.

    - it is a chemical substance that is added in a constant amount to all samples, to compensate for variability in the analytical procedure.

    - It should behave similarly to the analyte but generate a response that can be distinguished from that of the analyte. (eg. ibuprofen and ketoprofen)

    - next step is that the the peak area of the samples is divided by the peak area of the internal standard. In this way less variability is obtained for constant measurements.
  • What are quantification limits?
    These limits are the highes and the lowest analyte concentration that can be determined with the required accuracy and precision.

    - It is typically determined on the basis of expected concentrations.

    LLOQ: lower limit of quantification
    ULOQ: upper limit of quantification

    The interval between the LLOQ and the ULOQ is called the concentration range.
  • The drug and the dose are defining the concentration range
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So, what is flow cytometry?
- it can use fresh whole blood
... frozen cells
... living cells
it studies the phenotypic characteristics and the functionality of cells.  

cells are analyzed in a fluid stream
What is the purpose of this lecture?
To review the analytical methodology and applications of flow cytometry in the development of new drugs.
What is the neutralizing capacity?
It is the fact if the ADA can neutralize the effect of the drug. This needs to be tested into a biological system (e.g. iLite)
e.g. build in the luciferase gene into the DNA of cells. 
if a certain antibody acts on the receptor and the whole cascade will be activated ending up into transcription of the gene of interest, the luciferase gene will be transcribed as well and as a result light will be produced. 
If the antibody is not active anymore, it cannot bind anymore to the receptor and no light will be produced. 

To test this, you treat with very high concentrations of drug until it becomes neutralizing.
What are other parameters that you need to take into account regarding to sample storage?
- bench-top (how long can you keep your sample on the bench before antibodies are gone? In principle antibodies are really stable. 
- freeze/thaw
- short term/ long term
- The matrix effect (patient matrix)
1) you might have negative individuals
2) You can overcome this by spiking at a low and a high QC level for all samples. 
3) this can be screened and confirmed.
What about precision?
= reproducibility
- 2 you need 2 control samples spiked at the low and the high level. 
- the low control is just above the cut point
- The high control is at the upper end of the linear part of the curve. 
- 6 runs can be done for a (relative response)
- a standard curve can be used (semi-quantitative)

- The QC's need to be always positive during screening
- The QC's need to be always confirmed during confirmation
- And a precision can be demonstrated with titration.
What is the case with drug tolerance?
Then you get a false negative screening. 
The question here is what is the concentration in ug/ml that still can give a positive response. 
If this concentration lies between 15-80 ug/ml then use the Low QC. This one is the most important one because this reflects low positive samples. 
- Here you use 2 different QC's. The highest QC contains a lot of ADA and therefore the signal will be high as well when the concentration is low (in a a competitive assay)
- the more drug you add the lower the signal will become.  

There is a certain cut-off value. is the response lower than this cut-off value, there is a drug tolerance.
How titration is performed?
- it is done with all the samples that are confirmed. 
- Here serial dilutions are performed and the ADA assay (same is with screening) is performed again with these dilutions. 
- The dilution that gives a negative signal is used to calculate the titer. 
- titer = log (dilution)
- The higher the titer the higher the amount of ADA. 
- pre-dose samples and post-dose samples  can be tested for evaluation.
How is confirmation performed?
- You use again the same samples from the naive matrices. 
- This time you add your drug as well and you perform a MSD e.g.
- The blank samples should not give a different result from the blank samples in the screening. 
- Here another cut-point is determined, the confirmation cut-point. 

- To your samples that scored positive during screening you add the extra amount of drug as well.  To your plate with labeled drug you add your sample. If the sample indeed contained the ADA, it will not be recognized anymore by the ELISA since it is blocked by the drug. Therefore the more ADA is present the lower the response. If the signal inhibition is more than the specificity cutpoint the positive value is confirmed. If not it is not confirmed.
How is screening performed?
- you have 50 individuals. You use their drug naive matrix.
- This will give a little response. 
- If you plot the frequency against the response, you have to determine a screening cut-off value with at least 5% false positives.   Everything on the left is negative and everything on the right is positive.  
- You will test your samples. 
- Every sample that is above the cutpoint is positive and every sample that is below will be negative.   

You need to take into account that your cutpoint might be too high or too low.
What is the strategy you use for immunogenicity?
- screening: Is the sample positive or negative?
- confirmation: Does the sample have a specific positive signal?
- titration:   How much specific is that positive signal?