BIOPHARMACEUTICALSAn overview

Dr. Nitin C. Salvi,

Haffkine Bio-Pharmaceutical Corporation Limited,

Pimpri, Pune- 411 018.

9552274460 / nitinvibha@yahoo.com

CONTENTS

Biopharmaceuticals

Quality Assurance

Research & Development

Quality Control

Production

CONTENTS

Biopharmaceuticals

Biopharmaceuticals!!!

Biopharmaceuticals or Biotherapeutics or Biologicals

Biologically significant compounds like Hormones, Proteins (eg.antibodies) & Nucleic Acids (DNA & RNA)

Biopharmaceutical drugs are large, complex molecules derived from living cells.

Obtained from biological source and produced through industrial biotechnology

Useful for treatment of variety of human health disorders, fight cancer, viral infections, diabetes, hepatitis and multiple sclerosis

Biopharmaceuticals!!!

These are medical drugs produced using biotechnology especially

genetic engineering or

hybridoma technology or via

biopharmaceutical techniques such as recombinant DNA technology, gene transfer

antibody production methods

Conventional vaccines

Insulin-

First Recombinant Biopharmaceutical pdt

Classification of Biopharmaceuticals

First Generation Biopharmaceuticals unengineered murine monoclonal antibodies or simple

replacement proteins displaying an identical amino acid sequence to a native human protein.

Second Generation Biopharmaceuticals -engineered, products. alteration of amino acid sequence, glycocomponent of a

glycosylated protein,

covalent attachment of chemical moieties such as polyethylene glycol.

alter immunological or pharmacokinetic profile of protein, or in order to generate novel fusion products

Types of Biopharmaceuticals

Antisera

Cytokines – Interferon, Interleukins

Enzymes -

Hormones

Clotting factors

Vaccines

Monoclonal antibodies

Cell therapies

Antisense drugs

Peptide therapeutics

Types of Biopharmaceuticals

Enzymes – Activase, alteplase, TPA (dissolves blood clots)

Pulmozyme, dornase alfa , (a recombinant DNAse I that digests DNA in the mucous secretions in lungs)

Cerezyme, imiglucerase, (a recombinant glucocereborsidasefor Gaucherûs disease, bone destruction and enlargement

of the liver and spleen)

Alphanine SD, Benefix, Bebulin VH, Profilnine SD, Proplex T Factor IX, belonging to peptidase family S1, is one of the serine proteases of the coagulation system. Deficiency of this

protein causes hemophilia B

Types of Biopharmaceuticals

Horomones –

Insugen, Humulin, Novolin -- Insulin

Ascellacrin, Crescormon --human growth hormone

Ovidrel– gonadotrophins

Types of Biopharmaceuticals

MONOCLONAL ANTIBODIES

Produced by using Hybridoma Technology

Popularly known as “Magic bullets”

Used for the treatment of Rheumatoid Arthritis , cancers

Antibody produced by a single clone of cells

Types of Biopharmaceuticals

VACCINES

Biological preparation that improves immunity to a particular disease

They can be prophylactic or therapeutic

e.g. HBV vaccine--a Subunit Vaccine composed of only surface proteins, produced in the yeast

to respond to a human influenza pandemic.

to respond to a human influenza pandemic.

BiopharmaceuticalsUSAN/INN Trade

Name

Indication Technology Mechanism of

Action

abatacept Orencia rheumatoid arthritis

immunoglobin CTLA-

4 fusion protein

T-

cell deactivation

adalimumab Humira rheumatoid

arthritis, ankylosing

spondylitis, psoriatic arthritis,

psoriasis, Ulcerative

Colitis, Crohn's disease

monoclonal antibody TNF antagonist

alefacept Amevive chronic plaque psoriasis immunoglobin G1

fusion protein

incompletely

characterized

erythropoietin Epogen anemia arising from

cancer chemotherapy, chroni

c renal failure, etc.

