Amgen Intro to Bio by uri703

10/20/2021

Introduction to Biopharmaceutical Manufacturing October 20‐22, 2021

Instructors

 Beth Zielinski‐Habershaw, Ph.D., PDI, The University of Rhode Island  Sharon McGuire, MS, PDI, The University of Rhode Island  Cahil McGovern, Ph.D., PDI, The University of Rhode Island  Malcolm Pluskal, Ph.D., Landrau Scientific Innovations

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Day 1

Industry Status Update 2021

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2018 was a record year for new drug approvals FDA Expedited Pathways and Other Approval Attributes 2017 and 2018 Designation or Attribute

2017 (N = 46)

2018 (N = 59)

Orphan Designation

18 (39%)

34 (58%)

Fast Track Designation

18 (39%)

24 (41%)

Breakthrough Therapy Designation

17 (38%)

14 (24%)

Priority Review

28 (61%)

43 (73%)

Accelerated Approval

6 (13%)

4 (7%)

First‐Cycle Approval

39 (85%)

56 (95%)

First Approved in US

36 (78%)

42 (71%) Source: https://www.raps.org

What about 2020?

Source: fda.gov/news‐events Source: https://www.raps.org

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Registered Studies as of February 4, 2020 ClinicalTrials.gov currently lists 329,173 studies with locations in all 50 States and in 209 countries

Location

Number of Registered Studies and Percentage of Total (as of February 4, 2020)

Non‐U.S. only

161,669 (49%)

U.S. only

111,377 (34%)

Both U.S. and non‐U.S.

17,133 (5%)

Not provided

38,994 (12%)

Total

329,173 (100%) Source: https://clinicaltrials.gov

Registered Studies as of February 4, 2021 ClinicalTrials.gov currently lists 364,943 studies with locations in all 50 States and in 209 countries

Location

Number of Registered Studies and Percentage of Total (as of January 24, 2021)

Non‐U.S. only

183,505 (50%)

U.S. only

120,000 (33%)

Both U.S. and non‐U.S.

18,754 (5%)

Not provided

42,864 (12%)

Total

364,943 (100%) Source: https://clinicaltrials.gov

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Clinical Studies include:  Biologicals  New molecular identities  Small molecules  Cell Therapy*  Gene Therapy*  Medical Devices  Combination products  Biosimilars

Intense Efforts

Along with the Biologicals, things to watch on the horizon….  CART Cell Therapies….  Chimeric antigen receptor T cells are T cells that have been genetically engineered to produce an artificial T‐cell receptor for use in immunotherapy.  Kymriah CAR‐T therapy (Novartis): $475,000  Yescarta CAR‐T therapy (Gilead): $373,000

Developmental areas: logistics: cryopreservation and thawing

 Autologous vs. Allogeneic (treating individuals vs. treating populations)  Forecast  Can anticipate 50 new cell therapy approvals in the next 2‐3 years

 Cell therapies‐ others    

Blood vessels Skin 3D printed organs Vaccines

Source: https://weillcornell.org/

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Autologous vs. Allogeneic Therapies  Autologous (treating individuals‐the starting material and finished product are the same cells)  Immunogenic benefits  Patient variability  Small scale manufacturing lots

 Allogeneic (treating populations)  Less complex in nature‐use of characterized cell banks  Potential immunogenicity  Potential tumor formation

Autologous Manufacturing Process Source: BioProcess International April, 2019 page 16

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Gene Therapy….  Gene Therapies….  Replacing faulty genes with the corrected version using viral delivery systems  Luxturna (Spark): $425,000 (per eye)  Zolgensma (Novartis): $2,100,000  LentiGlobin/Zynteglo (Bluebird): $1,800,000

 Candidates in development include:  Hemophilia (Phase 3‐Spark/Pfizer)  ALS (Phase 1‐ALS Association)  Retinitis Pigmentosa (SKYLINE; Phase 2)

Source: http://www.alsa.org/

Pharmaceutical Products vs. Biological Products  Pharmaceutical Products are more commonly known as medicines or drugs which are chemically produced and have low molecular weights. Typically, these have simpler structures.  Biological Products are more complex products which typically are produced through living systems, such as microorganism, plant cells, or animal cells and are often more difficult to characterize than traditional pharmaceuticals. These also have high molecular weights and have complex structures (primary through tertiary structures).

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Pharmaceutical Products vs. Biological Products

Source: https://www.google.com/search?q=small+chemical+molecule+vs+complex+biologic

Example: Ibuprofen vs. CNTF  Ibuprofen (C13H18O2)

 anti‐inflammatory  molecular mass of about 206 Da

 CNTF (C1018H1598N288O298S5)

 neuroprotective molecule  approximately 200 amino acids  molecular weight of 23,000 Da (relatively small compared antibodies which are in the 100‐150 KDa range, bifunctional antibodies are even larger (500 KDa)).

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Example: Ibuprofen vs. CNTF

Ibuprofen

Source: https://www.researchgate.net/

CNTF Source: https://en.wikipedia.org/

Top 200 Brand‐ Name Drugs by Retail Dollars 2006 Source: https://njardarson.lab.arizona.edu Njardarson Group

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Top 200 Pharmaceutical Products by Retails Sales in 2018 Source: https://njardarson.lab.arizona.edu Njardarson Group

Top 200 Pharmaceutical Products by Retails Sales in 2019 Source: pharmaexcipients.com

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Top 10 Biologicals (updated June 23, 2020) Name

Manufacturer AbbVie

Launch Date 2001

Global Sales $18.4B

Humira

Rituxan

Roche

1997

$9.2B

Enbrel

Amgen/Pfizer

1998

$7.9B

Herceptin Avastin

Roche Roche

1998 2004

$7.4B $7.1

Remicade

J&J/Merck

1998

$7.1

Lantus Neulasta Avonex Lucentis

Sanofi Amgen Biogen Roche/Novartis

2000 2002 1996 2006

$5.7 $4.7 $2.1B $5.1B

Indications Rheumatoid arthritis, plaque psoriasis, Crohn’s disease, ulcerative colitis, ankylosing spondylitis, psoriatic arthritis, polyarticular juvenile idiopathic arthritis Non‐Hodgkins lymphoma, chronic lymphocytic leukemia, rheumatoid arthritis Rheumatoid arthritis, plaque psoriasis, psoriatic arthritis HER2+ breast cancer Breast, colorectal, kidney, non‐small‐cell lung, glioblastoma, ovarian cancers Rheumatoid arthritis, Crohn’s disease, ankylosing spondylitis, psoriatic arthritis, plaque psoriasis, ulcerative colitis Diabetes Neutropenia related to cancer chemotherapy Multiple sclerosis (MS) Age‐related macular degeneration Source: verywellhealth.com

