Biology 116-Biotechnology

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Biology 116-Biotechnology

• Ralph M. Sinibaldi, Ph.D.
• .

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Course Goals

• Technical training for research, development or
  production positions in biotech
• Conceptual training in molecular biology and
  biotechnology
• Biotech Industry overview
• Soft skill training
• Resumes
• Interviews
• Project teams and teamwork

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Learning Outcomes

• Describe the science of biotechnology and
  identify its product and company domains
• Give examples of careers and job responsibilities
  associated with biotechnology
• Understand and apply safety considerations and
  lab etiquette
• Describe how scientific methodologies are used to
  conduct experiments and develop products
• Understand and apply rules of documentation and
  intellectual property
• Describe what intellectual property is and why it
  is important in biotechnology
• Understand regulatory compliance and what
  agencies are responsible for it
• Describe the Human Genome project and be able to
  discuss its implications

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Vocabulary

• Insulin a protein that facilitates the uptake
  of sugar into cells from the blood
• DNA abbreviation for deoxyribonucleic acid, a
  double-stranded helical molecule that stores
  genetic information for the production of all of
  an organisms proteins
• Recombinant DNA (rDNA) technology cutting and
  recombining DNA molecules
• Polymerase chain reaction (PCR) a technique
  that involves copying short pieces of DNA and
  then making millions of copies in a short time
• Cloning method of asexual reproduction that
  produces identical organisms
• Fermentation a process by which, in an
  oxygen-deprived environment, a cell converts
  sugar into lactic acid or ethanol to create
  energy
• Diabetes a disorder affecting the uptake of
  sugar by cells, due to inadequate insulin
  production or ineffective use of insulin
• Proteases proteins whose function is to break
  down other proteins
• Antibodies proteins developed by the immune
  system that recognize specific molecules
  (antigens)
• Pharmaceutical relating to drugs developed for
  medical use

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Vocabulary

• Research and development (RD) refers to the
  early stages in product development that include
  discovery of the structure and function of a
  potential product and initial small-scale
  production
• Pure science scientific research whose main
  purpose is to enrich the scientific knowledge
  base
• Virus a particle containing a protein coat and
  genetic materials (either DNA or RNA) that is not
  living and requires a host to replicate
• Applied science the practice of utilizing
  scientific knowledge for practical purposes,
  including the manufacture of a product
• NIH abbreviation for National Institutes of
  Health the federal agency that funds and
  conducts biomedical research
• CDC abbreviation for Centers for Disease
  Control and Prevention national research center
  for developing and applying disease prevention
  and control, environmental health, and health
  promotion and education activities to improve
  public health
• DNA fingerprinting an experimental technique
  that is commonly used to identify individuals by
  distinguishing their unique DNA code

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New technology is neither inherently good or
harmful, this is determined by how man chooses to
use the technology
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What is Biotechnology?
Biology
Technology
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Defining Biotechnology
Biotechnology is defined as the study and
manipulation of living things or their component
molecules, cells, tissues, or organs.
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Biotechnology Business and Business Strategy
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Fact

• Most new Biotech Companies Ultimately Fail

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Domains of Biotechnology. The major domains of
biotechnology include 1) industrial and
environmental 2) medical/pharmaceutical 3)
agricultural and 4) diagnostic/research
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Types of Companies

• Product Development
• Advantages
• Therapeutic products with large markets
• Patent protection
• High gross margins
• Disadvantages
• High risk
• Long development times
• Platform Technologies
• Advantages
• Shorter development times
• Lower risk
• Disadvantages
• Highly competitive with ever changing technology

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Types of Companies

• Reagent
• Advantages
• Short development time
• High profit margins
• Disadvantages
• May not be proprietary
• Manufacturing costs driven
• Service
• Advantages
• No manufacturing
• Can be highly profitable
• Disadvantages
• Can underestimate costs

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Type of companies

• Equipment or Instruments
• Advantages
• Proprietary
• Can bundle with associated reagents
• Disadvantages
• Significant capital investment
• Lower margins on instruments

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Starting a Company

• An Idea or a Technology
• Projected product(s) or service(s)
• Market Analysis
• Business Plan

