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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5397_Библиотеки_им_академика_М_И_Перельмана
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Abbreviations
APIs Active pharmaceutical ingredients
AQbD Analytical quality by design
CE Capillary electrophoresis
CMPs Conjugated microporous polymers
ELISA Enzyme-linked immunosorbent assay
GC Gas chromatography
HPLC High-performance liquid chromatography
MOFs Metal-organic frameworks
MS Mass spectrometry
PAT Process analytical technology
PCR Polymerase chain reaction
QbD Quality by design
QC Quality control
TLC Thin-layer chromatography
WB Western blotting
4.1 Introduction
M. Aslam et al.
Pharmaceutical chemical analysis is an essential area of study since medications
have a signicant impact on the well-being of people and animals. The pharmaceutical sector, biomedical research facilities, hospitals, and other industries have a
signicant demand for dependable, accurate, precise, and delicate analytical procedures. Analytical procedures are commonly used in the production of novel pharmaceuticals, degradation products, contaminants, biomarker determination,
pharmacokinetic investigations, and diagnosis or analysis of therapeutic drugs in
biological samples, among other things (Fanali et al. 2023; Klatte et al. 2017).
Several analytical methods, which include supercritical uid chromatography (SFC)
(Gros etal. 2023; Cobo-Golpe etal. 2022), gas chromatography, high-performance
liquid chromatography (HPLC) (Lo Faro etal. 2023; Lin etal. 2021; Zou et al.
2023; Aspromonte et al. 2019), and microuidic techniques such as nano-liquid
chromatography (nano-LC) (D’Orazio etal. 2019), capillary electrophoresis (CE)
(Woźniakiewicz and Wietecha-Posłuszny 2023; Krait et al. 2021), and capillary
electrochromatography (CEC) (Fanali etal. 2021; Chankvetadze etal. 2003), are
now utilized in pharmaceutical companies and healthcare analysis. Bioanalysis is
employed in the pharmaceutical sector throughout the initial phases of the discovery
and development of drugs to give evidence to determine the metabolic and pharmacokinetic destiny of drug prospects. Bioanalysis is still employed in the preclinical
and clinical stages of discovering drugs, and in certain circumstances, clinical therapeutic drug evaluation (Pandey etal. 2010). Parallel to this, the issue of pharmaceutical contamination has grown in prominence previously in the past. There are
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serious threats to human health and the ecosystem when there is persistent contamination from medicines in the water, food, and land in certain parts of the world
(Patel etal. 2019; Rehman etal. 2015). The preliminary treatment of samples before
analytical analysis is critical in the study of dietary, ecological, biological, and
medicinal substances. Effective separation and accurate detection of complex and
trace materials can be achieved using sample pretreatment methods (Wen et al.
2014). A number of substances exhibit excellent properties and potential use in
separating and analysis of various pharmaceutical samples. Due to their functional
diversity and porous structure, researchers have developed an interest in organic
molecule-based framework porous materials. Different types of porous organic
frameworks were documented, including conjugated microporous polymers
(CMPs), covalent organic frameworks (COFs), hyper-crosslinked polymers (HCPs),
metal organic frameworks (MOFs), and covalent triazine frameworks (CTFs)
(Zhang etal. 2018). Because of its label-free and real-time detecting process, ease
of use, and excellent detection, plasmonic-based nanosensors are among the most
powerful tools for detecting and quantifying low amounts of molecular analytes.
Plasmonic sensors are also useful for detecting a variety of other applications,
including biosensors, environmental monitoring, medicines, and food control
(Yesudasu etal. 2021; Tseng etal. 2017; Dissanayake etal. 2019; Muneer etal.
2020; Mauriz 2020).
For quantitative researchers, identifying and determining drugs, pollutants, compounds, and biomarkers in various matrices has become a challenging undertaking.
Therefore, developing analytical tools to evaluate drugs, their derivatives, and biomarkers in biological samples is always necessary.
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4.2 Importance ofBiotechnology inPharmaceutical
Product Analysis
Biotechnology is critical in pharmaceutical product analysis, contributing to product safety, efcacy, and quality control of drugs. Here are some key reasons why
biotechnology is relevant in this context.
4.2.1 Analytical Techniques
Biotechnology allows researchers access to cutting-edge analytical tools like
genomics, proteomics, and metabolomics, which allow them to thoroughly study
drug compounds at the molecular level. Pharmaceutical research has traditionally
had an important role in the advancement of pharmaceutical development, directed
by pharmacological and clinical research and supported by the eld of chemistry.