Biosimilars (are not generics)  Biosimilars (also known as follow‐on biologic or subsequent entry biologic) is a biologic medical product that is almost an identical copy of an original product that is manufactured by a different company.  Biosimilar approvals have stalled (somewhat) despite 22 approvals (as of October 2019).  Not anymore (February 2021)  In the US 26 are approved/17 on the market  Sales expected to triple ($2.4B to $6.5B)

 Zarxio  Amjevita  Ixifi

Neupogen Humira Remicade

Source: https://www.zarxio.com/biosimilars

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Bi‐Specific Antibodies  Large molecule  Fusion protein  Two separate and distinct variable regions  Can be cellularly produced  AMG 420  Myeloma and T cell binder

 Manufacturing hurdles (can be overcome) Source: https://www.amgenscience.com/

Bi‐Specific Antibodies Under Investigation

Source: Amgen’s BITE Engager, 2020

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Other Trends to watch for.......  Combination Products (combination of device and cells or slow release biologicals)  Biodegradables  Slow releasing polymers

 Living implants  Artificial organs  Encapsulated cell implants  3D printed organs/bioprinting

 Reservoirs (delivery systems)

Let’s begin……

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Therapeutic Gene R&D and Product Development

Typical Biological Manufacturing Process Flow Diagram (Mab)

Expression Vector / Host Cell Line Development

ICH Q5A Q5B Q5D Q6E

Clone

Research Cell Bank (RCB) Cell Banking

Upstream Manufacturing

Master Cell Bank (MCB) Working Cell Bank (WCB)

USP Drug Substance Cell Culture Scale‐up N‐2, N‐1, N

Cell Culture / Seed Train / Bioreactor

Downstream Manufacturing

Harvest / Clarification

ICH Q5A Q5C Q5E Q6B Q11

• • • • • •

Purification Capture Viral Inactivation UF / DF Polish Viral Filtration UF / DF

Bulk Drug Substance Formulation & Fill

Clarified Bulk

ICH=International Conference on Harmonization Guidance USP=United States Pharmacopeia A typical biologic manufacturing process flow diagram with appropriate ICH guidance steps. (Source: Cytovance Biologics Inc. John Conner, 2013.)

DSP Process Drug Product

Drug Product Formulation & Sterile Formulation & Fill

Q5E, Q6B, Q8

Therapeutic Gene R&D and Product Development

Typical Biological Manufacturing Process Flow Diagram (Mab)

Expression Vector / Host Cell Line Development

ICH Q5A Q5B Q5D Q6E

Clone

Research Cell Bank (RCB) Cell Banking

Upstream Manufacturing

Master Cell Bank (MCB) Working Cell Bank (WCB)

USP Drug Substance Cell Culture Scale‐up N‐2, N‐1, N

Cell Culture / Seed Train / Bioreactor

Harvest / Clarification

Downstream Manufacturing

YOU! Q7

ICH Q5A Q5C Q5E Q6B Q11

• • • • • •

Purification Capture Viral Inactivation UF / DF Polish Viral Filtration UF / DF

Bulk Drug Substance Formulation & Fill

Clarified Bulk

DSP Process Drug Product

Drug Product Formulation & Sterile Formulation & Fill

Q5E, Q6B, Q8

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Identifying Potential Therapeutic Molecules

 You have a target and let’s say that it is known to cause inflammation  You have a known mechanism of action A

Inflammation

No Inflammation B

 You have the sequence of a protein that you know binds to protein A in such a way that it interferes with the mechanism of action  You know both the protein sequence and genetic (DNA) sequence  So you want to make lots of your molecule but to get there, a few steps are needed……

Monoclonal Antibodies  Common production molecules  High specificity (low occurrence of cross reactivity)  Lock and key mechanism

 Can be easily expressed in cell lines  Favorable half life (compared to other molecules)

Source: https://en.wikipedia.org/

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Wait………  First, everyone needs to realize that biomanufacturing is 100% a team sport.  What you do has a ripple effect…..  You rely on the people upstream from you (R&D, PD, etc.) to do their job.  Likewise, the people downstream from you (clinicians, patients, etc.) rely on you to do your job.

Most important Source: https://www.nepatriots.com

Vector Design and Creation of Cell Lines Objective: create a vector that can be used to stably transfect a cell line which has the quality attributes of genotypic and phenotypic stability 32

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Step 1: Vector Design  Characteristics of plasmids     

Selection markers (vary) Amplification strategy Polyadenylation signals MARS sequences Multiple cloning sites (MCS)  ECO RI  Bam HI

Source: https://www.invivogen.com/pcpgfree

Gene Amplification  Genes can be amplified once they have been incorporated into the cell pCpG free plasmids  Decreased methylation  MARS sequences

 DHFR/Methotrexate (or GS/MSX system)  Found in plasmids like pNUT  Methotrexate    

Targets DHFR (dihydrofolate reductase) an enzyme that is essential for thymidylate biosynthesis Glycine synthesis Inhibits purine and pyrimidine synthesis (plays an important role in DNA and RNA synthesis) Placing the gene of interest close to DHFR and culturing cells in increasing amount of MTX followed by selection.

Source: https://www.albert.io/blog/g1‐g2‐phases‐cell‐cycle

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How does this happen? TargetGene Antibody B Genetic code

 Cut open (digest) plasmid using enzymes for the target gene (creating sticky ends)  Ligate plasmid and target gene  Transform bacteria and grow up the plasmid

Source: https://www.khanacademy.org

Step 2: Identifying Your Workhorse Cell Line  Mammalian Cells  Insect Cells  Yeast  Bacteria Mammalian cells (adherent CHO‐K1)

Bacteria Source: https://www.phe‐culturecollections.org.uk Source: https://www.researchgate.net Source: https://www.quora.com

Yeast 36

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Why use CHO cells?  Advantages     

Easy to manipulate genetically Adaptable to serum/protein free media Large‐scale suspension culture Insusceptible to human pathogens Capable of human like glycosylation

 Disadvantages  Genomic/phenotypic instability  Potential decreases in productivity

Suspension CHO cells https://www.ncbi.nlm.nih.gov/books/NBK100915/figure/ionchannel.F14/

Methods of Transfection  Methods     

Microinjection Gene gun Electroporation Viral transduction Lipofection

Source: https://www.sciencedirect.com

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Methods of Transfection

Source: https://www.google.com/search?q=DNA+uptake+by+cells&rlz

Getting your gene into the cells  Cells must be able to take up and incorporate the DNA into it’s genome  Demonstrated by reporter genes (i.e. GFP)  **Notice differences in intensity‐what does this mean????

