Objective of the presentation: • To give clue about recent applications that are added to pharmaceutical industry Introduction • Biotechnology is a broad field that can be incorporate into agriculture, industry, environment, food science, and pharmaceuticals. • Pharmaceutical companies use biotechnology in the advancement of humankind in terms of healthcare such as: ✓ Manufacturing drugs ✓ Vaccinations ✓ Pharmacogenomics* ✓ Gene therapy ✓ and Others *Pharmacogenomics, also known as pharmacogenetics, is the study of an individual’s genes and how it responds to certain medicines. A vaccine is a biological preparation to establish a type of immunity known as artificial active immunity to a particular disease. The main objective used to improve immunity, the antigen is known as a vaccine Introduction cont… 25 January 2023 4 • There are various classes of biotechnology based products that are produced for the treatment or prevention of different pathological conditions like ➢ Growth factors ➢ Hormones ➢ Vaccines and ➢ Monoclonal antibodies ➢ Antibiotics ➢ Blood factors ➢ Cytokines ➢ Enzymes ✓In the future, biopharmaceuticals may be used against the AIDS, different types of cancer, asthma, Parkinson’s and Alzheimer’s disease Introduction conti… 25 January 2023 5 • The conventional pharmaceutical formulations are relatively simple molecules manufactured mainly through • trial and error technique for treating the symptoms of a disease or illness. • Since recent biotechnology applications are incorporated into pharmaceutical companies • This trial and error technique has been overtaken and production of biopharmaceuticals are oversimplified. 6 Introduction cont… • Some of the recent applications in pharmaceutical biotechnology are listed below: 1. High - Throughput Screening 2. In Silico Pharmacology 3. Microarray Technology 4. Chemical Proteomics 5. HTP RNAi Screening 6. Nanotechnology ❖ OTHERS… 25 January 2023 The primary purpose of screening is to identify members of a chemical library that interact in a defined way with a selected system 25 January 2023 7 • HTS, as the name indicates, is a drug discovery process that enables a biochemical or cellular event to be reproducibly and rapidly tested against chemical entities many hundreds of thousands of times. • HTS utilizes robotics, liquid handlers, data processing, considerable software, and sensitive detection systems, ▪ To quickly conduct vast numbers of biochemical, genetic, or pharmacological tests 1. High - Throughput Screening (HTS) 8 HTS cont… • The objective of HTS is to rapidly identify active compounds (hits) that modulate a particular target, pathway, or biochemical/cellular event. • These are used as starting points for medicinal chemical optimization during pharmacological probe or

1. University of Gondar
College of Veterinary Medicine and Animal Science
Department of Veterinary Pharmacy
Recent Applications in Pharmaceutical
Biotechnology
25 January 2023
Sewagegnegu Getachew

2. 25 January 2023
2
Objective of the presentation:
• To give clue about recent applications that are added to
pharmaceutical industry

3. Introduction
3
• Biotechnology is a broad field that can be incorporate into agriculture,
industry, environment, food science, and pharmaceuticals.
• Pharmaceutical companies use biotechnology in the advancement of
humankind in terms of healthcare such as:
 Manufacturing drugs
 Vaccinations
 Pharmacogenomics*
 Gene therapy
 and Others
*Pharmacogenomics, also known as pharmacogenetics, is the study of an individual’s genes and how it responds to certain medicines.
A vaccine is a biological preparation to establish a type of immunity known as artificial active immunity to a particular disease.
The main objective used to improve immunity, the antigen is known as a vaccine

4. Introduction cont…
25 January 2023
4
• There are various classes of biotechnology based products that are produced
for the treatment or prevention of different pathological conditions like
 Growth factors
 Hormones
 Vaccines and
 Monoclonal antibodies
 Antibiotics
 Blood factors
 Cytokines
 Enzymes
In the future, biopharmaceuticals may be used against the AIDS, different
types of cancer, asthma, Parkinson’s and Alzheimer’s disease

5. Introduction conti…
25 January 2023
5
• The conventional pharmaceutical formulations are relatively simple
molecules manufactured mainly through
• trial and error technique for treating the symptoms of a disease or illness.
• Since recent biotechnology applications are incorporated into
pharmaceutical companies
• This trial and error technique has been overtaken and production of
biopharmaceuticals are oversimplified.