• Funding
• Seed round
• Friends Family
• Early Venture Capital Investor
• Angel Investor(s)
• A round
• Venture Capital
• Angel Investors
• B Round
• Venture Capital
• Corporate Investors or Partners
• C Round
• Exit Strategy
• IPO or Acquisition

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Business Plan

• Summary-two pages
• Market Opportunity
• Company background- stage type
• Market
• Market analysis
• Competitors
• Technology
• Proof of concept
• Similar technologies
• Expert opinions

• Intellectual property
• Patent applications
• Potential conflicts
• Development Plan
• Marketing Plan
• Distribution
• Management
• Org chart
• Bios of Principals
• Appendices

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Role of People

• Corporate structure
• Skill base of employees
• Building the right team
• Human resources system

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Technology

• Publications
• Patents
• Proof of concept for components
• Breadboard
• Full Working prototype

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Types of Finance

• Debt Financing
• Loans
• Credit
• Equity Financing
• Private stock
• Friends family
• Private investors
• Angel Investors
• Venture Capital funds
• Corporate partners

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Other Sources of Funding

• Grants
• SBIR
• NIH, NSF, USDA, NASA, NIST
• Stage I- 100,000.00
• Stage II- 750,000.00 to 1 million
• ATP- 2 million up to 32 million
• DARPA- national defense applications
• Corporate partnerships
• Marketing Distribution relationship
• Equity

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What appeals to investors

• Technology
• Business Plan
• Management Team
• Multiple Products

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Compensation

• Salary
• Bonus -10 to 30 of salary
• Must achieve aggressive goals
• Stock options
• Founders
• Employee

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Corporate Structure Hierarchical
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Corporate Structure-Matrix
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Corporate Structure-Hybrid

• Hierarchical Matrix Combined
• Departmental Organization
• Multidisciplinary Project Teams

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Decision Making

• Technology-based
• Research
• Manufacturing
• Resource-based
• Marketing-based

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Sustainable Business

• Reducing Chances
• Large and Unpredictable Capital Requirements
• Long Product Development Cycles
• Regulatory Issues with Product
• Rapidly Changing Market Forces
• High Probability of Late Stage Product Failure
• Rare Instances of Sustained Profits

• Increasing Chances
• Capital Requirements Kept Low
• Well-Defined, Predictable Business Milestones
• Clear, Market-Oriented Business Plan
• Critical Mass to Successfully Compete
• Experienced Management Relevant to Strategy Being
  Pursued

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Evolution of Company

• Production-based
• Technology-based
• Market-based

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Marketing SWOT analysis

• Strengths
• Weaknesses
• Opportunities
• Threats

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Safety
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Safety Values

• Safety is not just a priority but a value
• Safety is an unwritten rule, a special norm, the
  workers should follow in all circumstances
• It is a value that is never questioned or
  compromised

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Safety Habits

• Safe for you and me
• Prevent accidents by noticing at-risk situations
  and behaviors
• Live safely at home, at work, and everywhere you
  go
• Teach an attitude, promoting safety

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Personal Safety

• Right to work in a safe workplace
• Responsibility
• Protect your circle of safety and know how it may
  influence others
• Illness and Injury prevention program

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Work Environment

• Organize safety for everyone
• Remove tripping hazards
• Do not store heavy items up high where they may
  fall
• Do not rush or run in the workplace
• Cleanup any liquid spills immediately
• Report any potential hazards

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Stress can lead to accidents

• Recognize personal burn-out
• Get enough sleep
• Get professional help
• Respect emotions of coworkers
• Develop active listening skills
• Develop positive, healthy relationships with
  coworkers

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Emergencies

• Medical response
• Earthquake
• Fire
• Chemical spills
• Regional disasters

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What to do

• Know emergency numbers-911 etc.
• Be prepared and have a plan
• Follow plan
• Stay calm
• Consider immediate need and response
• Communicate with others
• Know safety procedures, tools escape routes

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Neighborhood or regional disaster

• Home communication plan
• Know alternative routes
• Know who are your neighbors
• Be a good citizen
• You may have to stay where you are

