The Basics of Bioanalysis

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B.TARAK PRASADAssistant Manager-BioanalyticalACT
IMUS BIOSCIENCES PVT LTD

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

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Introduction

• Bioanalytics is an essential tool in drug
  discovery and development for determining the
  concentration of drugs and their metabolites as
  well as various pharmacodynamic biomarkers in
  biological fluids.
• In these analyses, scientists use developed and
  validated methods to quantitatively detect
  analytes and metabolites within biological
  matrices such as blood, serum, plasma, urine,
  etc.
• A critical component of any bioanalytical program
  includes bioanalytical method validation,
  ensuring quantitative results demonstrate
  accuracy, precision, selectivity, and stability
  so the accuracy of sample analysis results can be
  justified.

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Table of Content

• Overview Definition
• Bioanalytical Method Development
• Bioanalytical Method Validation
• Sample Analysis
• Reporting
• Regulatory Guidelines

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Overview Definition

• Bioanalysis isnt 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.
• Its imperative to have the support and expertise
  of veterans, such as the team at Actimus BioLab,
  when the complexities of bioanalysis get
  cumbersome.
• Our scientists help navigate bioanalytical method
  validation following method development focused
  on the appropriate detection range for your
  assay, 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.

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schematic diagram of bioanalytical workflow
Background of analyte, available dosage forms
etc..
Literature Review
Physical state of analyte, Pka, solubility,
chemical properties and structure of analyte etc..
Physical chemical properties of analyte
On the basis of Cmax data dose of drug, range
of quantification is selected
Setting the range of quantification
On the basis of nature and chemical properties of
analyte, analytical instrument is selected
Selection of analytical instrument
Mass spectrophotometer
HPLC
Optimisation of mass parameters
Selection of mobile phase internal standard
Optimisation of chromatographic conditions
Experiment using final parameter of method e.g.
precision and accuracy batches , selectivity ,
sensitivity etc. as per requirement
Optimisation of Extraction procedure
Evalution of analyte recovery and stability
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Bioanalytical Method Development

• Steps involved in the Method Development
• Method selection and information of Sample
• Initial method conditions
• Processing the analytical method in aqueous
  standards
• Development and optimization of sample processing
  method
• Checking the analytical method in biological
  matrix
• Pre-validation

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1.Method selection and information of Sample

• Literature survey shall be conducted to have
  first hand information on drug profile and its
  pharmacokinetic properties.
• Collection of physicochemical properties of the
  analytes and the related compounds are essential
  for the development of the analytical method.
• Based on the drugs physicochemical properties
  such as molecular size, shape, structure,
  functional groups, polarity, partition
  coefficient, solubility, dissociation constant
  etc.,
• choose the internal standard having comparable
  molecular structure and physicochemical
  properties with respect to the analytes. Same
  molecule with different isotopes like deuterium,
  C13 and N15 will be a better alternative for
  internal standards.

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2. Selection of initial method conditions

• Setting the initial method conditions include
  diluent selection based on the solubility of the
  drug, drug metabolites and internal standard and
  compatibility with analytical method.
• The lowest concentration to be quantified shall
  be assessed using aqueous solutions during this
  phase.
• Run time and resolution between the peaks should
  be taken care during this phase

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3. Parameters to be optimized

• 1.Mass parameters
• 2.LC parameters
• Mode of separation
• Selection of stationary phase
• Selection of mobile phase