recombinant protein

stimulation of red

blood cell

production

etanercept Enbrel rheumatoid arthritis,

ankylosing spondylitis,

psoriatic arthritis, psoriasis

recombinant human

TNF-receptor fusion

protein

TNF antagonist

infliximab

Remicad

e

rheumatoid arthritis,

ankylosing spondylitis,

psoriatic arthritis, psoriasis,

Ulcerative Colitus, Crohn's

disease

monoclonal antibody TNF antagonist

trastuzumab Herceptin breast cancer

humanized monoclon

al antibody

HER2/neu (erbB2

) antagonist

ustekinumab Stelara psoriasis

humanized monoclon

al antibody

IL-12 and IL-23

antagonist

denileukindiftito

x

Ontak cutaneous T-cell lymphoma

(CTCL)

Diphtheria toxin

engineered protein

combining Interleukin-

2 and Diphtheria toxin

Interleukin-

2 receptor binder

golimumab Simponi rheumatoid arthritis, psoriatic

arthritis, ankylosing

spondylitis, Crohn's disease

monoclonal antibody TNF antagonist

Advantages of Biopharmaceuticals

Highly effective

Highly specific

Fewer side effects

Not carcinogenic

Safe

Easy commercial production

Global Scenario

Indian Scenario

2,345 crore rupees -- 2002-2003

20,441 crore rupees --2011-2012

Domestic sales have exceeded the export revenues.

Indian Scenario

Company Segment Revenue 2011-2012 (crore rupees)

Serum Institute of India Biopharmaceutical 1708

Biocon Biopharmaceutical 1676.40

Nuziveedu seeds Bio-agriculture 745

Reliance Lifesciences Biopharmaceutical 693

NovoNordisk Biopharmaceutical 647.28

Future Trends Regulatory Trends

Increasing Government Support

IP Protection Enforcement

Regulatory Reforms (innovation, restrict imitation, outsourcing, VC)

Drug Price Cuts

Technology Trends (novel tchnologies/pdts)

Enterprise Development Trends

Strategic Alliances

Cluster Development

Increasing R & D investment (Estb Pdt Pipeline)

More International Clloaboration

Mammalian Cell Expression Drug Development

Key Factor

The main driver for FUTURE GROWTH

The key factor to compete and proper in the 21st century GLOBAL ECONOMY

INNOVATION

Components of any Biopharmaceutical Industry

Production

Quality Control

Quality Assurance

Research & Development

CONTENTS

Production

PRODUCTION

Upstream Protein Separation

Cell expansion Fermentation

Clarification --------------------

Downstream Centrifugation

Chromatography

Ultrafiltration

Antiserum or Antivenom Preparation

Venom !!!

Biotoxins - highly complex or relatively small protein. Two functions

Vary greatly in functions& mechanism

Predation –snakes, spider, scorpion, jellyfish, wasp

Defense – bee, ant, honeybee, frog, termite

Typically injected into prey or aggressors by biting or stinging or other sharp body feature.

Snake Bite

Snake bite is a acute life threatening time limiting medical emergency a occupational hazard often faced by farm labourers and farmers. It is in endemic form all over tropical countries like India.

WHO -Snake bite Neglected Tropical Disease.

GRAVITY

2.5 lakh snake bites per year in India. 35,000 to 50,000 deaths per year due to

snake bite in India. High mortality in Maharashtra, up to 5000

deaths per year High mortality in rural population. Death figure may be high. 3000 species of snakes are distributed

worldwide. 500 are venomous species. 52 venomous species are found in Indian subcontinent.

Common Indian Cobra (Nag)(Naja naja)

Avg. Venom Yield per

snake per milking

Liquid—(0.098-1.56 ml)Lyophilised(56.4-514.9mg)Avg. 126 mg

(Manyar)(0.033- 0.250 ml(1.25-18.89 mg) Avg. 8 mg

Russell’s Viper (Vipera russelli),GHONAS(0.192 -1.356 ml20 -277.5 mg)Avg. 76 mg

And the FIFTH PIT VIPER

KING COBRA

Medically Important Snakes

Dangerousness of snake species

Venom Characteristics

Lethality of the venom

Key Variables

Frequency of medical attention after a bite

Local or systemic envenomation

Fatal Bites

Long term consequences

Availability of antivenoms

Size of population at risk

Snake Venoms

Complex mixture of proteins and peptides

80-100 or more proteins

Small number of superfamilies

Enzymes

Nonenzymatic proteins

Snake Venom

Contains number of toxins and enzymes. It is a clear transparent, amber tinted fluid and contains.

