Bioanalysis is the quantitative measurement of drugs and metabolites in biological systems. It plays an important role in drug discovery and development from lead optimization through clinical trials. Liquid chromatography coupled with tandem mass spectrometry is a key technique used in bioanalysis due to its high sensitivity and specificity. Metabolic stability screening and toxicokinetics studies incorporating bioanalysis help evaluate candidate drugs' pharmacokinetic properties and safety.

1. Dr. Manoj Ramesh Kumbhare
Professor and H.O.D.
Department of Pharmaceutical Chemistry,
SMBT College of Pharmacy, Dhamangaon Tal-Igatpuri,
Dist. Nashik (M.S.) India
Mob-09850232594 mrkumbhare@rediffmail.com
Kathmandu University, Dhulikhel 45200
Significance of Bioanalysis in drug discovery and
development
“Significance of Bioanalysis in drug discovery and development”
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2. Contents
 What is bioanalysis ?
 Overview of Drug discovery process
 Combinatorial Chemistry
 Automation High- throughput screening LC-MS
 Drug discovery – Lead optimization
 Screening in-vivo & in-vitro for pharmacokinetic properties
 Preclinical ADME and metabolite identification and characterization
 Therapeutic drug monitoring
 Bioanalytical test in marketing services
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3. Bioanalysis is a sub-discipline of analytical chemistry covering the quantitative measurement
of xenobiotics (drugs and their metabolites, and biological molecules in unnatural locations or
concentrations) and biotics (macromolecules, proteins, DNA, large molecule drugs,
metabolites) in biological systems.
What is bioanalysis
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4. Overview of Drug discovery process
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5. Drug discovery process
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 Step 1: Discovery and Development.
 Step 2: Preclinical Research.
 Step 3: Clinical Research.
 Step 4: FDA Drug Review.
 Step 5: FDA Post-Market Drug Safety Monitoring
Modern drug discovery involves the identification of screening hits, medicinal
chemistry and optimization of those hits to increase the affinity, selectivity (to
reduce the potential of side effects), efficacy/potency, metabolic stability (to
increase the half-life), and oral bioavailability.

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7. Preclinical development
 Preclinical studies or nonclinical studies: Drug safety data are collected, typically
in laboratory animals.
 Goals: To determine a starting, safe dose for first-in-human study and assess
potential toxicity of the product, which typically include new medical
devices, prescription drugs, and diagnostics.
 Statistics: To illustrate the risks in preclinical research.
 Average, only one in every 5,000 compounds that enters drug discovery to the
stage of preclinical development becomes an approved drug.
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8. Clinical development
 It includes drug discovery / product development, pre-clinical research
(microorganisms/animals) and clinical trials (on humans).
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9. “Significance of Bioanalysis in drug discovery and development”
Manufacturing Drugs
 There is a prompt need for a regulation, which in India can be found in the
Drugs and Cosmetics Act of 1940.
 This act explores the norms pertaining to the manufacture, sale and
distribution of drugs and cosmetics.
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10. Combinatorial chemistry
 Generation of a large array of structurally diverse
compounds, called a chemical library, through
systematic, repetitive and covalent linkage of
various “building blocks”.
 Chemical synthetic methods that make it possible
to prepare a large number (tens to thousands or
even millions) of compounds in a single process.
 These compound libraries can be made as
mixtures, sets of individual compounds or
chemical structures generated by computer
software.
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11. Combinatorial chemistry
 These compound libraries can be made as mixtures, sets of individual compounds
or chemical structures generated by computer software. Combinatorial chemistry
can be used for the synthesis of small molecules and for peptides.
 Strategies that allow identification of useful components of the libraries are also
part of combinatorial chemistry. The methods used in combinatorial chemistry are
applied outside chemistry, too.
 Even though combinatorial chemistry has been an essential part of early drug
discovery for more than two decades, so far only one de novo combinatorial
chemistry-synthesized chemical has been approved for clinical use by FDA
(sorafenib, a multikinase inhibitor indicated for advanced renal cancer
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12. Automation: High-Throughput Screening
 Small-molecule drug discovery can be viewed as a challenging
multidimensional problem in which various characteristics of compounds —
including efficacy, pharmacokinetics and safety — need to be optimized in
parallel to provide drug candidates.
 Identify the approaches and technologies that could be implemented
robustly by medicinal chemists in the near future and to critically analyse the
opportunities and challenges for their more widespread application.
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13. Automation: High-Throughput Screening
 HTS uses automated liquid handling devices, multi-mode readers and
other devices paired with data processing and control software, to
perform biological or chemical tests at scale.
 HTS is the use of automated equipment to rapidly test thousands to
millions of samples for biological activity at the model organism, cellular,
pathway, or molecular level.
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14. Automation: LC-MS-MS
 Liquid chromatography coupled with tandem mass spectrometry (LC-MS-
MS) is widely used for highly selective and sensitive bioanalysis of small
molecules. However, large molecule bioanalysis presents challenges
including the need for extensive and complex sample preparation for LC-
MS-MS.
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15. Automation: LC-MS-MS
 It is the preferred method for the fast and sensitive quantitation of small
molecules, peptides, and proteins in complex matrices including plasma, blood,
urine, feces, and tissue.
 MS is more sensitive and significantly more specific than other LC detectors such
as UV-Vis, fluorescence, and refractive index routinely achieving nano- to
picogram-per-milliliter detection.
 LC/MS/MS can analyze compounds that lack a suitable chromophore and can
identify components in unresolved chromatographic peaks, reducing the need for
ideal chromatographic conditions.
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16. LC-MS-MS- Method for Bioanalysis
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17. Metabolic stability screening
 One of the most commonly performed in vitro ADME
assays during the lead generation and lead
optimization stage of drug discovery is metabolic
stability evaluation.
 Metabolic stability is typically assessed in liver
microsomes, which contain Phase I metabolizing
enzymes, mainly cytochrome P450 enzymes (CYPs).
The amount of parent drug metabolized by these CYPs
is determined by LC/MS/MS.
 The metabolic stability data are typically used to rank
order compounds for in vivo evaluation.
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18.  Practicing Toxicologist/Study Director in small molecule drug development.
 The importance of toxicokinetics (TK), and the bioanalysis that supports TK,
to nonclinical toxicity testing and the integration of TK and bioanalysis into
the design, conduct, and evaluation of toxicology studies.
 TK is an extension of pharmacokinetics (PK) in animal safety studies.
 Before scheduling a toxicity-TK study and finalizing the protocol, the Study
Director must check the status of the bioanalytical method because it can
be a rate limiting step in the study schedule.
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Metabolic stability screening: Toxicity testing