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Mass parameters

• DP (declustering potential) -The declustering
  potential (DP) is a voltage applied to the
  orifice that helps to prevent the ions from
  clustering together.
• EP (entrance potential) -The rods in Q0 do not
  act as mass filters but serve to guide and focus
  the ions into the mass spectrometer. It is here
  that the entrance potential (EP) is applied.
• CE (collision energy) -The collision energy (CE)
  refers to the rate of acceleration as the ions
  enter quadrupole 2 (Q2.). The ions undergo a
  thermal interaction with the collision gas and
  fragment.
• CXP (collision cell exit potential)-The Collision
  Cell Exit Potential (CXP) focuses and accelerates
  the ions out of Q2 and into Q3.
• CAD (collision gas) -Collisionally Activated
  Dissociation (CAD) is the process of colliding
  precursor ions (parent ions) with a neutral gas
  to break the molecule into fragment ions. The
  neutral gas used during this demonstration is
  nitrogen.
• IS (ionspray voltage)
• TEM (temperature of ion source)
• GS1 (nebulizing gas)
• GS2 (drying gas)

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LC parameters

• Mode of separation
• In reverse phase mode, the mobile phase is
  comparatively more polar than the stationary
  phase. For the separation of polar or moderately
  polar compounds, the suitable mode is reverse
  phase. The nature of the analyte is the primary
  factory in the selection of mode of separation.

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Selection of stationary phase

• Selection of the column is the first and the most
  important step in method development, because the
  column is the heart of separation process.
• The appropriate choice of separation column
  includes different approaches
• ? Column dimensions
• ? Nature of packing material
• ? Shape of the particles
• ? Size of the particles
• ? Surface area
• ? Pore volume
• ? End capping

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• The optimum length of the column required for a
  particular separation is dictated by the number
  of theoretical plates needed to give the desired
  resolution. If the column is too short, then the
  clearly the column will not have enough
  resolving power to achieve the separation and
  if it is too long, then analysis time is
  needlessely extended. The most common column
  lengths used in regular analytical HPLC are 10,
  12.5, 15 and 25 cm, with 15 cm columns being
  perhaps the most popular.
• Currently, most HPLC separation are carried out
  with 5µm diameter packing materials. Columns with
  5µm particle size give the best compromise of
  efficiency, reproducibility and reliability.
• As the particle size decreases the surface area
  for coating increases. Generally high specific
  surface area will increase the retention of
  solutes by increasing the capacity factor.
• Reverse phase mode of chromatography facilitates
  a wide range of columns like dimethylsilane (C2),
  butylsilane (C4), octyl silane (C8), octadecyl
  silane (C18), cyanopropyl (CN), nitro, amino etc.

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Selection of Mobile phase

• The primary objective in selection and
  optimization of mobile phase is to achieve
  optimum separation of all the individual
  impurities and degradants from each other and
  analyte peak.
• The following are the parameters to be considered
  during selection and optimization of mobile
  phase.
• ? Buffer
• ? pH of the buffer
• ? Mobile phase composition

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• Buffer - Buffer and its strength play an
  important role in deciding the peak symmetries
  and separations. The retention time depends on
  molar strength of buffer. Molar strength is
  proportional to retention time. In order to
  achieve better separation the strength of the
  buffer can be increased.
• pH of the buffer- pH plays an important role in
  achieving the chromatographic separation as it
  controls the elution properties by controlling
  the ionization characteristics. A different
  concentration of buffer was chosen to achieve
  required separations. It is important to maintain
  the pH of mobile phase in the range of 2.0 to 8.0
  as most of the columns does not withstand out of
  this range 12. As Siloxane linkages are cleaved
  below pH 2 and at above pH 8 silica dissolves.

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4.Checking the analytical method in aqueous
standards

• Before going to analyze a method in biological
  matrix, first check the analytical method in
  aqueous standards.
• Prepare aqueous calibration curve standards, at
  least with four concentrations, including the
  highest and lowest. Concentration of the highest
  standard shall be based on Cmax and lowest
  standard shall be tentatively fixed based on the
  preliminary studies. Make injections of each
  calibration curve standard and find the
  correlation coefficient. Correlation co-efficient
  (r) should not be less than 0.99.
• If required, adjust the mobile phase, mass
  spectral parameters (if applicable) and
  chromatographic conditions such as mobile phase
  constituents, buffer strength, ratio, pH, flow
  rate, column, column oven temperature etc., to
  get the clear resolution with required
  sensitivity.