1. Neurotoxin (Predominant in Elapids) 2. Cholinesterase (Predominant in Elapids) 3. Haemolysins (Predominant in Viper) 4. Thromboplastin (Predominant in Viper) 5. Fibrinolysins 6. Proteolysins 7. Cardiotoxin 8. Agglutinins 9. Coagulase, Hyaluronidase etc. 10 out of 26 in each venom with seasonal & Regional

variations in potency.

Superfamilies of Enzymes

Phospholipase A2 enzymes

Proteases

Serine proteases

Metalloproteases

Others

Nucleotidases

Amino acid oxidase

Acetylcholinesterase

Non-enzymatic snake venom proteins

Other than enzymes, snake venom contains

numerous non-enzymatic proteins, which

play an important role in toxicity of the

venom.

Examples of non-enzymatic snake venom

toxins are- 1. Neurotoxins 2. Cardiotoxins

3. Haemorrhagins 4. Myotoxins

5. Haemolysins

Antivenoms(Therapeutic Antibodies)

Vaccination -Active immunization

Hyperimmune sera Passive immunization

Polyvalent

Monovalent

Homologous

Heterologous

What is Antivenom ?

Anti venoms are also known as antivenenum, antivenine or antivenin

Anti venoms are preparations of intact or fragmented immunoglobulin G used for treatment of human or animal suffering from severe envenoming from the bites & stings of various venomous animals.

IgG

Large molecules -150 Kda

Four peptide chains. A tetrameric quaternary struct.

Linked by disulfide bonds.

The Fc regions of IgGs bear a highly conserved N-glycosylation site.

History

Anti venom is in existence for over a century.

Dr. Albert Calmette in 1894 demonstrated that protection could be imparted against venoms by immunising animals with low doses of venom.

In 1895, he saved a life of a severely bitten person by using anti venom raised in horses.

In India, Central Research Institute,Kasauli & Haffkine Institute, Mumbai have been producing snake anti venoms since 1925.

Are Antivenoms Safe

Adverse Reactions

Early Anaphylactic reactions

After 10-180 mins.

Pryogenic (endotoxic) reactions

After 1-2 hrs

Late Anaphylaxis reactions

After 1-12 days (mean 7 days)

Process

Horse Procurement -Open market / Army

Quarantine Period -One month TTD, Mallein, Virus Scr, Wt, Tmp,CBC LFT, KFT.

Immunization

Primary-Increasing sublethal doses with adjuvants

8-9 months Antibody titre obtained

Secondary immunization phase

Bleeding & Booster Doses

Bleeding and Plasma separation

Antibody Separation

Enzymatic Digestion – Pepsin

Precipitation by Ammonium sulphate (14-17% W/V)Thermocoagulation (550c –1 hr.)Microfiltration & Ultrafiltration

Formulation, Sterile Filtration & Lyophilisation

Enzymatic Cleavage of IgG

WHY EQUINES ARE USED?

Easy to Handle

Large volume of blood/plasma can be collected at periodic intervals

Well established and validated purification process for over 100 years

Very sensitive for venoms/toxins, hence excellent immuno conversion

90% of commercial therapeutic sera are equine origin

Anti venoms available in India

Polyvalent Snake Antivenin,I.P.

Effective against the bites of Indian Cobra(Najanaja), Russell’s Viper(Vipera russelli), Indian Common Krait(Bangarus coeruleus) & Saw Scaled Viper( Echis carinatus ).

Monovalent Scorpion Venom Antiserum,I.P.

Effective against the stings of Red Scorpion ( Buthus tamulus ).