Active pharmaceutical ingredients (APIs) production and characterization, along
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with their evaluation to obtain provisional precautions and therapeutic efcacy
information, must be carried out before drug candidates can be identied for further
comprehensive research (Siddiqui 2017; Anon n.d.-a). Within the sector of pharmaceutical research, the analytical evaluation of large quantities of drug items, intermediary substances, medications, formulations of drugs, contaminants and
compounds from degradation, and biological specimens comprising pharmaceuticals and metabolites of them is essential.
Titrimetric methods are being shown to be useful in kinetic evaluations, which
can be then utilized for determining rates of reaction through the development of
functional group evaluation procedures (Rahman et al. 2005; Basavaiah and
Prameela 2003). Titrimetry was formerly used to estimate pharmacological product
breakdown as well as drug concentrations (Matei etal. 2008). Different analytical
techniques, including titrimetric, chromatographic, spectroscopic, and electrochemical techniques, are commonly employed to determine the quality and quantity of
the drug product as well as the materials utilized during its synthesis (Beccaria and
Cabooter 2020). Three main groups comprise the several techniques used to assess
antioxidant capacity: spectrometry, electrochemical tests, and chromatography
(Munteanu and Apetrei 2021).
M. Aslam et al.
4.2.2 Quality Control
Biotechnology is instrumental in establishing rigorous quality control standards for
pharmaceutical products. Quality control (QC) measures are essential in a pharmaceutical manufacturing control plan to ensure product quality (Deidda etal. 2018).
Companies submit QC procedures to regulatory agencies in each country where a
pharmaceutical item is supplied to assist in market licencing procedures (Åsberg
etal. 2016). The quality by testing concept, commonly known as trial-and-error, is
a conventional strategy for developing analytical methods. Such a technique surely
does not facilitate additional modications that may be required due to a lack of
understanding of the possible effect on method performance (Rozet etal. 2013).
Capillary or chip-based electrophoretic techniques used for separation have essentially superseded traditional slab-gel SDS-PAGE methods utilized for biological
impurity and purity testing. In QC labs, image capillary isoelectric focusing techniques have mostly superseded gel IEF techniques (Rogers etal. 2018). The quality
data of TCM preparations is restricted by the capabilities of current quality traceability technology, making them unsuitable for use in production or sales. One of
the key methods for quality assurance and evaluation of herbal medicines is the use
of chemical ngerprints, sometimes referred to as chemical markers (Liu et al.
2018). The denitions of Analytical Quality by Design (AQbD) and Quality by
Design (QbD) are comparable when it comes to manufacturing processes. As a
result, AQbD also consists of four primary phases: (a) calculating the necessary
analytical method performances (ATP), (b) identifying pertinent procedure parameters and quality standards (the screening stage), (c) dening the analytical DS (the
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Fig. 4.1 Comparison of Quality by Testing (QbT) approach (Left) and Quality by Design (QbD)
approach (Right) (Dispas etal. 2018)
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robust optimization) allows for method optimization and risk assessment, (d) application of a control technique to ensure the method is improved continuously
(Fig.4.1) (Dispas etal. 2018).
In addition to the digital revolution, modelling approaches for process understanding, prediction, and consistency have received more attention as a result of the
FDA and EMA’s support of Quality by Design (QbD) and Process Analytical
Technology (PAT) concepts. As a result, these paradigms allowed for a big deal of
exibility for selection and use cases of the quantitative methodologies (Narayanan
etal. 2020). The most effective method for quality control in routine procedures as
well as research projects is metabolomics. To track the metabolites present, untargeted metabolomic ngerprint can be utilized and statistical control charts can be
generated by multivariate statistical analysis to conrm that the composition is
maintained throughout the production process (Mattoli etal. 2023).