6. Introduction cont…
25 January 2023
6
• Some of the recent applications in pharmaceutical biotechnology are listed below:
1. High - Throughput Screening
2. In Silico Pharmacology
3. Microarray Technology
4. Chemical Proteomics
5. HTP RNAi Screening
6. Nanotechnology
 OTHERS…
The primary purpose of screening is to identify members of a chemical library that interact in a defined way with a selected system

7. 25 January 2023
7
• HTS, as the name indicates, is a drug discovery process that enables a
biochemical or cellular event to be reproducibly and rapidly tested against
chemical entities many hundreds of thousands of times.
• HTS utilizes robotics, liquid handlers, data processing, considerable software,
and sensitive detection systems,
 To quickly conduct vast numbers of biochemical, genetic, or
pharmacological tests
1. High - Throughput Screening (HTS)

8. HTS cont…
25 January 2023
8
• The objective of HTS is to rapidly identify active compounds (hits) that
modulate a particular target, pathway, or biochemical/cellular event.
• These are used as starting points for medicinal chemical optimization
during pharmacological probe or drug discovery and development.
• The output from a HTS campaign provides the basis upon which drug design
and elaboration are used
 To generate lead compounds with appropriate physicochemical properties
for therapeutic indications

9. HTS cont…
25 January 2023
9
• HTS allows rapid identification of disease genes, proteins, RNA, miRNA,
antibodies, and so on, from omic based experiments using microarray and
robotics.
• Similarly, HTS are frequently used as starting points in identification of
signaling pathways, drug targets, active compounds, and functional assays.

10. HTS cont…
25 January 2023
10

11. HTS cont…
25 January 2023
11
• HTS can screen 10k– 100k chemical cpds daily, in ultra high - throughput
screening greater than 100k.
• Therefore, both the HTS and uHTS are now an integral part of the drug
discovery pipeline for screening conventional and new chemical entities on
mass.

12. HTS cont…
25 January 2023
12
• HTS assays are performed in microtiter plates in 96-, 384-, 1536-, 3456 well
formats
• With the increases in library size over time, in addition to the increase in the
number of molecular targets,
►assay miniaturization, automation, and quality concerns with respect to
screening have become relevant.
• This has created a huge shift in the time process of HTS in terms of
• assay development, tool production, adaptation, detection tools, counter
and selective screening, quality assurance, hit - to - lead, and lead
optimization, and so on.

13. 25 January 2023
13

14. 25 January 2023
14

15. 2. In Silico Pharmacology and Virtual
Ligand Screening
25 January 2023
15
• The term in silico denotes the usage of computational technologies for
experimentation in a particular field of study .
 sometimes referred to as computational therapeutics/pharmacology
What is pharmacology?
• These computational methods are relevant in limiting the use of animal models
in pharmacological research,
 for aiding the rational design of novel and safe drug candidates, and
 for repositioning marketed drugs, supporting medicinal chemists and
pharmacologists during the drug discovery trajectory.

16. In Silico ….
25 January 2023
16
• It is basically the use of computers which include
→ databases, quantitative structure activity relationships, pharmacophores,
homology models and
→ other molecular modeling approaches, machine learning, data mining, network
analysis tools and data analysis tools.
• Is the science dealing with the use of biomedical information in creating
computational models or simulations that can be used for making predictions,
suggesting hypotheses, and hence,
 leading to discoveries or advances in medicine and therapeutics
• Pharmacological screening of drugs is an essential part of the drug discovery
process (pharmacodynamic & pharmacokinetic studies)

17. Quantitative Structure – A activity Relationship (
QSAR )
25 January 2023
17
• A clear relationship exists between the chemical structure and biological effect or activity
of a compound.
• As expected, the physicochemical properties of a compound are also a deciding factor of
its biological activity or range of biological activities.
• Some of the biological activities are beneficial in clinical scenarios (therapeutic activity)
and some are harmful (toxicity).
• In silico pharmacology exploits this relationship ( structure activity relationship , SAR ) to
construct mathematical models and enables the quantification of SAR.
• This is the main domain of in silico pharmacology – QSAR or the quantitative structure –
activity relationship .
• Over a period of the last 40 years or so, several QSAR models have been generated and
they are stored in the C - QSAR database.