CONTENTS

Quality Control

QUALITY CONTROL

Definition

Process or system for monitoring the quality of laboratory testing, and the accuracy and precision of results

Routinely collect and analyze data from every test run or procedure

Allows for immediate corrective action

QUALITY CONTROL

Designing a QC Program Establish written policies and procedures

Corrective action procedures

Train all staff

Design forms

Assure complete documentation and review

QUALITY CONTROL

Qualitative vs.Quantitative

Quantitative test

measures the amount of a substance present

Qualitative test

determines whether the substance being tested for is present or absent

QUALITY CONTROL (Biological)

Microbiology Environmental Water analysis FP,RM analysis: Sterility ,Pathogen testing

Biological Testing Potency Testing Abnormal toxicity Pyrogen testing BET

Inprocess Quality checks Stability testing

QUALITY CONTROL (Chemical)

Chemical and instrumental testing Water analysis WFI, PW, Steam and Raw water testing

FP,RM,PM analysis Inprocess Quality checks

Protein Estimation

Phenol testing

Stability testing Real time Accelerated studies

Process validation studies

QUALITY CONTROL

Data Analysis Select high quality controls Experimental Analysis Statistical analysis

Central tendency (Mean, Median Mode)

Variability (Range,Variance,SD, CV)

Develop Levey-Jennings chart

Monitor control values using the Levey-Jennings chart and/or Westgard rules

Take immediate corrective action, if neededRecord actions taken

QUALITY CONTROL Levey-Jennings Chart

QUALITY CONTROL

Quality Control is used to monitor both the precision and the accuracy of the assay in order to provide reliable results.

Accuracy and Precision

The degree of fluctuation in the measurements is indicative of the “precision” of the assay.

The closeness of measurements to the true value is indicative of the “accuracy” of the assay.

QUALITY CONTROL

Summary Establish written policies and procedures Assign responsibility for monitoring and

reviewing Train staff Obtain control materials Collect data Set target values (mean, SD) Establish Levey-Jennings charts Routinely plot control data Establish and implement troubleshooting and

corrective action protocols Establish and maintain system for

documentation

Purchasing & Inventory

AssessmentOccurrence

Management

Information Management

Process Improvement

Customer Service

Facilities & Safety

The Quality System

Organization Personnel Equipment

Documents & Records

Process Control

(QC & EQA) & Specimen

Management

CONTENTS

Quality Assurance

QUALITY ASSURANCE

Quality assurance -Wide ranging conceptcovering all matters that individually orcollectively influence the quality of a product.

It is the totality of the arrangements -ensurethat pharmaceutical products are of the qualityrequired for their intended use.

QA is the heart and soul of quality control QA = QC + GMP /Other Quality Systems

FINE QUALITY INPUT ONLY CAN GIVE YOU A FINE QUALITY OUTPUT.

QUALITY ASSURANCE

GMP (Good Manufacturing Practice)

It works in favor of Manufacturer Its focus is on Manufacturing

GMP consists of more technical operationsMandatory

ISO (International Organization for

Standardization) It work in favor of customer

Its focus is on product Quality ISO consists more Business operations

Optional

QUALITY ASSURANCE

The Five M’s of Quality Man

Material

Machinery

Manuals/Methodology ( SOP)

Motivation

QUALITY ASSURANCE

Activities of QA Technology transfer

Monitoring Production

Validation

Documentation

Assuring quality of products

Quality improvement plans

Training

Quality Risk Management

QUALITY ASSURANCE

Technology transfer

Checking and approval of documentsgenerated based on research centredocuments i.e. batch manufacturing record

Scale-up and validation of product

QUALITY ASSURANCE

Monitoring Production

All the production activities Utility Systems- HVAC, WFI, PSG, etc Personnel

Equipment, Operational & Process Qualification

Validation & calibration

QUALITY ASSURANCE

Validation Preparation of Validation Master plans for

facility/equipments/process Utility, Cleaning and all the sections of the validation

Approval of protocol for validation of facility/ equipment/product/ process/Utility

Team member for execution of validation of facility/equipment / product/ process

Final approval of the facility/ equipment/product/ process/Utility validation

QUALITY ASSURANCE

Documentation Standard operating procedures

Protocols of tests,

Results

Reports

Standard Operating Procedures An authorized written procedure giving instructions for

performing operations not necessarily specific to a given process, product or material (e.g. equipment operation, maintenance and cleaning; validation; cleaning of premises and environmental control; sampling and inspection).

