Adapted from K.R.S.S. RaoDepartment of BiotechnologyAcharya Nagarjuna University

AIM: Ability to define, describe

and utilize microbial application in biotechnology

and biological process specially in pharmaceutical and medical biotechnology

At the end of the lecture, the students should be able to:

name the type of biopharmaceuticals technology that and describe what is occurring in human cells

compare and contrast the various methods for biopharmaceutical

describe the various types of commercial production in medical biotechnology and explain the differences in their function

describe the influence of various environmental factors that effect the storage and suitability of biopharmaceutical.

OUTLINE

Production of Biopharmaceuticals – An Overview

Microbial Technology Commercial Production Insulin Vaccines Hepatitis B Alkaloids from Plants rDNA technology Challenges in Biopharmaceutical

What are Biopharmaceuticals

Biologically significant compounds like hormones and proteins useful

for treatment of variety of human health disorders, usually called as

Biopharmaceuticals or Biotherapeutics or Biologicals,

usually obtained from biological source and produced through

industrial biotechnology

Production of Biopharmaceuticals – An Overview

Microorganisms as tools for production of various Biotherapeutics

From genes to processes Gene resources

Gene diversity Methods of gene discovery

Expression of genes and production of gene products

Use of organisms and enzymes as catalysts

Environmental processes Fermentation processes Biotransformations

From Genes to Processes

Gene discovery

Cloning/expression

Production and scale-up

Engineering the catalyst

Application

Insulin- hormone which lowers blood sugar; used by diabetics

Interferon- class of cytokines, effective vs viral infections

Streptokinin - bacterial enzyme to dissolve blood clots in coronary arteries

Beta endorphins- pain suppressors

Important Biotherapeutics

Production of Biotherapeutics

Microbes as Factories for Biotherapeutics:

The technology is relatively simpler compared with the other systems

In Vitro maintenance does not require special components

Their unicellular nature

Their genomes are simpler

Their ecological distribution is very diverse

Why we have to choose this technology

Potential of a Microbes

Microorganisms are capable of growing on a wide range of

substrates and can produce a remarkable spectrum of

products

Which Microbes are useful 

   Several species belonging to the following categories of microorganisms are useful

 PROKARYOTIC  Unicellular:     Bacteria,

Cyanobacteria    Multicellular: Cyanobacter

 EUKARYOTIC    Unicellular:      Yeasts, Algae                         Multicellular:    Fungi, Algae

Some potential microbes used in Microbial Technology

Bacillus Sp. Actinimycetes Sp.

Extra reading:Bacillus sp: Peter C. B. Turnbull; Chapter 15Bacillus. Medical Microbiology. 4th edition. http://www.ncbi.nlm.nih.gov/books/NBK7699/

Escherichia coli

Saccaromyces cerevisiae(Yeast)

*What is the main application of yeast in biopharmaceutical?

Many different cell factories are used for the production of biopharmaceuticals, but the yeast Saccharomyces cerevisiae is an important cell factory as it is used for production of several

large volume products. Insulin and insulin analogs are by far the dominating

biopharmaceuticals produced by yeast, and this will increase as the global insulin market is

expected to grow from USD12B in 2011 to more than USD32B by 2018. Other important

biopharmaceuticals produced by yeast are human serum albumin, hepatitis vaccines and

virus like particles used for vaccination against human papillomavirus (HPV). 

Bioengineered. 2013 Jul-Aug;4(4):207-11. doi: 10.4161/bioe.22856. Epub 2012 Nov 12.Production of biopharmaceutical proteins by yeast: advances through metabolic engineering.

Nielsen J.

Biologicals vs Conventional DrugsBiologicals

• Protein or carbohydrate based product

• Extracted from living organism

• Complex physicochemical structure

• Less well-defined Macromolecule (> 500 kd) Tertiary structure Location, extent and type of glycosylation

• Heat- & Shear- sensitive

Conventional Drugs

• Synthetic, organic compounds

• Defined structure, physical & chemical characteristics Chemical

synthesis Micromolecules Stable

More than 30 recombinant therapeutics have been approved globally for commercial use and several are on the way

In India, 12-15 of these are presently being marketed. Many of these are being imported (excepting few like Hepatitis B vaccine, Insulin etc.) and consumed and now several are underway for indigenous production

Globally approved recombinant therapeutics are broadly categorized into blood factors, hormones, growth factors, interferons, interleukins, vaccines and other miscellaneous therapeutic products

Some approved Therapeutics

The products of rDNA technology Hormones

Growth hormone, Insulin, Calcitonin, FSH

CytokinesInterferons (Interleukins), EPO,

CSF, Neurotrophic factors Clotting factors

Factor VIII, Factor IX Vaccines

Hepatitis B, acellular pertussis vaccine (Bordatella pertussis, whooping cough)

Monoclonal antibodies

Human Insulin StreptokinaseErythropoietin Hepatitis B vaccineHuman growth

hormoneHuman interleukin .

