University of Indonesia / Jakarta

March 6th 2019

Biotechnology in Drug Development

Hans-Jürgen Mägert, Anhalt University of Applied Sciences, Köthen, Germany

Biotechnology in Drug Development

1. INTRODUCTION

What is Pharmaceutical Biotechnology, history, market potential of

pharmaceuticals

2. MAIN PART

Basics of Pharmaceutical Biotechnology

– Medical needs
– Target identification / validation
– Compound libraries
– Assay establishment and agent screening
– Lead optimization
– Recombinant production of pharmaceutical proteins
– Safety Aspects
 In Vitro Toxicity Tests

– For testing of drug candidates

– At the cell or tissue level

– Four important parameters

- Method of cell / tissue culturing

- Concentration of the substance to be tested (as a rule of thumb
maximum of 100 mg/ml, for drug candidates 100-
1000-fold of concentration post application)
- Duration of exposition and period of recovery
- Selection of "end points" for quantification of damages
 MTT-Test => Integrity of Mitochondria

MTT = tetrazolium salt, may act as an electron acceptor, in mitochodria and

peroxisomes (also cytosol) it is converted to insoluble blue product (formazan) by
oxidoreductases => may be extracted and photometrically quantified

– Transfer cells into microtiter plates, incubate for 24 h

– Aspirate medium, add 200 µl medium with substance to be tested per well (dilution
series) - also controls

– Cultivate for 24-36 h, then add 20 µl of a sterile MTT-solution to each well

– Carefully mix, then incubate for 2 h in CO2-incubator

– Aspirate supernatants and add 100 µl of a mixture of DMSO, acidic acid and SDS
per well

– Leave plates alone for 5 min, then mix for 5 min on shaker

– Measure in photometer and evaluate tests, suitable values may be obtained from
cell numbers of 200,000 - 300,000 per well
Reduction of MTT to formazan
 MTT-Test => Integrity of Mitochondria

MTT = tetrazolium salt, may act as an electron acceptor, in mitochodria and

peroxisomes (also cytosol) it is converted to insoluble blue product by
oxidoreductases => may be extracted and photometrically quantified

– Transfer cells into microtiter plates, incubate for 24 h

– Aspirate medium, add 200 µl medium with substance to be tested per well (dilution
series) - also controls

– Cultivate for 24-36 h, then add 20 µl of a sterile MTT-solution to each well

– Carefully mix, then incubate for 2 h in CO2-incubator

– Aspirate supernatants and add 100 µl of a mixture of DMSO, acidic acid and SDS
per well

– Leave plates alone for 5 min, then mix for 5 min on shaker

– Measure in photometer and evaluate tests, suitable values may be obtained from
cell numbers of 200-300.000 per well
WST-1 (similar to MTT)
 Cytotoxicity

140

120

100

80
Viability (%)

P1
60
P2

P3
40
MBI-28
20

-20

0 100 200 300 400 500

Concentration (µg/ml)

Potential antimicrobial peptides exhibit

strong cytotoxic activity on human peri-
pheral mononuclear cells (PBMNs)
 CASY – Cell Analysis System

– Electrical resistance of cells is measured by means of a measuring

capillary and is digitalized

– Electrical resistance (impedance) of dead cells is lower compared to

living cells
Rule for animal experiments: „3 Rs“

– Replacement

– Reduction

– Refinement
ATC Method for Determination of Acute Toxicity
Permanent correspondence with the responsible

administration (FDA, EMEA, BPOM.....)

Biotechnology in Drug Development

1. INTRODUCTION

What is Pharmaceutical Biotechnology, history, market potential of

pharmaceuticals

2. MAIN PART

Basics of Pharmaceutical Biotechnology

– Medical needs
– Target identification / validation
– Compound libraries
– Assay establishment and agent screening
– Lead optimization
– Recombinant production of pharmaceutical proteins
– Safety Aspects