QUALITY ASSURANCE

Assuring Quality of products cGMP training

SOP compliance

Audit of facility for compliance

Line clearance

In-process counter checks

Critical sampling

Record verification

Release of batch for marketing

Investigation of market complaints

QUALITY ASSURANCE

Quality improvement plan Feedback received from the compliance team

Customer complaint history

Proposals for corrective and preventive actions

Annual Products review

Trend analysis of various quality parameters for products,

environment and water

Review of the Deviations, Change Controls, Out Of

Specifications and Failures.

QUALITY ASSURANCE

Training

Induction training program

On the job training

Quality Risk Management

Assessment of risk analysis of every

process/activity in production QC

Quantifying or grading the probablity of

occurrence of activities

Taking measures to minimize the occurrence of

risks

Everyone is Quality Assurance

Quality Assurance is a dynamic process

Research areas in Anti venom production

Chicken Egg Yolk Antibodies High avidity antibodies

Reduced cross reactivity

Doesn’t bind to rheumatoid factors

Applications mostly diagnostic rather than clinical.

Camelid Antibodies Antibodies Antibodies devoid of Light Chains

Less immunogenic- least complement activation

Least anaphylactic & serum sickness reactions

High Titres & Avidity

Thermostable- supply chain

Universal Antivenom

CONTENTS

Research & Development

Recent trends in Anti venom production

VHH antibobies / Nanobodies

First single-domain antibodies were engineered from heavy chain antibodies found in camelids; these are called VHH fragments.

Advantages

genetic manipulation

Increased functional size of immune libraries

production of multivalent formats

production of oligoclonal preparations from

single cells

High physicochemical stability

High solubility

Recognition of hidden antigenic sites

Rapid tissue penetration, fast clearance

Well expressed

Research areas in Anti venom production

Equine Health Mapping of CBC, LFT, KFT

Nutritional immunopotentiaters

Control of equine diseases R.equii, Glanders

Interlukine mapping studies

Venomics Study of venoms

Variability of venoms due to size, age, geographical location

Design of effective antivenom (Mono / polyspecific)

Antivenoms may have paraspecific effects

--Cross neutralization

Research areas in Anti venom production

Immunization Science + Art

Poorly immunogenic antigens – long immu. Sch.

Variability immune response

Toxic stress

Increased cost of production

Immunization protocols

Low Dose Low volume multisite pro.with adjuvants

Newer Adjuvants - Liquid, Emulsion, Nanoparticle

Safety & Immunogenicity

Research areas in Anti venom production

Plasma Processcing Less Protein Aggregates

Pyrogen Free Antisera

Virus Free Antisera

Chromatographically purified antivenoms

Least protein & more neutralizing antibodies

Safety & Efficacy

Formulations Search of best stabilizers, preservatives

Lyohilization

Research areas in Anti venom production

Quality Control Water Analysis – TOC, Rapid Tests

Enviromental Monitoring – Rapid Tests

Raw material analysis (Method val.)

Sterility –Rapid tests

Endotoxin testing

Animal Testing 3 R’s (cytotoxic / ELISA/ LFA)

Research areas in Anti venom production

Quality Control Animal Testing 3 R’s (cytotoxic / ELISA/ LFA)

(European Centre for the Validation for Alternative Methods)

Reduce

The number of animals used.

Refine

In vivo test methods/ techniques by

Lessening or eliminating pain or distress to animal.

Replace

Partially or totally the use of animals with in vitro assays.

For Antirabies serum

Two Tests Comply: ELISA and RFFIT

Summary of Research Areas for Antivenoms

Knowledge based composition design of venom mixtures used for immunization

The number of animals used.

Careful selection and adequate management of animals used for immunizations.

Well designed immunization protocols.

Sound innovations in plasma fractionation – recovery, tolerability & stability of antivenoms.

Use of recombinant toxins as immunogens.

Synthesis of engineered antibodies to substitute for animal derived antivenoms.

Summary of Research Areas for Antivenoms

Scientific studies in existing manufacturing steps towards inactivation or removal of viruses and other zoonotic pathogens

Introduction of novel quality control tests

Devp. of invitro assays to substitute invivo assays

Scientifically sound preclinical and clinical assesment of antivenoms

THANKS