Granulocyte macrophage colony stimulating factor Alpha-interferon, Gamma-Interferon, Blood factor VIII Follicle stimulating hormone Granulocyte colony stimulating factor (GCSF),

Therapeutic products approved in India

The commercial production of recombinant biological and therapeutics now became an important area in global industrialization

The process developed in production of therapeutics has to be taken to industrial scale for implementing these developed process in industrial scale and to get viable industrial production of these therapeutics or biological mainly through Fermentation technology

Commercial production of Therapeutics

And any Fermentation process should go for

scale-up from

laboratory scale to

Industrial level

Through

Pilot scale Fermentors

Pilot scale Fermenter

Industrial Scale up process

Marlow Foods, UK

*Extra reading:http://www.quorn.co.uk/

World largest (50 m tall and 155,000 litre capacity) airlift fermenter (1994)

Production of Quorn™ myco-protein

Commercial Production of some important

ofBiotherapeutics

Insulin is an important hormone which regulates sugar metabolism

An inability to produce insulin results in a form of diabetes, this disease can be treated by daily injections of insulin

Historically, insulin from pigs or cows is used, but known to produce immune reactions in some patients

Challenge: how to make human insulin to be used as a drug in cell systems or microbes?

Insulin - first recombinant protein to be produced

Idea: take the gene of human insulin, clone into a plasmid, introduce the plasmid into E. coli or cells, and use them E.coli as “Biological Factory” for insulin production

Amino acid sequence produced insulin (Contains 51 amino acids) and is identical to that of the “natural human protein” and it will not cause any immune reactions

Much more economical than attempts to produce insulin by chemical synthesis

So, how to do this?

Recombinant Insulin overcome many problems

Strategy for insulin production

Insulin crystals from the purification

process

https://www.youtube.com/watch?v=IrotOPgSkR4

Owen Mumford Ltd., UK

VaccinesVaccines effective against many viral infections and diseases require the cultivation and mass production of the virus followed by its attenuation衰减

The drawback in this is that virus requires a living medium to replicate and multiply. Rather than the traditional concept- “Sacrifice one life to save many”, Animal cell culture can be employed to mass produce the virus

Passively, Animal cell culture can be employed to reduce the virulence of particular virus strains by cultivating them on cells other than target cells, in which the virus infection followed by repeated passaging will be performed

The cell-culture process for vaccines offers high potential as an alternative method to egg-based production. Cell culture has the capability to offer a predictable, rapid and responsive method for production of well-tolerated and effective vaccines, with low levels of adverse events similar to egg-based vaccines

Cell-culture materials can be stored, so the production process can be initiated at any time. In addition, production can be scaled up in response to increased vaccine demand

Recombinant Hepatitis vaccine The hepatitis B virus (HBV)

vaccine Originally based on the

surface antigen purified from the blood of chronically infected individuals.

Due to safety concerns, the HBV vaccine became the first to be produced using recombinant DNA technology (1986)

Produced in bakers’ yeast (Saccharomyces cerevisiae)

Electron micrograph of the hepatitis B virus

Recombinant Hepatitis B Vaccine

One of the most recent developments is the production of a vaccine against hepatitis B using genetically modified yeast cells

Hepatitis B is a viral infection which attacks the cells of the liver. It can be very serious, causing chronic liver failure, liver cancer and death

Hepatitis B can be prevented by a vaccination, and in countries like India where it is relatively common

Problems With The Early Traditional vaccine

For many years the vaccine was produced by growing the live virus in animals and then inactivating it by chemical treatment

This led to the risk of infection during the manufacture, and in the delivery of the vaccine, as well as raising animal rights issues

to respond to a human influenza pandemic.

to respond to a human influenza pandemic.

                                                                           

Vaccine Production at industry level

Some viral vaccines currently available for human and veterinary use

Effects of alkaloids on humans

High biological activity Produce varying degrees of physiological

and psychological responses - largely by interfering with neurotransmittersothers interfere with membrane transport,

protein synthesis or other processes In large doses - highly toxic - fatal In small doses, many have therapeutic

valuemuscle relaxants, tranquilizers, pain killers, mind

altering drugs, chemotherapy

Production of Alkaloids from Plants

Stem Cells

Alzheimer’s Disease Parkinson’s Disease Various Leukemias

Hodgkin’s Lymphoma Non-Hodgkin’s Lymphomas Immune Deficiency Disease

Liver Failure Heart Disease Diabetes

Stroke Multiple Sclerosis Huntington’s Disease

Osteoarthritis Rheumatoid Arthritis Coeliac Disease

Crohn’s Disease Lupus Erythematosus Periodontal Disease

Sickle Cell Anaemia Thalassemia Psoriasis

Deafness Blindness Osteoporosis

Spinal Injuries Burns Blackfan Diamond Anaemia

Fanconi Anaemia

Challenges in biopharmaceutical:Biotherapeutics are delicate drugs

Much larger and more complex than traditional pharmaceuticals

Composed of unstable proteins with a precise structure

Easily damaged by unfavorable temperature history during storage

Even insulin has temperature problems

Insulin is a very temperature stable biotherapeutic

A graph of storage life vs temperature shows a “saw tooth” peak

The product dies at both temperature extremes

Insulin Shelf-life

0.1

1

10

100

1000

-20 0 20 40 60

Temperature (C)

Mon

ths

Effect of temperature storage extremes

< 0o C

Freezing Protein denaturation Formation of

aggregates Loss of functional

activity Formation of

potentially hazardous immunogenic byproducts

> 8o C

Chemical side reactions Protein denaturation Formation of

aggregates Loss of functional

activity Formation of

potentially hazardous immunogenic byproducts