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Emergency Evacuation Plan

• Assist those who need help to get to the
  protected area
• Know who is present and absent
• Communicate with other tenants
• Be prepared for first aid and medical responses

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Medical responses

• Immediate first aid
• Notify response teams, call 911
• Provide assistance and comfort
• Transport to trauma or urgent care facility

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Earthquake Safety

• Stay calm, shield yourself from falling objects
• Prevent falling objects by storing heavy objects
  low and tie down equipment
• Keep aisles and routes clear
• Follow evacuation plan

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Fire Safety

• Report fires immediately-response time is
  critical
• Know locations of fire fighting equipment
• Extinguishers
• Fire blankets
• Fire alarm
• Know when to evacuate get everyone out
• If smoke is present stay low, crawl if necessary
• Know evacuation route

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Fire Extinguishers

• Classification
• A- Ordinary combustible
• B- Flammable Liquid
• C- Electrical
• D- Combustible metal
• P-A-S-S
• Pull-Aim-Squeeze-Sweep
• Aim at the base of the fire and sweep
• Limited time and quantity of extinguishing
  material

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Personal Protection

• Actively work to prevent avoid accidents
• Protect working space
• Protect coworkers
• Secondary containment- create boundaries layers
  of safety appropriate for conditions and scale of
  work

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Working with hazards

• Create a safety zone, CONTAIN
• Know the hazard, PROTECT
• Protect yourself
• Protect those around you
• Protect environment around you
• Safe to touch, DECONTAMINATE
• Secondary tertiary zones reduce the chances of
  injury or disaster

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Personal safety attire

• Lab coat
• Safety glasses
• Closed-toed shoes
• Gloves when appropriate

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Chemical safety

• Know the hazards-MSDS sheets
• Specialized training may be necessary
• Proper storage of chemicals
• Use proven well thought-out protocols
• Additional personal protection attire may be
  required
• Face shield
• Chemical goggles
• Latex gloves and aprons
• Additional shielding
• Adequate ventilation
• Proper disposal of chemicals

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Radiation safety

• Proper training
• Shielding
• Monitoring equipment
• Geiger counter
• Wipe tests
• Proper storage and disposal of radioactive
  materials

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Radiation Safety

• Commonly used isotopes
• 14C, 35S, 32P, 3H, 125I, 131I
• Geiger counters
• Different probes
• Scintillation Counters
• Radiation exposure badges

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Lab Etiquette Lab Operation
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Common Courtesy

• Do not use the last of a reagent and not replace
  it
• Do not use other peoples equipment and reagents
  without asking
• Keep your work area and common work areas clean
  and orderly
• Do not play the radio/music without consulting
  others in the work area
• Be willing to work as a team on all projects
• Dress appropriately including avoiding excess
  perfume/cologne

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Levels of Operation

• Sterile reagents
• Liquids autoclave at 121º C for 15-20 minutes
  using slow exhaust. Alternatively, reagents can
  be filter-sterilized using a 45 or 22 micron
  filter
• Glassware autoclaved and cover with aluminum
  foil.
• Plastic ware is sterile
• Bottles/reagents may be needed to be flamed when
  opened or opened in a sterile environment
  (laminar flow hood)
• RNase-free
• Liquids sterilized for 1 hour or made with
  Rnase-free reagents and solvents.
• Glassware treated in an oven for several hours
  and covered with foil
• Reagents must be RNase-free
• Clean room conditions
• Dress and garb appropriately for the level of
  clean room
• May include no makeup and cologne

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Documentation
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Documentation System

• Corporate Policy Procedures
• Department Policy Procedures
• Quality System Requirements
• Management Control
• Traceability, Records Archival

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Quality System

• Each manufacturer shall establish and maintain a
  quality system that is appropriate for the
  specific medical device(s) designed or
  manufactured, and that meets the requirements of
  this part

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Quality System Requirements

• Management responsibility
• Quality Policy- commitment to quality that is
  understood, implemented and maintained at all
  levels
• Organization- assigned responsibility and
  independent authority, adequate resources,
  effectively establish, effectively maintain,
  review, quality plan, quality procedures
• Quality Audit- independent documented
• Personal- qualifications training
• Made aware of device defects which may occur from
  improper performance of theirs specific jobs
• Made aware of defects errors in verification
  validation