18. Virtual Ligand Screening
25 January 2023
18
• Virtual ligand screening is the process of scoring and ranking molecules in large chemical
libraries as per their probability of having an affinity for a specific target.
• It can also be considered to be an attempt to extend the concept of QSAR along the chemical
dimensions as appropriately defined by existing synthesized as well as theoretically
synthesizable molecules.
• Virtual screening gained much popularity in the late 1990s when experimental (HTS) techniques
were displaying poor performances and higher costs in comparison with what had originally
been anticipated for the concept.
• The pharmaceutical industry has accepted virtual screening as an efficient complement to HTS.
• The industry belief in virtual screening has grown to such an extent that today it has become an
integral part of lead generation and drug discovery processes.
• Virtual screening is basically a knowledge - driven process and requires structural information
on either the ligand or the target protein, hence the virtual ligand screening

19. Virtual Affinity Profiling
25 January 2023
19
• Virtual ligand screening has extended the concept of QSAR in the chemical sense and
virtual affinity profiling has extended it in the biological sense.
• Virtual affinity profiling has enabled the estimation of pharmacological effects of a single
molecule on multiple target sites.
• Both ligand - based and target - based methods are available for virtual affinity profiling

20. Data Visualization
25 January 2023
20
• Computational technologies can be used for generating predictions of pharmacological and
physicochemical properties for each molecule structure.
• The analysis of such data requires multidimensional methods and sophisticated
visualization tools for proper and effective data mining.
• Diva and Spotfire programs are widely used for analysis of ADME and physicochemical
properties

21. In Silico ….
25 January 2023
21
• There are several approaches to virtual screening of drug molecules
Quantitative structure – activity relationship ( QSAR )
1. Descriptor - based methods
2. Rule - based methods
3. Knowledge - based methods
Virtual ligand screening
1. Ligand - based methods
2. Target - based methods
Virtual affinity profiling
1. Ligand - based methods
2. Target - based methods
Data visualization

22. In Silico ….
25 January 2023
22
Table:- a partial list of available programs and databases for virtual screening

23. In Silico ….
25 January 2023
23
►Virtual screening has become popular in lead drug identification through
computational screening of a vast array of chemicals against protein targets.
►Fluorescently tagged drug molecules are successfully utilized for visualization of the
location of their receptor targets particularly G - protein coupled receptors GPCRs.
►Recently, a novel ligand - based screening process (MTree approach) has been
reported.
►This method utilizes a new concept of combining query molecules into a multiple
feature tree (MTree)
►These MTrees can be used to identify new leads for chemo - optimizations.
►MTrees have been generated for ACE enzyme and alpha 1a receptor.

24. 3. Microarray Technology
25 January 2023
24
• Microarray refers to a piece of glass, plastic or silicon with a large number of
biosensors at known, specific locations.
• These microarrays are often referred to as biochips or DNA chips.
• They can be used for testing a single biological specimen for a range of effects or
attributes.
• Several chemical moieties of variable nature can be used for detecting proteins.
• These protein detection molecules include ligands, dyes changing color,
fluorescing, or causing electronic signals when they come in contact with specific
protein molecules.
• If these protein - detector molecules are placed onto a microarray, protein
expression and expression levels of genes in the given biological specimen can be
evaluated

25. Cont….
25 January 2023
25
• They are utilized in the analysis of gene expression levels in an organism and
for comparing gene expression levels (e.g. diseased and healthy)
• This is accomplished by passing samples with DNA (e.g., in liquid) over the
array surfaces.
• As a result, mRNA hybridizes to its counter part DNA sequence.