Source: https://openlab.citytech.cuny.edu/

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Selection and Cloning  Your cells are “polyclonal”‐meaning the cells have the gene, some have more copies of the gene  You need to separate (i.e. select) these cells so that you have 1 cell type all containing the same number and location of the insert  There are different methods to do this….

Source: https://www.euromabnet.com/protocols/cloning

Screening Cell Line Candidates  ICH Q5D‐urges biomanufacturers to begin each process with a clonal cell substrate  Filter based on output  Selection criteria

250

200

cell output

 Looking for good output (PCD)  Cell morphology (i.e. comparable to parental cell line)

Clonal output 300

150

Original Polyclonal Output

100

0 90 98 57 24 75 17 79 37 59 44 35 34 20 33 30 28 51 67 66 50 10 83 7

6 41

clone designation

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Choosing a Clone  Screening cell line candidates  Good output (PCD)  Stable output over many passages  Normal doubling times (compared to parental)  Consistent cell morphology

Clonal Output 300

250

200

150

100

0 60

63 Passage 1

Passage 4

Passage 8

Passage 10

Cell Banking Operations Objective: create and stably store cell banks for continual manufacturing and safety testing 44

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Cell Banks Seed Bank (usually 10‐20 vials) Master Cell Bank* (usually 100‐150 vials) 3‐tier system Working Cell Bank* (X00‐X000 vials) End of Production Cell Bank* (50‐100 vials) *store at multiple locations… WHY? 45

Cell Bank Characterization  Adventitious Agent testing    

Viral (numerous tests‐in vitro, in vivo, PCR) Sterility (USP <71>) Mycoplasma (USP <63>, PtoC, PCR) Endotoxin (USP <85>)

 Cell Stability (viability) and Genetic Stability (loss of insert/rearrangement‐restriction digests)  Tumorigenicity (big for cell therapy applications)  Comparison of output (from Master to End of Production)  Morphology assessment (looking for consistency)  Doubling times (looking for consistency)  Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals (1993)  ICH guidelines (Derivation and Characterization of cell substrates used for the production of biotechnological/biological products)

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Why do all this development and characterization work?  Cell Banks are critically important‐Company value  Highly protected  Limited access

Source: Cord Blood Banking Processscbb.com.sg

Time for Regulatory Objective: successfully file a submission (i.e. and gain approval) with the FDA for the manufacturing of your investigational new drug (IND) for clinical use 48

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What is the FDA?  What is the FDA?  The FDA is a governmental body that provides guidance for bringing a drug, biologic or medical device to market

 What does the FDA do?  The FDA enforces rules governing the process for bringing a drug, biologic or medical device to market  Combination product?

 What authority does the FDA have?  Where is the FDA?

Ready for your IND  Preclinical Testing  Pharmacology and toxicology studies (is the drug safe for humans)

 CMC (Drug Substance/Drug Product)  Composition, manufacturer, stability, controls used for manufacturing the drug, consistency, aseptic validation, assays and qualifications

 Investigator Information  Qualifications for the investigator(s) who oversee the administration for the experimental drug

 Clinical Trial Protocol  Detailed protocol for the proposed clinical study and exposure to any risks (emphasized and scrutinized)

 30‐day review period

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Clinical Trials and Beyond

Clinical Trials Objective: successfully enroll a series of clinical trials that will be used to demonstrate the safety and efficacy of your new drug 52

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CMC Development

Refer to Current Good Manufacturing Practice for Phase 1 Investigational Drugs Guidance for Industry JULY 2008 53

Ready for the clinic  Phase I (usually small:10‐50 patients depending on the indication)  Safety is the primary outcome  Sometimes requested by the FDA to be staggered (especially for new technologies)

 Phase II (larger population: 50‐X00’s patients)  Continued safety and efficacy

 Phase III (larger population: X00’s‐X000’s patients)  Continued safety and efficacy

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Where are you going to Manufacture your B Antibody?  Good question ‐This has to fit into previous 2 slides!!!  Site Identification  Facility Design‐Clean Spaces  Facility Construction  Validation  Process Performance Qualification (PPQ)  CFR part 211 Subpart C, Building and facilities [covers design and construction features, lighting, HVAC, plumbing, maintenance, etc.]

Source: https://pbn.com/

More Regulatory  Biologics License Application (BLA)     

Applicant information Product/Manufacturing Information Pre‐Clinical Studies Clinical Studies Labeling

Form 356H

 PAI (FDA inspection)‐”green light”  Commercial Manufacturing

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Utilities Objective: become familiar with different utilities within a biopharmaceutical manufacturing plant 57

Utilities  HVAC  Electrical  Compressed Air  Clean Compressed Air (CCA)  Instrument Air

 Water  Purified water  Water for Injection (WFI)

 Steam  Plant steam  Clean steam

 Compressed gases

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Utilities ‐ HVAC  Heating, Ventilation, Air Conditioning  Controls air borne particles, dust and microorganisms using high efficiency particulate air (HEPA) filters  Maintain room pressure– Areas that must remain “cleaner” than surrounding areas must be kept under a “positive” pressurization, meaning that air flow must be from the “cleaner” area towards the adjoining space (through doors or other openings) to reduce the chance of airborne contamination.  Provides adequate air changes/hour  Maintain space moisture (relative humidity)  Maintain space temperature

Utilities ‐ Electrical  Electrical  Various supplies are needed  110 volt to 480 volt  Single to triple phase

 Emergency back up power  To support critical equipment  Typically has short duration delay

 UPS  To support critical equipment during delays

Source: https://cfeedu.cfemedia.com

 Electrical supply is typically verified at points of use during validation

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Utilities ‐ Compressed Air  Clean Compressed Air Systems  Used to blow down systems  Oil free compressor  Requires Testing:  Oils/hydrocarbons  Vapor pressure dewpoint  Particulates