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Quality System Subparts

• Subpart B- Quality system requirements
• Subpart C- Design controls
• Subpart D- Document controls
• Subpart E- Purchasing controls
• Subpart F- Identification traceability
• Subpart G- Production process controls
• Subpart H- Acceptance activities

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Quality System Subparts

• Subpart I- Nonconforming product
• Subpart J- Corrective preventive action
• Subpart K- Labeling packaging control
• Subpart L- Handling, storage,distribution and
  installation
• Subpart M- Records
• Subpart N- Servicing
• Subpart O- Statistical techniques

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Subpart D- Document Controls

• Each manufacturer shall establish and maintain
  procedures to control all the documents required.
  The procedures shall provide for the following
• Shall designate an individual(s) to review for
  adequacy and approve prior to issuance
• Date and signatures of approval
• Available where needed and obsolete documents
  removed
• Changes reviewed, approve, documented, described,
  recorded, identity documents affected,communicated
  and effective date noted

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What is a Document?

• Legal perspective- any scrap of paper that has
  written information
• A memo, email,letter, note, meeting minutes
• Notebook entry, patent application, report
• Plan, protocol, written instruction, procedure,
  policy statement
• Label, tag, placard, sign, flowchart,
  blueprint,design description
• Formal documentation, contracts, licenses,
  publications, marketing ads, regulatory
  submissions

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Document Chain

• Quality requirement, quality procedure, corporate
  policy, Mfg process, records of work, history
  files, legal contracts, Dept specific procedures,
  communication, personnel, training, reports, etc.
• Request forms, Drafts, revision control,
  Identification, approval process,
  signatures,dates, archival, accessibility

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Material Chain

• Acceptable design and supply, vendor, identity,
  purchasing, receiving, inspection, acceptance,
  raw material, storage inventory, use, in-process,
  finished good, labeling, packaging,
  qualification, storage, distribution, customer,
  non-conformance, complaint,retention practices,
  disqualification, disposition, records

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Subpart M- Records

• All records required by this part shall be
  maintained at the manufacturing establishment or
  other location that is reasonably accessible to
  responsible officials of the manufacturer and to
  employees of FDA designated to perform
  inspections
• Such records, including those not stored at the
  inspected establishment, shall be made readily
  available for review and copying by FDA employees
• Such records shall be legible and shall be stored
  to minimize deterioration and to prevent loss
• Those records stored in automated data processing
  systems shall be backed up

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Confidentiality Retention

• The firm should be encouraged to mark records
  they feel are confidential to assist the FDA in
  determining what information may be disclosed
  under the freedom of Information Act (FOIA)
• Impress upon the manufacturers that marking all
  copies of records and documents confidential does
  not aid the FDA in making its FOIA determination
• Records required by the QS/GMP must be retained
  by the manufacturer for a period of time
  equivalent to the design and expected life of the
  device, but in no case less than 2 years from the
  date of release for commercial distribution by
  the manufacturer

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Records and Reports

• Final report
• Name address of facility performing study
• Objective and procedures in approved protocol
• Statistical methods, transformation of data,
  calculations
• Test articles control articles (include
  stability), test system, dosage
• Describe circumstances that may affect quality
  integrity of data
• Name study director, other professionals,
  scientists
• Signed and dated reports of each individual
• Location of data records, specimens, final
  report
• QA statement of completion
• Signature of study director
• Amendments to report, signed
• Storage, retention, retrieval, of records data,
  specimens

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Notebook Entry

• Title, date, who, witness (legal, patent)
• Purpose, materials methods
• TRACEABILITY- Identify equipment, and source of
  materials protocols used
• Factual Statements for observations and
  conclusions
• Avoid unsupportable claims or leading suggestions
  for follow-up

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Development Report

• Title, project identity, investigators, date,
  distribution
• Summarize, show linkage to records
• Objective and outcome
• Protocol test methods
• The facts- results and conclusions
• The importance (simple and realistic)

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Validation Report

• Title and identity, controlled document
• Reference approved validation protocol
• Object and outcome, clear conclusion
• Was the method, process, product validated?
• How?
• Results vs acceptance parameters
• Archive record, design history file

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How to use documents

• Use approved, effective documents, or documents
  identified for approved protocols
• Follow the procedure
• Indelible ink (black), legible, in designated
  fields for entering information
• No extraneous entries!! Record deviations from
  procedure by creating separate document
• Sign and date
• The job is not finished until documented!