26. 25 January 2023
26
★ Because gene expression only tells part of the picture.
★ By combining gene expression data with information from newer
microarray technologies scientists can begin to apply themselves to
many exciting applications, such as
○ finding cures for infectious diseases,
○ discovering the mechanisms involved in cancer and
○ driving the developments of new therapeutics.

27. DNA Microarray Fabrication
25 January 2023
27
• DNA microarray fabrication methods include inkjet and microjet deposition or
spotting technologies, in situ or on-chip photolithographic oligonucleotide
synthesis, and electronic DNA probe addressing processes.
• DNA microarray hybridization applications include gene expression analysis,
genotyping for point mutations, single nucleotide polymorphisms (SNPs) and
short tandem repeats (STRs)

28. Cont….
25 January 2023
29
• There are two commonly used and relatively simple approaches for
manufacturing DNA microarrays.
1. Using the reverse transcriptase polymerase chain reaction (RT - PCR),
 cellular mRNA is utilized to prepare approximately 500 – 5000bp long
segments of complementary DNA (cDNA) which are later attached to a
nylon or glass surface at known spots.
When hybridization of sample DNA takes place, the location of the spot
specifies the DNA in the sample.

29. 25 January 2023
30
Cell
mRNA Extraction
Reverse Transcription
Fluorescent Labelling
Fluorescent DNA
cDNA
Microarray
Hybridization
on
Microarray
Scanning Visual output

30. 25 January 2023
31
• In another method, oligonucleotides or peptide nucleic acids of known
sequence are attached at known spots on the nylon or glass surface.
• Then the biological sample with DNA (e.g., in liquid) is passed over this
surface to identify the DNA contained in the sample

31. Application of DNA Microarrays in HTS and Drug
Discovery
25 January 2023
32
• DNA microarrays have the distinction of being the first array technique that are
close to entering routine laboratory diagnostics.
• In the near future they may provide information about activated oncogenic
signaling pathways and other cell functions in malignant tissues.
• They may also help in predicting drug response or metastasizing potential.
• DNA microarrays can be used for a variety of applications in pharmaceutical
research (pharmacogenomics and drug discovery), toxicogenomics, diagnostics
(infectious, genetic disease, and cancer), and forensics (genetic identification).

32. Cont….
25 January 2023
33
• DNA microarrays and proteomics technologies can be combined together to
study the gene regulation process in diseases and identify drug discovery and
diagnostics targets.
• The combined application of these technologies holds the potential to
revolutionize areas of biology particularly in molecular medicine.

33. Cont….
25 January 2023
34
• DNA microarrays have proven their worth in
i. Anticancer
ii. Antibacterial therapy
iii. Prediction of drug toxicity.

34. 25 January 2023
35

35. Chemical Microarray
25 January 2023
36
• A chemical microarray is a form of chemical combinatorial peptide library and is a
powerful tool in drug screening and discovery.
• A large chemical library can be screened against hundreds of biological targets
using chemical microarrays or small - molecule microarrays in solution phase on an
automatic liquid handling platform.
• However, isolated wells are required for individual reactions when compounds are
of different structures and properties, and currently available liquid handling
systems are not capable of processing thousands of reactions individually in a
solution phase.
• Therefore, several versions of this technology are currently under development.

36. 4. Chemical Proteomics for Drug Discovery and Development
25 January 2023
37
• According to the human genome project (HGP) data there are 25k - 30k
protein coding genes present in the human genome, and
 the study of proteomics reflects the understanding of function, interaction,
modification, localization, and
 regulation of all protein expressed by a cell or in an organism.
• Typical expression levels of proteins maintain normal physiology and any
alteration in protein structure or expression causes diseased phenotypes.
• Therefore, functional and structural proteomics are very important.