 Instrument Air  Actuates valves in process lines/autoclaves

Source: https://www.airbestpractices.com

Utilities ‐ Water  Water for Injection (WFI)  Used for final rinses during cleaning  Product contact  Subjected to testing:    

Total organic carbon Bioburden Conductivity Endotoxin

 Purified Water  Used to feed stills and laboratory use  Total organic carbon  Bioburden  Conductivity Source: http://kremesti.com/water

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Utilities ‐ Steam  Clean Steam  Used in cGMP autoclave for processing equipment and humidification  Clean steam condensate meets WFI standards  Generated from purified water using a clean steam still

 Plant Steam  Used for heating purposes (environmental conditions)  Heat exchangers  Potable water supply

Utilities ‐ Compressed Gases  Carbon Dioxide  Nitrogen  Liquid Nitrogen  Subjected to testing  Purity

Source: https://en.wikipedia.org

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Lock Out/Tag Out  Safety concerns  Do not operate  Request information

Source: https://safetymanagementgroup.com/

Gowning: Contamination Control Objective: successfully don gowning in a manner than substantially decreases the likelihood of contamination. 66

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Manufacturing Biopharmaceuticals in Cleanrooms  A room in which the concentration of airborne particles is controlled and which is constructed and used in a manner to minimize the introduction, generation, and retention of particles inside the room and in which other relevant parameters, e.g. temperature, humidity, and pressure, are controlled as necessary.

Source: https://www.gotopac.com/art‐cr‐iso‐cleanroom‐classifications

Gowning Do’s and Do Not’s  Do gown‐in according to SOP  Do not gown‐in if:  You have an illness or medical condition that adversely affects the safety or quality of the drug product  You have been exposed to the animal facility, wastewater treatment plant or laboratories that test for live viruses or mycoplasma (on the same day).  You are wearing cosmetics of any kind or jewelry (excluding medical alert identification items)

 No eating, drinking, smoking or wearing personal outer wear inside classified production areas and controlled unclassified spaces or utility areas

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Gowning  Levels of gowning  Level 1  Scrubs, lab coat, hairnet, booties, safety glasses (ISO 8)

Source: https://www.panelbuilt.com

 Level 2  Scrubs, hairnet, booties, safety googles followed by hood, coverall, knee high boots, sterile gloves, goggles (ISO 7)

 Level 3  Level 2 with sterile sleeves, a second layer of sterile gloves (ISO 5)

 Gowning Qualification (testing and frequency) Source: https://www.aramarkuniform.com/

Why gown?  Requirement (both EU and FDA)  Overall reduction of bioburden shedding  Less microbial load in clean rooms  Enhances microbial monitoring results  Per Guidance for Industry. Sterile Drug Products Produced by Aseptic Processing‐ Current Good Manufacturing Practice  A program that establishes, both initially and on a periodic basis, the capability of an individual to don the complete sterile in an aseptic manner.  Plating operators (qualification)

Source: https://www.dphu.org

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Assessment

Break

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Biomanufacturing Overview

BioManufacturing Overview

Lyophilization

Source: https://www.researchgate.net/figure/The‐biopharmaceutical‐manufacturing‐technology‐flowchart

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Therapeutic Gene R&D and Product Development

Typical Biological Manufacturing Process Flow Diagram (Mab)

Expression Vector / Host Cell Line Development

ICH Q5A Q5B Q5D Q6E

Clone

Research Cell Bank (RCB) Cell Banking

Upstream Manufacturing

Master Cell Bank (MCB) Working Cell Bank (WCB)

USP Drug Substance Cell Culture Scale‐up N‐2, N‐1, N

Cell Culture / Seed Train / Bioreactor

Downstream Manufacturing

Harvest / Clarification

ICH Q5A Q5C Q5E Q6B Q11

• • • • • •

Purification Capture Viral Inactivation UF / DF Polish Viral Filtration UF / DF

Bulk Drug Substance Formulation & Fill

Clarified Bulk

ICH=International Conference on Harmonization Guidances USP=United States Pharmacopeia A typical biologic manufacturing process flow diagram with appropriate ICH guidance steps. (Source: Cytovance Biologics Inc. John Conner, 2013.)

DSP Process Drug Product

Drug Product Formulation & Sterile Formulation & Fill

Q5E, Q6B, Q8

First and Foremost: cGMP/Good Documentation Practices  GDP is included in cGMP     

Standard operating procedures (SOP) Specifications Validation documents Batch records Product and sample/Status labels

 The FDA can inspect the facility at any time….Surprise!!!!!

Good Documentation Practice ...presentationeze.com

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Media Prep A water‐based nutrient solution that contains defined amounts of salts, buffers, sugars and proteins in which the cells live. Nutrient media is added to all single use systems and stainless‐steel bioreactors Objective: Manufacture media for cell growth (inoculum through final bioreactor) 77

Media  Types of Media:  MEM

 MEM is a modification of Basal Medium Eagle (BME) which contains a higher concentration of amino acids and vitamins, as well as additional supplementary components

 DMEM–Dulbecco's Modified Eagle Medium  Hams F12

Source: https://www.fishersci.com

 Supplements

 Glutamax in place of glutamine  Protein source (plant Vs animal)

 Cell and Gene Therapy vs. Large scale

 Bottles vs. prepared on site from powders  Both scenarios involve a hold period  For large volumes, media trains utilize filtrations as a means to sterilize the media (housings constructed of 316Lss) Source: https://www.jobvector.ch

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Did you know???....  The raw materials used to prepare media are assessed for quality and sterility prior to being sent to upstream laboratories for media preparation and use.

Supplier and Raw Materials ...bioprocessintl.com

Media Prep: Process Parameters  Hold storage conditions  WFI temperature  pH  Osmolality  Why are these important????

Microbial Control

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Media Prep: Performance Indicators  pH  Pre‐filtration osmolality  Pre‐filtration bioburden  Why are these important????

