Post on 15-Jul-2015
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BIOPHARMACEUTICALSAn overview
Dr. Nitin C. Salvi,
Haffkine Bio-Pharmaceutical Corporation Limited,
Pimpri, Pune- 411 018.
9552274460 / nitinvibha@yahoo.com
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
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
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
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
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
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 ).
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
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
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
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
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