37. Cont….
25 January 2023
38
• Recently, new technologies such as
 Shotgun proteomics
 Reverse proteomics
 Top - down proteomics and
 Chemical proteomics have been developed to elevate this intimidating
task.

38. 25 January 2023
39
• Proteomic approach of drug discovery includes finding an unstable protein that
is causing an undesirable affect and then usage of a molecule to modify its
effect
• Chemical proteomics is a powerful tool for isolating and identifying novel
cellular receptors at the level of enzymatic activity using
• both in vitro and in vivo chemical probes, and
• thereby has potential to accelerate the discovery of new drug targets and
• often providing precious selective information regarding biochemical and
cellular process.
4. Chemical Proteomics for Drug Discovery and Development

39. Cont….
25 January 2023
40
• It is emerging as a powerful tool for drug discovery as it only requires a known
drug to start with and
 can be defined as application of synthetic organic chemistry, bioinformatical
analysis, biochemistry, and mass spectrometry to the field of proteomics.
• Chemical proteomics can help us design specific protein modifying reagents
that can be used for functional studies of a distinct enzyme family within a
proteome.
• These chemical probes are designed to covalently modify a target enzyme in
such a way that it can be subsequently identified and/or purified.

40. Cont….
25 January 2023
41
• Chemical proteomics parallel functional proteomics except that a tagged small molecule is
used to isolate a single protein or a family of proteins from an entire proteome.
• The tag can consist of either a radioactive/fluorescent label to allow visualization of bound
protein on an electrophoresis gel, or a solid phase bead/surface, to allow affinity
purification of the protein.

41. 25 January 2023
42
• There are two strategies for chemical proteomics techniques:
 Activity - based probe profiling (ABPP), which is based on enzymatic
activity of a particular class of enzyme, and
 The fragment - based approach, which facilitates the assembly of a large
library of diverse molecules from a smaller number of building blocks

42. Applications of Chemical Proteomics
25 January 2023
43
• Chemical proteomics is a new and promising tool for drug discovery,
• as it requires only a known drug as the starting point, and leads to the identification of a
suitable target whose biological activity can be directly linked to a pathological process.
• Minor imbalance in enzyme activities are known to cause debilitating diseases and even
promote cancer or tumor metastasis.
• In this modern age more emphasis are giving to natural druggable compounds, for modern
pharmaceutical research chemical proteomics again seem to be a useful tool for the
prediction of suitable interacting targets
• Identification of new drug target is also essential for solving drug resistance problems

43. 5. HTP RNAi Screening for Targeted Drug Discovery
25 January 2023
44
• Genes involved in phenotypes of interest can be directly identified using
functional genomics approaches.
• RNA interference (RNAi) provides a powerful tool for screening genes based
on loss of function.
• Thus this technology can also be used in HTS of genomic targets for drug
development and discovery, based on
• the understanding that induced expression of screened genes using a
suitable antagonist will overcome the defective phenotype due to the loss
of function of the screened genes that cause the disease.

44. Cont….
25 January 2023
45
• RNAi is a widely accepted technology for various purposes such as gene therapy,
know out experiments, and so on.
• In this technique, a small single stranded non - coding RNA (ncRNA) molecule
that is complementary to certain mRNA can bind to the mRNA,
• therefore inhibiting the translation process of the particular mRNA so that the
corresponding protein is ultimately involved in expressing a certain specific
phenotype.
• Owing to the immense potential of RNAi, today it is also used for HTP
screening.

45. HTS Using RNAi Libraries
25 January 2023
46
• Various RNAi libraries are available based on experimental systems, or
experiments or diseases of interests.
• Various forms of ncRNAs that are mainly used in HTP screening include:
• siRNAs or shRNAs are designed specific to mRNAs to be used in RNAi and
they may be natural but are mostly artificially synthesized.
 small interfering RNA (siRNA)
 small nucleolar RNA (snoRNA)
 short hairpin RNA (shRNA)
 microRNA (miRNA) and
 Piwi - interacting RNAs (piRNA)

46. Cont….
25 January 2023
47
• Loss of function HTP screening using RNAi can be performed using either pool
based library selection or array based screening approaches.