4.2.3 Pharmacokinetics andPharmacodynamics
Biotechnology aids in studying the pharmacokinetics (the manner in which drugs
are absorbed, metabolized, distributed, and excreted) and pharmacodynamics (manner in which drugs show interactions within the body) of pharmaceuticals
(Chowdhury et al. 2021). Studies on the metabolism and pharmacokinetics of
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potential medications have become a signicant aspect of drug discovery and development programmes, and they typically begin concurrently with biological activity
screening (Üëá etal. 2011). Throughout the early learning period of preclinical drug
discovery, a comprehensive and rigorous PK/PD programme might serve as an
intermediate connecting preclinical development and drug discovery (Meibohm and
Derendorf 2002). The link involving a drug’s dose and its biological effects is examined by pharmacodynamic analysis. To ascertain the drug’s potency, maximum
response, and minimum effective concentration, concentration-effect curves may
need to be established (Crommelin etal. 2020). By combining the two, pharmacokinetic/pharmacodynamic modelling forecasts the drug’s impact according to its
pharmacokinetic prole. Optimizing dosage tactics can benet from this (Sarkar
et al. 2022). There are multiple potential benets to developing and applying
mechanism- based PK/PD modelling in different animal populations: (a) It characterizes the dosage concentration effectiveness relation accurately and thoroughly,
(b) it provides effectiveness and intrinsic performance measurements using concentrations instead of doses, (c) it enables research into the role of inuencing physiological and pathological factors, in addition to resistance in conditions (Felmlee
etal. 2012). The genetic variations of drug-metabolizing enzymes and transporters
have a signicant impact on the pharmacokinetics of natural products. This has
garnered increasing attention in recent years, but despite the wide range and widespread applications of natural products worldwide, there is still a deciency of
knowledge in this area (Rao etal. 2019). The volume of distribution (Vd) and clearance (CL) are the two most signicant PK parameters for pharmaceuticals. A proportionality constant known as the Vd is described as the relation among the
observed plasma concentration and the dosage, or amount of drug supplied. The
volume of blood or plasma from which a certain medication is entirely eliminated
per unit of time (hours or minutes) is known as CL, which is a measure of drug
elimination (van den Anker etal. 2018).
M. Aslam et al.
4.2.4 Drug Discovery andDevelopment
Biotechnology techniques such as recombinant DNA technology and gene expression proling are used to identify prospective therapeutic targets, nd novel medication candidates, and improve their properties. The Human Genome Project has so
far had a substantial effect on new drug discovery (Boulnois 2000). The development of microarray technology, which allows for the simultaneous study of the
expression of tens of thousands of genes, as well as the increasing sophistication of
proteome analysis, has created numerous opportunities for those working in drug
discovery (Lander 1999).
Currently, biosensors are employed in assay development, validation, lead optimization, target identication, excretion, absorption, metabolism, distribution, and
toxicity (Grimm etal. 2004). Proteomics is becoming increasingly signicant in the
target identication and validation stages of the drug discovery process.
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Nanotechnology innovations are being used to improve drug delivery, drug discovery, and pharmaceutical manufacturing (Jain 2009).
Medical biotechnology is critical in today’s climate for understanding the molecular factors that cause health problems, enhancing diagnostic processes, and generating tailored treatments. This approach enables disease therapy with an intriguing
new pharmacological module that targets a milieu of causes (Anwar etal. 2022).
Gram staining can distinguish among both kinds of bacteria and continues to be
utilized in clinical specimens that include cerebrospinal uid or biopsies for early
bacterial infection detection since it is a simpler and faster way of identifying bacteria compared to the cultivation of cells (Lokko etal. 2018). However, due to the
precision of such surgeries made possible by robots, they have been expanded to
meet the needs of more people. It allows an experienced surgeon that is a recognized
specialist in the discipline to operate on an individual in a different region of the
world (Anwar etal. 2022). Traditionally, medicinal chemistry has taken advantage
of any opportunity to contribute to the identication of new therapeutic molecules.
Although medicinal chemistry as a profession has improved optimization into a
process free of substantial unidentied barriers, the task of lead identication has
yet to reach a comparable level of maturity (Venuti 1989; Anon n.d.-b).
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4.3 Biotechnological Techniques inPharmaceutical
Product Analysis
Biotechnological techniques have revolutionized pharmaceutical product analysis,
providing powerful tools for researchers and analysts to assess the safety, efcacy,
and quality of drugs and biopharmaceuticals. Here are some of the most important
biotechnological approaches used in pharmaceutical product analysis.
4.3.1 Polymerase Chain Reaction
Recent advancements in methodology have made it possible to enhance an organism’s genetic abnormalities in DNA or RNA invitro using polymerase chain reaction (PCR), perhaps eliminating the need for culture. Primer annealing, primer
extension, and denaturation of double-stranded DNA comprise the fundamental
three- step cycle process of PCR (Schochetman etal. 1988). Certain DNA fragments
of interest are surrounded by two oligonucleotide primers that combine to adjacent
strands in the polymerase chain reaction (PCR) in culture technique for enzymatic
synthesis of specic segments of DNA (Erlich 1989). Through a cyclic process
called PCR, practically any sequence of nucleic acids found in a complex substance
can be processed, producing a large number of identical copies that are simple to
assess (Kubista etal. 2006).