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Technical Writing

• DELIVER THE MESSAGE- communicate the objective,
  scope and outcome
• DELIVER THE HOW- communicate the means, source of
  records, raw data and conclusions
• DELIVER THE SO WHAT- communicate the importance
  of the findings, the relevance to the business,
  project, process or system

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Intellectual Property and Compliance
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Intellectual Property

• Laboratory notebooks
• Content Witnessing
• Disclosures of invention
• Priority dates
• Confidential Information
• Trade secret vs Patent
• Patents
• Compositions of matter, Process or procedure,
  Articles of Manufacture, Machines and
  Improvements
• Types of Patents
• Utility, Design and Plant
• Patent Criteria
• Conception, Reduction to practice, Utility,
  Novelty, Obviousness

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Proper Research Notebooks

• Physical requirements
• Bound notebook ( no removable pages)
• Permanent ink ( Blue or Black)
• Content
• Purpose of experiment
• Materials and Methods
• Results
• Pictures and graphs pasted in have to be signed
  across
• Discussion and Conclusions
• New inventions are recorded
• Witnessing
• Who should witness and how often?

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Witnessing Lab Notebooks

• Who ?
• Someone familiar with the research
• It should not be a colleague working on the same
  project
• Why not? They may be an inventor if they have
  contributed know how
• How often?
• Every week or two weeks

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Disclosure of Invention

• Some companies require as the second step in
  pursuing a patent
• Refers to initial notebook entry
• Can include a brief mention of related technology
  and prior art
• Who is the inventor or inventors?
• Inventors must contribute to the conception of
  the idea
• People or staff who perform the experiments are
  not inventors unless they contribute
  intellectually

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Trade Secret or Patent

• Trade secret
• When the process or formulation is not novel
• When it can be easily used by competitors without
  the knowledge of inventor
• Can last indefinitely
• Patenting is publishing exactly how something is
  made or produced
• Patent to protect the inventor from others using
  his invention or idea
• Patents can be licensed to others for a fee
  and/or royalty
• Patents are not intended to create a monopoly
• Patents last 20 years

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What can be patented

• Compositions of matter
• A new chemical entity produced from a combination
  of two or more compounds
• Common in agricultural pharmaceutical research
• Process or procedures
• A series of steps that are followed to synthesize
  a new compound or make a new product
• Articles of manufacture
• Nearly every man-made object
• Machines
• Any mechanical or electrical apparatus/device
• Improvements on any of the previous

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Types of Patents

• Utility Patent
• Most common and most difficult
• Functional characteristics of machines, devices,
  compounds
• Exhaustive description of how to make and use the
  invention including drawings
• Duration is 20 years
• Design Patent
• Protects the shape and ornamental design of an
  article
• 14 year duration
• Plant Patent
• New plant variety awarded for 20 years

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Patent Criteria

• Conception
• Formulation of the invention detailed enough to
  allow a person knowledgeable in the field to make
  and use the invention
• Reduction to practice
• Inventor makes or constructs the invention to
  demonstrate its usefullness
• Utility
• Invention must be useful or have utility
• Novelty or prior art
• Must not be a copy or a repetition of an existing
  invention
• Obviousness
• The invention should not be obvious to some one
  well-practiced in the field

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Filing a Patent

• Filing fee
• The applicant is required to pay a fee for the
  processing of the application
• Search examination
• The examiner will conduct a prior art search to
  ascertain novelty and evaluate the claims to
  establish the scope of the invention
• Publication
• Sucessful applications will be published
• Maintenance fees
• Applicant must pay periodic maintenance fees