19. TK applies the principles and ideas to hypothetical studies using a -
 Systemic toxicology studies,
 Single-dose studies, Repeated-dose studies,
 Reproductive toxicology studies, Male fertility studies, Female reproduction
 Local toxicology studies,
 Hypersensitivity studies,
 Genotoxicity studies,
 Carcinogenicity studies, etc.
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Metabolic stability screening: Toxicity testing

20. Lead optimization
 Lead optimization is a critical process
that culminates in the identification
of a preclinical candidate.
 The most promising hit series, once
they are identified through hit-to-
lead efforts, advance into the lead
optimization stage of drug discovery.
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21. In Vitro and In Vivo Assessment of ADME and PK Properties
 Assessment of the pharmacological properties of small molecule
 Selection or identification of a chemical lead
 Lead optimization to elucidate the Structure-Activity Relationships (SAR) &
Structure Property Relationships (SPR)
 Investigational New Drug (IND)-enabling studies
 ADME of compounds affects their ultimate pharmacokinetics (PK)
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22. Preclinical ADME and metabolite identification and characterization
 Metabolite identification plays a critical
role in the preclinical and clinical phases
during drug development
 It can contribute to efficacy, toxicity, and
drug-drug interaction
 Importance of identifying metabolites in
the of drug development
 Role of metabolites in
pharmacokinetics/pharmacodynamics
(PK/PD) analysis
 Exposure of metabolites contribute to drug
toxicity
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23. Preclinical ADME and metabolite identification and characterization
 It plays a critical role in the phases during drug development.
 Drug metabolites can contribute to efficacy, toxicity, and drug-drug interaction.
 Thus, the correct identification of metabolites is essential to understand the behavior of
drugs in humans.
 Drug administration authorities (e.g., FDA, EMA, and NMPA) emphasize evaluating the
safety of human metabolites with exposure higher than 10% of the total drug related
components.
 Scenarios include the role of metabolites in pharmacokinetics/pharmacodynamics
(PK/PD) analysis, disproportional exposure of metabolites that contribute to drug
toxicity, changes in metabolite exposure in renal-impaired patients, covalent tyrosine
kinase inhibitors (anticancer drugs), and metabolite identification of drug candidates
from natural medicines.
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24. Prediction and analysis of ADMET properties
 For safety and effective permeability, drugs are required to exhibit ideal absorption,
distribution, metabolism, excretion and toxicity (ADMET) properties.
 Simplified Molecular Input Line Entry System (SMILES) representation of drugs to
predict their ADMET properties.
 By feeding molecular descriptors (as global features) to traditional machine learning
models, ADMET properties of drug molecules can be predicted with an accuracy.
 Two-dimensional descriptors can better represent absorption, distribution and
excretion properties than the fingerprints widely used.
 However, they fail to distinguish metabolism and toxicity properties.
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25. Prediction and analysis of ADMET properties
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26. Prediction and analysis of ADMET properties
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27. Therapeutic drug monitoring (TDM)
 TDM measures the amount of certain
drugs in blood
 It signifies the amount of drug is safe and
effective
 TDM is useful only for drugs that have a
poor correlation between dose and clinical
effect (high pharmacokinetic variability)
 Clearly, if dose alone is a good predictor of
pharmacological effect, then measuring
the plasma concentration has little to
contribute.
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28. Therapeutic drug monitoring (TDM)
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Process for reaching dose decisions with TDM