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Recent improvements in PCR amplication methods have inuenced crucial
characteristics such as misincorporation rate, specicity (target versus non-target
amplication), and PCR product maximal length (Mfuh etal. 2019). The number of
amplication items is evaluated by many PCR techniques, oligonucleotide probe
typing, or direct sequencing; therefore infrequent mis-incorporated nucleotides are
not found (Erlich etal. 1985).
Additionally, it has been claimed that novel PCR-based techniques improve the
assay’s sensitivity and specicity. Furthermore, real-time Reverse TranscriptasePCR is more sensitive than traditional RT-PCR assay, which can be very helpful in
early infection diagnosis (Shen etal. 2020).
M. Aslam et al.
4.3.2 Enzyme-Linked Immunosorbent Assay (ELISA)
ELISAs are immunoassays through which an enzyme acts as the “signal generator”
or “reporter” while one reactant remains stationary on a solid surface. Enzymelinked immunosorbent assays (ELISA) and solid-phase immunoassays technically
simplied antigen quantication and antibody detection (Butler 2000).
An indirect ELISA to identify particular antibodies and a direct competitive
ELISA to identify soluble antigens are two of the six unique ELISA methods that
demonstrate the diversity of ELISAs. Soluble antigens can be detected using an
antibody-sandwich ELISA, specic antibodies can be found using a double
antibody- sandwich ELISA, and cell-surface antigens and antibodies specic to surface antigens can be found using two cellular ELISAs (Hornbeck 1992). The antigen that has coated the well is attached by a primary detection antibody with a
tagged enzyme in a direct ELISA (Schots etal. 1988). Sandwich ELISA is used to
analyse more complicated solutions. The capacity of a conguration to t an antigen between two antibodies is where the test gets its name (Katsurada etal. 2007).
A competitive ELISA differs from sandwich, direct or indirect ELISAs in that it
employs the method of efcient interactions (Libeau etal. 1995). Any antigen present is attached to the primary antibody when it comes into contact to an unpuried
sample. Complexes of antigen and antibody are formed in proportion to the number
of antigens in the material being examined (Starr and Tessier 2019). Lesser number
of antibodies are able to attach to the antigen in greater number of antigen-antibody
complexes that develop (Sittampalam etal. 1996).
4.3.3 Mass Spectrometry (MS)
Proteomics is the methodical analysis of every protein present in a given tissue or
cell, with mass spectrometry at the heart of most proteomic techniques. For a long
time, mass spectrometry was limited to tiny and thermostable compounds due to a
lack of adequate procedures for softly ionizing and transferring ionized molecules
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from the condensed phase to the gas phase without signicant fragmentation
(Domon and Aebersold 2006). Mass Spectrometry is unique among analytical techniques utilized for an astonishingly wide range of applications by analysing the
molecule’s inherent mass property with exceptionally high accuracy (Mann
etal. 2001).
Currently, there are three key applications of MS in proteomics. MS is the preferred technique for characterizing and controlling the quality of recombinant proteins and other macromolecules, which is a signicant task in the eld of
biotechnology (Glish and Vachet 2003). It is also widely utilized in protein identication, whether in traditional biochemical research or large-scale proteomic ones
(Zubarev and Makarov 2013). Finally, because MS assesses a protein’s molecular
weight, it is the method of choice for detecting and characterizing posttranslational
modications and has the potential to identify any covalent change that alters the
mass of a protein (Cooks etal. 2006).
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4.3.4 High-Performance Liquid Chromatography
Pharmaceutical analysis often entails conducting trials to determine how much of a
medication is present in a pharmaceutical formulation of therapeutic substances.
This method needs to be tried out extensively, and even after it is established and
proven, there may be situations where it still needs to be tried out again (Ganorkar
and Shirkhedkar 2017). Multidimensional column chromatography (also known as
column switching, multiphase, multicolumn, or coupled column chromatography)
is an effective method for separating multicomponent materials. The approach has
been used in thin-layer chromatography (TLC) for many years, although in a somewhat different way (Milroy etal. 2011).