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Parts of a Patent

• Title
• Inventors
• Assignee- the company or entity who is assigned
  ownership of the patent
• Abstract
• Summary of invention
• Detailed description of invention
• Figures and drawings
• Claims
• Establish scope of the invention

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Patent Strategy

• Patenting life forms and genes
• Easier following 1980 US Supreme court ruling,
  Diamond vs Chankrabarty
• Reach-through patents
• Patenting of genes based on their sequence but
  having no idea about their function
• Patent stacking
• Situation where more than one scientist has filed
  a patent on a gene

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Making Money on Patents

• Assignment- patent or patent application of
  invention can be sold or assigned to another
  party
• License- the patent may be licensed to another
  party. This may include a licensing fee and
  royalties
• Cross licensing- a situation where multiple
  patents cover the same or similar areas exist and
  the owners of such patents may have to cross
  license each others patents to exploit the
  invention

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Regulatory Compliance
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Regulatory Compliance

• US agencies their roles
• Food and Drug Agency (FDA)
• GLP and GMP
• Standard Operating Procedures (SOPs)
• United States Dept of Agriculture (USDA-APHIS)
• Environmental Protection Agency (EPA)
• National Institutes of Health (NIH)
• Office of Recombinant DNA
• Drug Development
• GLPGMP
• ISO 9000

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US Regulatory Oversight in Biotech
Agency Products Regulated
US Dept of Agriculture Plant pests, plants and veterinary biologics
Environmental Protection Agency Microbial/plant pesticides,new uses of existing pesticides, novel microorganisms
Food and Drug Administration Food, feed, food additives, veterinary drugs, human drugs, medical devices, diagnostics
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USDA and APHIS

• APHIS is authorized to regulate the interstate
  movement importation and field testing of
  organisms and products altered or produceds
  through biotech processes that are plant pests or
  suspected of being so.
• Permit for movement and importation
• Organism, origin and its intended use
• Permit for release into environment
• Oversight of field testing of biotech products
• Genes and gene products, origin, purpose of test,
  experimental design,and precautions to prevent
  escape
• Courtesy permits
• Involves non regulated plants
• Can involve intrastate movement

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FDA

• Unexpected effects- unexpected genetic effects
• Known toxicants
• Nutrient level
• Allergenicity
• New Substances
• Antibiotic resistance selectable marker
• Plants developed to make specialty nonfood
  substances
• Issue specific to animal feed

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Research and Development
Vocabulary

• Reagent chemical used in an experiment
• Efficacy the ability to yield a desired result
  or demonstrate that a product does what it claims
  to do
• Large-scale production the manufacture of large
  volumes of a product
• Clinical trials a strict series of tests that
  evaluates the effectiveness and safety of a
  medical treatment in humans
• FDA abbreviation for the Food and Drug
  Administration the federal agency that regulates
  the use and production of food, feed, food
  additives, veterinary drugs, human drugs, and
  medical devices
• Cystic fibrosis (CF) genetic disorder that
  clogs the respiratory and digestive systems with
  mucus
• Therapeutic an agent that is used to treat
  diseases or disorders
• EPA abbreviation for the Environmental
  Protection Agency the federal agency that
  enforces environmental laws including the use and
  production of microorganisms, herbicides,
  pesticides, and genetically modified
  microorganisms
• USDA abbreviation for United States Department
  of Agriculture the federal agency that regulates
  the use and production of plants, plant products,
  plant tests, veterinary supplies and medications,
  and genetically modified plants and animals

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Good Laboratory Practice (GLP)

• A very consistent way of performing and
  documenting research development work
• All documented experiments are performed in a
  consistent fashion and are witnessed in a timely
  and consistent fashion
• Procedures are validated
• Reagents are validated and listed
• Instruments and equipment that are utilized in
  experiments are routinely calibrated and
  validated
• FDA monitored

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Good Manufacturing Practice (GMP)

• All procedures used in manufacturing are
  consistent, fully validated and witnessed
• Use Standard Operating Procedures (SOPs)
• Reagents, chemicals and equipment are specified,
  validated and calibrated
• Testing equipment specified and routinely
  calibrated
• Some drugs need to be produced in a sterile
  environment
• The sterility of the manufacturing environment
  needs to be monitored and documented
• FDA monitored