Microbial Control

Courtesy of Amgen, Inc

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Inoculum Objective: successfully initiate a culture‐ this is likely an “open system” and requires training. 83

Cell Thaw: Inoculum  WCB vial is removed from cryostorage unit (‐196˚C)  Rapidly thawed (important‐why?)  Removal/dilution of cryoprotectant  Resuspended  Cell count and viability assessment (automated systems available for determining viability and cell number)  Viability is historically measured using trypan blue exclusion methodology

Source: www.gmi‐inc.com Source: www.planar.com

Source: www.fishersci.com

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Cell Thaw: Inoculum  Open system (risk)  Work is done in a Biological Safety Cabinet (ISO 5) or isolator  High air changes/hour  HEPA filtered air  Protects operator and the product  Use of flasks or other culture vessel (i.e. bottle, spinner)

Source: www.triumvirate.com

The LFH protects only the product in the hood such as sterile media, while the BSC protects the user and the product in the cabinet from bacterial contamination. Note direction of airflow

Source: www.fishersci.com Source: www.fishersci.com Source: www.fishersci.com

Inoculum: Process Parameters  Temperature  Gas  Agitation  Culture duration  Why are these important????

Microbial Control

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Inoculum: Performance Indicators  Final viable cell density  Final viability  Why are these important????

Microbial Control

Lab Aseptic Processing Cell Viability

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Aseptic Processing‐ Cell Thaw  Equipment and Raw Materials  Gowning  Trained Staff  Cell Thaw  Personnel Monitoring  Environmental Monitoring of BSC/LFU

Equipment and Raw Materials  Examples: Equipment ID BSC ‐001 BSC‐002

Certification Date 12/30/2020 12/31/2020

Certification Due Date 12/30/2021 12/31/2021

Minihelic Reading 0.25” 0.25”

Initial/Date CM 10/3/2021

Media Type Touch Plates

Manufacturer Remel

Media Lot Number 9280464

Date of Expiration 12/05/2021

Initial/Date CM 10/3/2021

Cell Culture Media IMX

Manufacturer

Media Lot Number

Date of Expiration

Initial/Date

Amgen

9280464

12/05/2021

CM 10/3/2021

What do you think it takes to use these?

100% Team Sport

10/20/2021

Typical Gowning Scenarios ISO 7 ISO 5

ISO 8

https://www.lifescienceleader.com

https://industry24h.com https://www.criticalenvironmentsolutions.co.uk

Why go to the trouble of gowning? Requirement (both EU and FDA) Overall reduction of bioburden and particulate shedding Less microbial load in clean rooms Enhances microbial monitoring results Gowning training and qualifications are always a favorite request of FDA inspectors and QPs (EU).  Important to have up to date training records

Amgen Training Workshop September, 2019

Baseline Reading SAS sample (200L)

Forced Failure (3‐5 second beard scratch) SAS sample (200L)

10/20/2021

Aseptic Cell Thaw (video)

Cell Viability/Cell Count Differentiate between live and dead cells Trypan Blue mechanism of action Manual vs. Automated

Source: www.fishersci.com Source: www. https://custombiotech.roche.com

10/20/2021

Determining Cell Number  Count cells as follows:  Clean, thoroughly dry, and assemble the hemocytometer with the cover slip.  Transfer a small amount of cell suspension to the edge of each of the two counting chambers. Allow the cell suspension to be drawn into the counting chamber by capillary action.  Place the hemocytometer under an inverted microscope and view the cells under magnification.  Focus on the quadrants, labeled 1, 2, 3, and 4.  Record the number of cells in each section. Average the number of cells, and multiply by the dilution factor. If the cells have not been diluted, this factor will be 10⁴ cells/mL. Any dilution of the sample after it was removed from the cell suspension, such as using vital stain, needs to be included in the calculation.

Source: https://www.hemocytometer.org/

Determining Viable Cell Number  Quick Calculation 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑉𝑖𝑎𝑏𝑙𝑒 𝐶𝑒𝑙𝑙𝑠 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑉𝑖𝑎𝑏𝑙𝑒 𝐶𝑒𝑙𝑙𝑠 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑁𝑜𝑛𝑣𝑖𝑎𝑏𝑙𝑒 𝐶𝑒𝑙𝑙𝑠

 From the hemocytometer:

𝑋 100

live

 Number of Viable Cells: 69  Number of Nonviable Cells: 13 Dead

69 69

𝑋 100

 Viability = 84%

10/20/2021

Cell Viability (video)

Image (Cell Viability)

10/20/2021

Calculation (Cell Viability)  Quick Calculation  From the hemocytometer count XX live cells and XX dead cells  Determine Viability x 100%

 The cell preparation is XX % viable

Calculation (Cell Viability)  Quick Calculation  From the hemocytometer we counted 20 live cells and 2 dead cells  Determine Viability x 100%

x 100%= 91%

 The cell preparation is 91% viable

100

10/20/2021

Calculation (Cell Count)  Given Information  Sample removed from a 20 ml cell suspension  A 1:10 dilution of the sample was prepared and counted (50µL cell suspension into 450µL trypan blue)

 Determine cells/mL and total cells x 104 x dilution factor

x 104 x 10 = 5.0 x 105

 Now multiply by the total cell suspension volume (i.e., 20 mL) to get total cell number: 5.0 x 105

x 20 mL= 10 x 106 cells 101

101

Lunch

102

10/20/2021

Cell Scale Up Objective: successfully increase your cell numbers of sequential cell culture operations to seed a final production reactor. 103

103

Cell Scale Up

Source: www.eppendorf.com

Source: www.americaninstrument.com

 After defined time, cells are processed (may mean trypsinization for adherent cell lines), and enumerated to get a total cell count.  Typically a multistep process  Following this, cells are used to seed the production reactor(s).  Both transfers from inoculum through scale‐up are typically (not always) closed systems (emphasis on closed systems).

Source: www.murraycompany.com

104

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Scale Up: Process Parameters  Target seed density  Temperature  Gas  Agitation  Culture duration  Why are these important????

Microbial Control 105

105

Scale Up: Performance Indicators  Final viable cell density  Final viability  Why are these testing important????

Microbial Control 106

106

10/20/2021

Bioreactors  Key Features:  Materials of Construction  Probes  Temperature  pH (indicative of cellular waste)  Dissolved oxygen (DO)

 Agitation  Antifoam addition  Sampling ports  SIP system‐closed  Nutrients (glucose, protein, etc.)