47. Pool based library
25 January 2023
48
• A viral vector mediated small library of RNAi (shRNA having inducible promoters) is
transfected to a pool of cells.
• The pool is then divided into two sub - pools and in one sub - pool the expression of shRNA is
induced. Thereafter, each sub - pool is subjected to stress, drug treatments, and so on.
• After a certain period of culture, both the sub - pools are harvested and differential screening of
gene expression in terms of barcodes is generated using microarray and PCR.
• Increased representation of a barcode from an induced sub - pool is indicative of selection of
the corresponding shRNA, and therefore the target gene that is responding to the stress.
 If the stress correlates to a disease condition, an antagonist to the gene might be a good
candidate drug for the disease.
 Similarly, if an shRNA induces some gene expression (causative to a disease) by targeting an
inhibitor of the gene, the target of the shRNA can be antagonized to induce the inhibitor gene
expression to inhibit the expression of disease causing gene.

48. Array based
25 January 2023
49
• Array based RNAi screening is the most common method, and multiple
different trigger deliveries are possible.
• In general, each well of a microtiter plate with a single RNAi trigger is
incubated with a transfection medium and cells.
• After a defined incubation period, flow cytometry analysis, high content
imaging, ELISA, and various homogeneous endpoint assays are performed to
identify the loss of gene function.
• RNAi technology is a cost - effective and rapid process.
• RNAi libraries have successfully been used to discover opportunistic drug
targets

49. 6. Nanotechnology
25 January 2023
50
• Nanotechnology can play an important role in development of proper
formulations that address the drug delivery issues related to new molecular
entities with poor biopharmaceutical properties, such as
• Nanotechnology can also achieve desirable pharmacokinetic and toxicological
properties that aid in the accelerated development of the new molecular
entities.
⁃ poor solubility
⁃ poor permeability across the intestinal epithelium
⁃ enzymatic or nonenzymatic degradation/metabolism
⁃ complexation with chelating ligands or metal cations
⁃ intestinal efflux, and poor transport properties

50. Nanotechnology
25 January 2023
51
• Nanoparticulate drug delivery systems are being used to alter the drug’s
biopharmaceutics and pharmacokinetics.
• Although it is expensive and time consuming it can significantly assist in the
accelerated development of new molecular entities with inadequate drug like
properties and
• can assist the pharmaceutical companies to add more lead molecules into
their pipeline.

51. Nanotechnology
25 January 2023
52
• Nanoparticle-based drug delivery has a significant advantage over the
conventional drug delivery systems in terms of
• It is important to include the research on nanoparticulate delivery systems in
the pre-formulation development of drug products.
• Specific platforms that include the use of liposomes, polymeric nanoparticles,
micelles, and dendrimer nanostructures are addressed.
o possibility of use with different routes of drug administration, and
o the capability to transport both hydrophilic and hydrophobic molecules.
o high drug carrying capacity,
o controlled release,
o high stability,
o high specificity,

52. 25 January 2023
53
• An example of the application of nanobiotechnology is the development of
cancer vaccines carriers.
• Cancer vaccines are used in tumor immunotherapy, which involves activation
of the patient’s immune system and
• allowing it to recognize the tumor cells and kill these cells, which are
normally not recognized by the immune system.
• This method of treatment can be used as an alternative treatment to the other
traditional methods of cancer treatment or adjuvant to them

53. Others
25 January 2023
54
 Lipomic Profiling
 Integrative Genomics
 Toxicogenomic
 High - Throughput Screening with Stem Cells

54. Summary
25 January 2023
55
• To sum up, through the development different advanced technologies, today
understanding the structure of proteins, enzymes and different target molecules
have become oversimplified.
• This promises for the searching of different agents to cure cancer, cardiovascular
diseases, for drug resistance, and better health safety
• It is important to incorporate such technologies in pharmaceutical campanies
• Additional research is necessary

55. 25 January 2023
56
THANK
U