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Standard Operating Procedure

• Detailed specific protocol
• Steps may be monitored or witnessed
• Reagents specified
• Grade
• Source or manufacturer
• Equipment specified
• Manufacturer
• Model number
• Equipment calibration
• Calibration method
• Calibration frequency
• Calibration log
• Calibrations are witnessed

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Iso 9000 or above

• Standard ways of doing business and documenting
  it
• In addition to manufacturing practices it can
  include
• Shipping
• Maintenance of plant and equipment
• Order taking
• Customer and technical service
• Handling of complaints
• Communications
• Needed for world marketing and distribution

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Scientific Method

• Codefined and promoted in 17th century by Rene
  Decartes and Francis Bacon
• Steps involved in scientific method
• Make observations
• Ask questions
• Make educated guesses about possible answers
• Base predictions on the guesses
• Devise ways to test predictions
• Draw conclusions

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Scientific Method

• Hypothesis educated guess based on
  observations and questioning
• Predicted result occurs hypothesis is most
  likely correct
• Individuals using scientific method should be
  objective and unbiased

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The Scientific Method
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Scientific Method
Original Hypothesis
Devise method to test hypothesis
Analyze results
Results support hypothesis
Results support hypothesis but suggest minor
refinements
Results do not support original hypothesis but
fall within range that could be expected if
original hypothesis is slightly modified
Results are so unexpected that they do not
support original hypothesis and require a new
hypothesis
Retest using minor refinements of process
Test new hypotheses
Test using slightly modified hypothesis
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Observe
Ask Questions
Formulate Hypothesis Derive Predictions
Test Hypothesis Perform Experiments Analyze
Data
Evaluate outcome
No
Hypothesis supported
No
New Hypothesis
Curiosity satisfied
Move onto another topic
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Scientific Method Experimental Design

• Testable hypothesis
• One variable at a time
• Positive controls
• Negative controls
• Background determinations
• Data Normalization

100
Human Genome Project
101
The Human Genome Project

• Determining the human DNA sequence
• Understanding the function of the human genetic
  code
• Identifying all of the genes
• Determining their functions
• Understanding how and when genes are turned on
  and off throughout the lifetime of an individual

102
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HGP (1990 2003 ) Participants

• US DOE
• NIH
• UK Medical Research Council and Wellcome Trust,
  UK
• 18 countries including France, Japan, Germany and
  China

104
Goals of HGP

1. Identify all the approximate 25,000 genes in
   human DNA.
2. Determine the sequences of the 3 billion chemical
   base pairs that make up human DNA.
3. Store this information in databases
4. Improve tools for data analysis,
5. Transfer related technologies to the private
   sector and
6. Address the ethical, legal, and social issues
   (ELSI) that may arise from the project.

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Methodology

• DNA Source
• Mapping
• Genetic Linkage Map
• Physical Map
• DNA sequencing
• Clone by clone sequencing
• Whole Genome Shotgun sequencing
• Assembling

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Genetic Linkage Map

• Distance between markers (genes) are determined
  by meiotic recombinational frequencies between
  the markers (or genes).
• Gives only an estimate of the distance between
  markers or genes
• Unit of measurement cM (centiMorgans)

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Construction of Genetic Linkage Map
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Physical Map

• Constructed from information obtained from the
  chemical characteristics of the DNA itself and
  not from the genetic recombination analysis.
• Unit of Measurement bp (basepair). Hence, more
  precise and exact in pinpointing the location and
  distance of the genes.