Source: https://pediaa.com/difference‐between‐bioreactor‐and‐fermentor

107

The process would look like this……  SUS (wave or SUB)  Can simplify the process  Efficient  Shorter runway  Utilizes prevalidated (cleaning) materials

BioProcess International, March, 2015

108

10/20/2021

Single Use Systems (SUS)  Batch to the Future: Stainless Steel Continues to Shine (Bioprocess International West, March, 2019)  Samsung BioLogics  Celltrion  Fujifilm Diosynth Biotechnologies

Recently build facilities using stainless steel

 Cited concerns include:    

Particulates (BioProcess International, April, 2019) Leachable (compromise cell viability and growth) “Close it up”….impact on COGs Leaks (manual connections)

109

Single Use Systems (SUS)  Still a strong movement towards SUS (paradigm shift)    

WuXi AGC Bio Patheon Catalent

Recently built facility using SUS

 Can a 1000L SUS equal to 10,000L 316L ss bioreactor (g/L to 10g/L)  Yes, the smaller facility footprint will work  Historically, add bioreactor suites to increase output  More recently, optimization of production media (multiple grams/L)  Enhancers added to media

 Now, attention has shifted from media to the cell line (10 g/L)  Characteristics which increase cell viability and improve expression titers

110

10/20/2021

Impact on Facility…….  Construction time….15 months  Cost….quarter of the capital cost  Highly sustainable environmental footprint  80% less energy and water  75% reduction in carbon dioxide emission levels

 75% smaller than a conventional facility  Still able to deliver the same quantity and quality of products

Source: www.amgen.com

111

Regardless of 316Lss or SUS…..  Process parameters:  Temperature (maintain 36°C)  pH (maintain around pH 7.0)  Nutrients (maintain [glucose], [protein], etc.)  Dissolved oxygen  Antifoam  Target seed density  Culture duration

Microbial Control

Source: https://pediaa.com/difference‐between‐bioreactor‐and‐fermentor

112

10/20/2021

Regardless of 316Lss or SUS…..  Performance indicators:  Final viable cell density  Final viability

Microbial Control

Source: https://pediaa.com/difference‐between‐bioreactor‐and‐fermentor

113

Types of Manufacturing Processes

Source: https://www.semanticscholar.org

114

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Characteristics of Batch, Fed Batch and Perfusion Systems  Batch  Add materials at the start, production is lower (1X)  Dominated the industry  Originally used for smaller quantities

 Fed Batch  Media addition to culture increasing yield up to 2X‐3X  Increase production with modest increases in COGs

 Perfusion  Perfusion cell culture to increase production up to 10X  Cost effective

115

Lab Bioreactors

116

10/20/2021

Introduction to Bioreactors  Cells (adherent vs. suspension)  T‐flasks, Roller Bottles, Cell Factories/Cubes, Culture Bags, and Shaker Flasks

 Expression of POI (protein of interest)  Freestyle Cell Line and media 117

117

Bioreactors  Key Features:  Materials of Construction  Probes  Temperature  pH  Dissolved oxygen (DO)  Agitation  Antifoam addition  Sampling ports  SIP system‐closed  Nutrients (glucose, protein, etc.)

Source: https://www.labcompare.com

Source: https://www. www.b2bcentral.co.za

118

10/20/2021

Cell Bioreactors (Video)

119

Recent BioFlo Run (1001 Data) BioFlo Run 1001‐ Temperature 38

Temperature (deg C)

37.8 37.6 37.4 37.2 37 36.8 36.6 36.4 36.2 36 94 91 88 85 82 79 76 73 70 67 64 61 58 55 52 49 46 43 40 37 34 31 28 25 22 19 16 13 10 7 4 1 Time (hours)

Process Value Setpoint

120

10/20/2021

Recent BioFlo Run (1001 Data) BioFlo Run 1001‐ pH 8 7.8 7.6 7.4

7.2 7 6.8 6.6 6.4 6.2 6 94 91 88 85 82 79 76 73 70 67 64 61 58 55 52 49 46 43 40 37 34 31 28 25 22 19 16 13 10 7 4 1 Time (hours)

Process Value

Setpoint

121

Recent BioFlo Run (1001 Data) BioFlo Run 1001 Dissolved Oxygen 38

Dissolved O2 (%)

32 94 91 88 85 82 79 76 73 70 67 64 61 58 55 52 49 46 43 40 37 34 31 28 25 22 19 16 13 10 7 4 1 Time (hours)

Process Value Setpoint

122

10/20/2021

Lab Mixing

123

Mixing  Importance of Mixing  Media/Cell Suspension/Buffers/Product Pools  Vessel Shape  Impellor Type  Speed  Cell Shear  Foam Generation  Vortex

124

10/20/2021

Impellers:

Gentle Marine Blade Impellers Pitched Blade Impellers

Source: BioProcess International; January, 2009

Rushton Impellers

What do you think the difference is? 125

125

Mixing Study (Video)

126

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Break

127

Cell Harvest Objective: successfully separate your workhorse cell line from the production media. 128

128

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Cell Harvest

Courtesy of Amgen, Inc.

129

Courtesy of Amgen, Inc.

130

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Cell Harvest  Cell separation is achieved by filtration or centrifugation.

Celeros APD‐250 Centrifuge

 challenges

 Different commercially available methods  In the end, the media is what we want  Contained in a harvest tank  May require additional processing before concentration

Source: www.celeros‐separation.com

131

Product Concentration  The membrane retains the larger macromolecules while allowing the flow of smaller molecules, media, and media components.  Substantially decreases the overall volume of the suspension decreasing the volume that will go over the column in subsequent steps.

Depth Filtration System Source: BioProcess International, May, 2012

132

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Process Parameters  Filtration  Filter area  Flow rate  Differential pressure during filtration

 Why are these important????

Microbial Control

133

Neutralization: Performance Indicators  Neutralization  pH  Acidic peaks  Bioburden

 Why are these important????

Microbial Control

134

10/20/2021

Filtration: Performance Indicators  Post‐harvest bioburden  Why is this important????