29.  As a tribute to the 10-year anniversary of the journal-
 It gives a high-level overview of the most important developments and trends
in the field, which have appeared on the pages of Bioanalysis in the past
decade.
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“Significance of Bioanalysis in drug discovery and development”
Summary for Bioanalysis journal

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 The global bioanalytical testing services market size was estimated at USD 3.64 billion in 2021 and
is anticipated to register a compound annual growth rate (CAGR) of 8.6% over the forecast period.
 The bioanalytical testing method is used for the quantitative determination of drugs or
metabolites in biological matrices, such as serum, blood, plasma, tissue, urine, and skin samples.
Bionalytical testing services : US Market

31. Global market has been segmented
into
 (ADME),
 Pharmacokinetics (PK),
 Pharmacodynamics (PD),
 bioavailability,
 bio-equivalence, etc
 The bioavailability segment
accounted for the highest share
of more than 18.5% of the
global revenue in 2021 owing to
the high demand for such
services in generic drug
development.
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“Significance of Bioanalysis in drug discovery and development”
Bionalytical testing services: Global Market

32. Bioanalytical testing market services
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 The global bioanalytical testing services market size was estimated at USD 3.64 billion in 2021 and is
expected to reach USD 3.9 billion in 2022.
 The global bioanalytical testing services market is expected to grow at a compound annual growth
rate of 8.6% from 2022 to 2030 to reach USD 7.65 billion by 2030.
 North America dominated the bioanalytical testing services market with a share of 46.2% in 2021.
This is attributable to the fact that it is one of the top manufacturing hubs of highly reliable, complex,
and high-end pharmaceuticals.
 Some key players operating in the bioanalytical testing services market include PPD; ICON plc;
Covance Inc.; LabCorp; Charles River Laboratories International, Inc.; inVentiv Health; SGS SA;
Toxikon, Inc.; and Intertek group.
 Key factors that are driving the bioanalytical testing services market growth include the increasing
frequency of outsourcing R&D activities by the major pharmaceutical companies to focus on their
core competencies.

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 HR-MS is increasingly being introduced in laboratories for quantitative bioanalysis and with these
instruments becoming more sensitive, robust and affordable, more scientists will likely try to move
from digestion of a protein and indirect quantification via a surrogate peptide to direct analysis of
the intact analyte.
 In this respect, technical advances are needed when it comes to stationary phases for LC and it is
hoped that the increased interest in intact protein analysis will stimulate the development of
materials with improved separation properties for protein forms.
 Similarly, user-friendly software solutions for robust deconvolution of the highly complex mass
spectra, in compliance with regulatory expectations.
 Finally, protein bioanalysis is a truly interdisciplinary science and much of its success is due to the
collaboration of researchers with very different backgrounds.
 It is our hope and expectation that the bioanalytical community will continue to work together to
help advance our knowledge in this fascinating field.
Bioanalysis Future perspective

34. Challenges in bioanalysis
 The development of bioanalytical methods has become more and more challenging over the
past years due to very demanding requirements in terms of method reliability, sensitivity,
speed of analysis and sample throughput.
 LC–MS/MS has established itself as a method of choice for routine analysis of biological
materials. A development of such method consists of several steps including sample
preparation and clean-up step, efficient chromatographic separation, sensitive and selective
detection of analytes in complex matrices, a choice of convenient data processing and
calibration approach and finally method validation.
 Each of these steps has its own constraints and challenges.
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35. Challenges in bioanalysis
 Structural, bioanalytical, characterization, and quality control studies are critical for
successful drug development.
 These studies must be as accurate, sensitive, and selective as possible, and liquid
chromatography coupled to tandem mass spectrometry (LC–MS–MS) has been the
technique of choice for many areas of small molecule analysis for the past 30 years.
 During that time, rapid improvements in analytical technologies have supported the
development of more sensitive and robust methods. However, the pharma and biopharma
industry continues to need more powerful instruments and more diverse methods,
particularly as therapeutics have expanded to include large molecules.
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36. Conclusion
 Lack of suitable analysis of pharmacokinetic data
 Bioanalysis emerges as an significant means throughout drug discovery and
development
 The highest impact of bioanalysis measure of the drug or its metabolites for the
study of pharmacokinetics, toxicokinetics, bioequivalence and exposure-response
like pharmacokinetic/ pharmacodynamic studies.
 The global bioanalytical testing services market is expected to grow at a compound
annual growth rate of 8.6% from 2022 to 2030 to reach USD 7.65 billion by 2030.
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