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2 Types of Physical Maps

• Low resolution
• Chromosomal (Cytogenetic) map
• cDNA map
• High resolution
• Top-Down Mapping
• Bottom-up Mapping

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Top Down Bottom Up
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Genetic Map VS. Physical Map
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Automated sequencers ABI 3700 and MegaBACE
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AUTOMATED SEQUENCE GEL
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AUTOMATED DNA SEQUENCE
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Methodology

• DNA Source
• Mapping
• Genetic Linkage Map
• Physical Map
• DNA sequencing
• Clone by clone sequencing
• Whole Genome Shotgun sequencing
• Assembling
• GigAssembler

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Result by the numbers

• The human genome contains 3164.7 million chemical
  nucleotide bases (A, C, T, and G).
• The average gene consists of 3000 bases, but
  sizes vary greatly, with the largest known human
  gene being dystrophin at 2.4 million bases.
• The total number of genes is estimated at 20,000
  to 25,000much lower than previous estimates of
  80,000 to 140,000.
• Almost all (99.9) nucleotide bases are exactly
  the same in all people.
• The functions are unknown for over 30 of
  discovered genes.

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Results Contd.

• Less than 2 of the genome codes for proteins.
• Repeated sequences that do not code for proteins
  ("junk DNA") make up at least 50 of the human
  genome.
• Repetitive sequences are thought to have no
  direct functions, but they shed light on
  chromosome structure and dynamics.
• During the past 50 million years, a dramatic
  decrease seems to have occurred in the rate of
  accumulation of repeats in the human genome.

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Genome Facts

• The human genome's gene-dense "urban centers" are
  predominantly composed of the DNA building blocks
  G and C.
• In contrast, the gene-poor "deserts" are rich in
  the DNA building blocks A and T.
• Genes appear to be concentrated in random areas
  along the genome, with vast expanses of
  non-coding DNA between.
• Stretches of up to 30,000 C and G bases repeating
  over and over often occur adjacent to gene-rich
  areas, forming a barrier between the genes and
  the "junk DNA.
• Chromosome 1 has the most genes (2968), and the Y
  chromosome has the fewest (231).

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Area Goal Achieved Date Achieved
Genetic Map 2- to 5-cM resolution map (600 1,500 markers) 1-cM resolution map (3,000 markers) September 1994
Physical Map 30,000 STSs 52,000 STSs October 1998
DNA Sequence 95 of gene-containing part of human sequence finished to 99.99 accuracy 99 of gene-containing part of human sequence finished to 99.99 accuracy April 2003
Capacity and Cost of Finished Sequence Sequence 500 Mb/year at lt 0.25 per finished base Sequence gt1,400Mb/year at lt0.09 per finished base November 2002
Human Sequence Variation 100,000 mapped human SNPs 3.7 million mapped human SNPs February 2003
Gene Identification Full-length human cDNAs 15,000 full-length human cDNAs March 2003
Model Organisms Complete genome sequences of E. coli, S. cerevisiae, C. elegans, D. melanogaster Finished genome sequences of E. coli, S. cerevisiae, C. elegans, D. melanogaster, plus whole-genome drafts of several others, including C. briggsae, D. pseudoobscura, mouse and rat April 2003
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Endeavors after HGP
Transcriptomics - involves large-scale analysis
of messenger RNAs transcribed from active genes
to follow when, where, and under what conditions
genes are expressed. Proteomics - can bring
researchers closer to what's actually happening
in the cell than gene-expression
studies. Structural genomics - initiatives are
being launched worldwide to generate the 3-D
structures of one or more proteins from each
protein family, thus offering clues to function
and biological targets for drug design.
Comparative genomics - analyzing DNA sequence
patterns of humans and well-studied model
organisms side-by-sidehas become one of the most
powerful strategies for identifying human genes
and interpreting their function.
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Summary of Human Genome Project

• Introduction
• Background and History of HGP (1990-2003)
• Methodology
• DNA source
• Genome Map
• Genetic Linkage Map
• Physical Map Low Resolution and High Resolution
  
• DNA sequencing
• Clone-by-clone sequencing
• Whole Genome shotgun sequencing
• Assembling
• GigAssembler
• Results
• Completed Human Genome Sequencing
• Identified 15,000 genes
• Future
• Applications in the field of Medicine, Forensics,
  Environment etc.,
• Further research in the fields of
  Transcriptomics, Proteomics, Structural and
  Comparative Genomics

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Resourceful information available at

• DOE website www.doegenomics.org
• National Human Genome Research Institute
  www.genome.gov
• Joint Genome Institute www.jgi.doe.gov
• National Center for Biotechnology Institute
  www.ncbi.nlm.nih.gov