Microbial Control

135

Downstream Purification

136

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Therapeutic Gene R&D and Product Development

Typical Biological Manufacturing Process Flow Diagram (Mab)

Expression Vector / Host Cell Line Development

ICH Q5A Q5B Q5D Q6E

Clone

Research Cell Bank (RCB) Cell Banking

Upstream Manufacturing

Master Cell Bank (MCB) Working Cell Bank (WCB)

USP Drug Substance Cell Culture Scale‐up N‐2, N‐1, N

Cell Culture / Seed Train / Bioreactor

Downstream Manufacturing

Harvest / Clarification

ICH Q5A Q5C Q5E Q6B Q11

• • • • • •

Purification Capture Viral Inactivation UF / DF Polish Viral Filtration UF / DF

Bulk Drug Substance Formulation & Fill

Clarified Bulk

DSP Process Drug Product

Drug Product Formulation & Sterile Formulation & Fill

Q5E, Q6B, Q8

137

Buffer Prep Solutions in which the pH remains relatively constant even when small amounts of acid or base are added: Objective: Manufacture buffers for purification operations 138

138

10/20/2021

Did you know???  The raw materials used to prepare buffers are assessed for quality and microbial control prior to being sent downstream for buffer preparation and use.

Amgen, Inc. 139

139

Why Use Buffers?  Buffers are used to protect the protein from changes in pH by buffering or absorbing changes in pH.  A well‐buffered solution will maintain its pH in spite of variations in processing, containers, and raw materials.  Proteins are sensitive to changes in pH, which can lead to denaturation, aggregation, and fragmentation. Types of buffers  Phosphate, citrate, acetate and tris‐based buffers (as examples)  Each have different buffering (pH controlling) capacities and are used at different steps in the biomanufacturing process.

140

10/20/2021

Buffers  Below is a partial list of buffers used at Amgen, Inc.  Growth promoting buffers are those that may be more susceptible to bacterial growth!!

Courtesy of Amgen, Inc.

141

Buffer: Process Parameters  Buffer Preparation  Temperature  pH and/or conductivity  Agitation

 What are you testing for, and when to ensure control?  Bioburden, endotoxin, conductivity, and pH  Close to use

Microbial Control

142

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Protein Purification Objective: successfully separate your protein from all the other components in the production media. 143

143

Courtesy of Amgen, Inc.

 Please note that the order of the chromatographic processes may change depending upon the protein that is being purified.  Typically, purification begins with Protein A chromatography 144

144

10/20/2021

Protein Purification  What is Column Chromatography?  Column chromatography is a common technique used to separate individual compounds from a mixture.

 A volume is layered over the column, washed, and collected (i.e. eluted)

Source: https://www.thermofisher.com

145

Types of Chromatography Type of Chromatography Affinity

MOA

Binds with:

A specific interaction Non competing ligand

Ion Exchange

Net surface charge

Low ionic strength

Hydrophobic Interaction

Hydrophobicity

High ionic strength

Size Exclusion

Hydrodynamic radii

Separation based on hydrodynamic size

Elute with:

Competing ligand (specific); conditions that disrupt protein/protein interactions (non‐ specific) High ionic strength; Increased (cation exchange) or decreased (anion exchange) pH Low ionic strength

146

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The Stationary Phase  Stationary Phase:  Chromatography occurs with a solid support (often called a resin or matrix) packed into a column forming the stationary phase.  Protein A (can be native or recombinant)  Similar Fc regions to IgG but recombinant has been engineered to include a C‐ terminal cysteine that enables a single point coupling to Sepharose increasing binding capacity.

Source: https://en.wikipedia.org

147

The Mobile Phase  Mobile Phase:  The mobile phase, a solution containing a mixture of molecules, is moved through the column from one end to the other.  The chemical and physical differences in the molecules leads to different rates of passage through the column matrix, leading to separation.

Source: https://en.wikipedia.org

148

10/20/2021

Wash Buffers  Wash Buffer Characteristics:  Once the binding interaction occurs, the support is washed with additional buffer to remove non‐bound components of the sample.  [Nonspecific (e.g., simple ionic) binding interactions can be minimized by moderate adjustments to salt concentration in the binding and/or wash buffer].  Can also be a change in pH.

 Typical wash buffers:  Phosphate buffers  Tris buffers Source: https://en.wikipedia.org

149

Elution  Elution Buffer Characteristics:  Break the binding interaction of the target molecule and releases it  Dissociate binding by changes in:  pH (low or high)  high salt (ionic strength)

 Subsequent dialysis or desalting is required to exchange the purified protein from elution buffer into a more suitable buffer for storage or downstream analysis.  Typical Elution Buffer:  100mM Sodium Acetate, pH 3.7

Source: https://en.wikipedia.org

150

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Monitoring  Methods for Monitoring:    

SDS page of fractions FTIR HPLC UV Detection

Monitoring the presence (or absence) of peaks is key to determining if the therapeutic protein has been separated properly! Know what to look for!! Source: https://www.researchgate.net

151

Protein Purification: Chromatography  Process parameters (examples)    

Load Factor Flow Rates Volumes Number of Cycles

 Performance indicators (examples)     

Bioburden Endotoxin SE‐HPLC Impurities (Host Cell Protein, DNA) Leached Protein A

Microbial Control

152

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Assessment

153

Day 1 Wrap Up and Evaluation

154

10/20/2021

Day 2

155

Biochemistry 101 & Downstream Purification 156

156

10/20/2021

Background Biochemistry 101 What is a Protein? What are its structural components? Linkage between protein amino acid sequence and 3D shape? Size‐Charge‐Hydrophobicity distribution in complex samples? Changes in protein structure – denaturation? Protein Aggregation?

157

Four levels of protein structure

Amino Acid Sequence

B‐Sheet α‐Helix

3D folded Tertiary structure

Multi‐subunit Quaternary 3D Structure

158

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Know your protein!

Human Serum Albumin

Primary Amino Acid Sequence MKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGE EHFKGLVLIA FSQYLQQCPFDEHVKLVNEL TEFAKTCVAD ESHAGCEKSL HTLFGDELCK VASLRETYGMADCCEKQEP ERNECFLSHK DDSPDLPKLK PDPNTLCDEFKADEKKFWGK YLYEIARRHP YFYAPELLYYANKYNGVFQE CCQAEDKGAC LLPKIETMRE KVLTSSARQR LRCASIQKFG ERALKAWSVA RLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDR ADLAKYICDN QDTISSKLKECCDKPLLEKS HCIAEVEKDA IPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRR HPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKL KHLVDEPQNL IKQNCDQFEKLGEYGFQNAL IVRYTRKVPQ VSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLIL NRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETY VPKAFDEKLF TFHADICTLPDTEKQIKKQT ALVELLKHKP KATEEQLKTV MENFVAFVDK CCAADDKEACFAVEGPKLV WSTQTALA

MWt. (molecular weight) = 69,000 Daltons (69 kD) pI (isoelectric point) = 5.82 Hydrophobicity index = ‐ 0.395 159

159

Protein chemistry ‐ MWt. distribution

160

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Protein Chemistry – Charge distribution

161

Protein Chemistry – Hydrophobicity

162

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Loss of native structure (denaturation)

163

Protein Aggregation

164

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Downstream Processing

165

How do you purify a product?