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5. Development and optimization of sample
processing method

• When the instrumental method is concluded with
  aqueous standards, prepare matrix sample. Based
  on the literature survey data on analyte and
  internal standards physicochemical properties
  like structure, functional groups, pH, partition
  coefficient, dissociation constant, polarity and
  solubility, set and optimize the sample
  preparation technique like
• protein precipitation
• liquid-liquid extraction
• solid phase extraction

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Bioanalytical Method Validation

• Method validation is a part of GLP study and it
  is to ensure the quality of analytical method or
  it is a process of demonstrating that analytical
  method is suitable for its intended use. Method
  validation is applied to ensure the method relia
• A typical Full Bioanalytical Method Validation
  should include determination of
• 1. System suitability 2. Selectivity 3.
  Sensitivity 4. Precision and Accuracy i)
  Inter-Day Precision and Accuracy ii) Intra-Day
  Precision and Accuracy 5. Recovery 6. Calibration
  Curve 7. Matrix effect 8. Haemolyzed effect 9.
  Lipemic effect 10. Dilution integrity 11.
  Stability 12. Auto sampler carry over 13.
  Reinjection reproducibility 14. Ruggednessbility,
  quality and reproducibility.
• Acceptance Criteria of an Analytical Runs
• For Blank sample and Blank IS sample
• If any peak area is present at the
  retention time of analyte in Blank or Blank IS
  sample, its area response should be lt 20.00 of
  analyte area response of LLOQ standard. If any
  peak area is present at the retention time of
  Internal standard in Blank sample, its area
  response should be lt 5.00 of the IS area
  response of LLOQ standard.
• At least 75 of calibrators must pass 15 of the
  nominal concentration (20 at the LLOQ)
• RSD for LLOQ QC level Not More Than 20.00
• RSD for samples other than LLOQ QC Not More
  Than 15.00
• Accuracy for LLOQ QC level 80.00 to 120
• Accuracy for samples other than LLOQ QC 85.00
  to 115.00
• At least 67 of total QC samples and 50 at each
  concentration level should comply with above
  mentioned criteria of Accuracy.

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Analysis of Study Samples

• Analytical Run (Batch)-Samples which are
  subsequently processed without interruption in
  time by the same analyst with the same reagents
  under homogeneous conditions.
• Acceptance Criteria
• Reanalysis of Samples
• Integration (Peak integration and re-integration
  described in an SOP)
• Incurred Samples Reanalysis (ISR) - verifies the
  reliability of the reported study sample analyte
  concentrations -Two concentration levels around
  the expected Cmax in the elimination phase
• Analytical Report

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Reanalysis of Samples

• Reanalysis of Samples Possible reasons defined
  in the protocol or SOP
• Examples
• Batch rejected (acceptance criteria for
  calibrators/QCs not met)
• IS response in study sample significantly
  different from calibrators/QCs
• Improper sample injection, malfunctioning
  equipment
• Sample concentration above ULLQ
• Quantifiable concentrations in pre-dose samples
• Poor chromatography
• Not acceptable in BE studies
• Pharmacokinetic reasons (irregular profile)

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• Reporting
• 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.
• Regulatory Guidelines
• With our team of dedicated scientists, we develop
  and optimize bioanalysis methods on which we then
  perform assay validation as per FDA guidelines
  using Good Laboratory Practices (GLP), regulated
  under US 21CFR part 58 and the bioanalytical
  method validation guidance for industry.

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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
  Actimus BioLab include testing drug and
  metabolite exposures, bioavailability and
  bioequivalence in various studies conducted
  during preclinical (animal) and clinical (human
  volunteers) phases of drug development.
• For expert support in developing and
  validating bioanalytical methods for your drug
  development and research, contact us at Actimus
  BioLab about our bioanalytical laboratory
  services. We seek contentment in serving our
  clients towards their noble mission of bettering
  the current standards of treatments.

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Thank you