Chemicals dissolved in solution can be separated and quantied using a particular chromatography technique. Based on the mechanism of action, chromatography
systems are frequently classied into four types: adsorption, partition, ion-exchange,
and size exclusion (Kupiec 2004). When it comes to bulk medications and pharmaceutical formulation quality control, this method is crucial. It encompasses various
tasks such as analysing active pharmaceutical ingredients (API), characterizing
impurities, guring out enantiomeric purity, and discovering breakdown of substances to evaluate product stability. Additionally, the byproducts of drugs in biological specimens are identied using it (Yabré etal. 2018).
4.3.5 Western Blotting
Northern and Southern blotting gave rise to Western blotting (WB), commonly
referred to as immunoblotting or protein blotting (Southern 2004; Alwine et al.
1977). The nature of the gel, molecular mass of proteins being transferred, and the
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membrane being utilized are all crucial factors in ensuring the effective migration of
proteins from a gel to a solid membrane support (Kurien and Scoeld 2006).
Researching the regulatory molecular mechanisms underlying protein turnover,
energy metabolism, and chronic physiological adaptations can benet greatly from
the extensive range of applications provided by the Western blot (WB) (Bass etal.
2017). A usual approach for identifying proteins and subsequent modications on
proteins is the western blot, which can provide numeric or indirect information on
the target protein in both straightforward and complicated biological materials
(Vallejo-Illarramendi etal. 2013). One popular method for analysing proteins is
western blotting (He and Herr 2010). Purication and concentration of proteins
determined by the extracts of cell or tissue are usually the rst steps in this multistep
process, protein size separation on a nitrocellulose or polyvinylidene uoride
(PVDF) and polyacrylamide gel containing sodium dodecyl sulphate (SDS)
membrane- mediated mobilization protein isolation, obstructing membrane proteins
without specicity, conjugation of a secondary antibody with a labelled uorescent
or chemiluminescent molecule, identication of a signal indicating the binding of
an antigen or antibody densitometry analysis using software to identify relevant
protein bands (Mishra etal. 2017; Murphy and Lamb 2013).
M. Aslam et al.
4.4 Applications inDrug Development andQuality Control
Biotechnology plays a crucial role in the pharmaceutical sector for both drug development and quality control. Here are some examples of major applications in both
domains. The development of quick and precise drug safety analysis methodologies
is necessary since current approaches and techniques are unable to keep up with the
demands of the industry (Chen etal. 2022). This section provides an overview of the
development of biological approaches used to analyse medication safety (Fig.4.2).
4.4.1 Target Identication andValidation
Target validation and identication are the initial crucial steps in the therapeutic
research process. Discovering new targets usually proteins, whose modication
could halt or reverse the course of a disease, is the aim of target discovery (Howbrook
etal. 2003). Nucleic acid microarrays have signicantly improved our knowledge of
both normal and abnormal elds of biochemistry, which in turn has inuenced the
choice of targets for medication development (Vernell etal. 2003; Wang etal. 2004).
It enables intuitive and accessible analysis of the data that links targets and diseases, while also giving methods for the inquiry of specic illness concept (Koscielny
etal. 2017). Relevant information linking a target to a disease is stored on the Target
Evaluation Services for all potential human targets, including proteins and RNA
molecules (Malone etal. 2010).
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Fig. 4.2 A summary for drug assessment by biological methods (Chen etal. 2022)
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4.4.2 Structural Biology
The earliest documented success stories reported in the early 1990s with structural
biology as a therapeutic research technique date back to the mid-1970s (Beddell
etal. 1976; Erickson etal. 1990). Nowadays, many corporate pharmaceutical development initiatives depend heavily on structural biology and SBDD, and these areas
of study are also the focus of research for numerous university groups (Van Duin
etal. 2003). This is the stage for the medication approach to research where SBDD
has most potential to make a difference, via inuencing the main components’ modication in chemicals (Scapin 2006). Known as in-cell or in situ structure commitment, the viewing of cellular frameworks in their native surroundings is still one of
structural biology’s long-term objectives. Indeed, the viability of this strategy is demonstrated by recent developments in mass spectrometry, chemical crosslinking, data
processing, cryo-electron tomography, and data processing (Cramer 2021).
4.4.3 Bioprocess Monitoring
HTRF (Homogeneous Time-Resolved Fluorescence) technology is based on the
principle of uorescence resonance energy transfer (FRET) where non-radiative
energy is transferred from a donor uorophore (rare-earth lanthanides) to an
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