166

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What protein properties can you exploit in developing a separation?

167

Factors influencing Size/Shape based separations – Stokes Radius effects Impact on charge state can lead to unfolding and change in shape Neutralize ionic interactions leading to a change in shape Promote Protein‐Protein interactions leading to aggregation Can promote aggregation Change protein diffusion rate and can impact kinetics Loss of disulfide bonds can lead to unfolding Can aid in solubility (membrane proteins) but also lead to denaturation Promote unfolding and denaturation

168

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Factors influencing a charge‐based separation

169

Factors influencing hydrophobic interaction (HIC) based separation Direct influence on the overall hydrophobicity of the target protein – membrane proteins Neutralize ionic interactions at high ionic strength Can impact protein solubility and HIC separation Increase in temperature promotes HIC interactions Can lead to changes in the hydration state of a protein due to un‐ folding of protein structure, Can impact kinetics of protein interaction with the stationary phase and local diffusion limitations. HIC surfaces can offer a range of hydrophobicity leading to varying degrees of retention/elution. 170

170

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Assessment

171

Break

172

10/20/2021

Chromatography I Modes

173

Chromatography Modes Size exclusion/gel permeation (SEC) Ion exchange (IEX) Hydrophobic interaction (HIC) Biospecific Recognition – Affinity Chromatography Mixed mode

174 174

174

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Size exclusion/gel permeation

175

Variables influencing IEX Chromatography • Choice of IEX chemistry, • pI range of sample, • pH of equilibration buffer, • Ionic strength, • Presence on non‐ionic components (glycerol, urea, detergents etc), • Residence time in contact with IEX media (flow rate, column geometry). 176

176

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Ion exchange (IEX) mode

177

Example ‐ IEX fractionation of plasma Basic pH optimization with NO salt elution

178

10/20/2021

Hydrophobic interaction (HIC) HIC ligands

Hoffmeister Series of Counter ions

179

Example of a HIC separation

180

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Mixed Mode Separation

181

Example of Mixed Mode ‐ Flow through polishing of a mAb after Protein A capture

182

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Bio‐specific Recognition (Affinity)

183

Components of an Affinity Resin

184

10/20/2021

Affinity Chromatography Workflow

185

Affinity Activation Chemistries

186

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Affinity Ligands

187

Protein Purification Applications Monoclonal Antibody (mAb) capture;  rProtein A affinity‐based purification of antibodies

from cell culture medium,  Direct capture on cation exchange resin.

Polishing of captured mAb to remove impurities;    

IEX chromatography, HIC, Hydroxyapatite, Mixed mode (AEX/HIC)

188

10/20/2021

rProtein A Affinity Capture

189

Recent improvements to rProtein A

190

10/20/2021

Direct Capture by Cation IEX

191

Capture‐polishing process integration ‐ Protein A capture followed by IEX and mixed mode polishing

192

10/20/2021

Please note that the order of the chromatographic processes may change depending upon the protein that is being purified. Typically, purification begins with Protein A chromatography

193

Overview of AMGEN multistep purification process for a mAb – Capture from cell culture

194

10/20/2021

Overview of AMGEN multistep purification process for a mAb ‐ Polishing  Polishing Step 1 – low pH viral inactivation,  Polishing Step 2 – Mixed mode AEX (Capto Adhere MMC);     

157 L column volume, mAb Flow Through mode, High mAb recovery (> 80%), Slight increase in volume, Removes CHO‐HCP, dsDNA, leached Protein A, mAb aggregates, viruses and endotoxin

 Polishing Step 3 – Cation Exchange (Fractogel‐S CEX) chromatography;  308L column volume,  Bind and elute mode,  Removes mAb dimers, aggregates and CHO‐HCP.

 Polishing Step 4 – Viral filtration.  UF/DF to condition the final mAb product for formulation 195

195

Assessment

196

10/20/2021

Lunch

197

Chromatography II Applications

198

10/20/2021

Trends in Bioprocess

199

Bead types improvements in mass transport

200

10/20/2021

Membrane Absorbers improvements in mass transport and flow rate balanced with lower capacity

201

Hybrid Membrane Absorbers improvements in mass transport, flow rate and capacity

202

10/20/2021

Cross‐linked Agarose

203

Fibrous Membrane Absorbers improvements in mass transport, flow rate and but still lower capacity?

204

10/20/2021

Properties of Large‐Scale Bioprocess Chromatography Media

205

Continuous Bioprocessing

206

10/20/2021

Multicolumn Bioprocessing

207

Summary

208

10/20/2021

Assessment

209

Break

210

10/20/2021

Lab Protein Purification

211

Day 2 Wrap Up and Evaluation

212

10/20/2021

Day 3

213

Viral Interaction/Removal Objective: successfully remove or inactivate any potential viruses in your protein. 214

214

10/20/2021

What are we removing?

Mammalian cells Used for protein production

Contamination Source: https://gut.bmj.com

Contamination

215

Viral Anatomy  Capsid (outer protein coat)  Genome  RNA viruses  DNA viruses

 Shape Variation  Helical, icosahedral, polyhedral, spherical

 Size variation    

Parvovirsuses‐20‐25 nm Circovirus‐18‐25 nm Retroviruses‐80‐100 nm Mimiviridae‐ 500 nm

Source: www.anipedia.com

Source: www.arstechnica.com

Source: www.sciencephoto.com

216

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Covid‐19  Enveloped  20 nm club shaped glycoprotein projections

 Genome  RNA viruses (positive sense, ssRNA)  Approximately 26‐32 Kbp

 Appearance  Halo/crown like

 Shape Variation  Spherical/pleomorphic form

 Size variation  80‐120 nm (variable references)

Source: S. Sikotra, Leicester Royal Infirmary, England

217

Viral Reduction  When does it occur?  At the very beginning…  Sourcing raw materials  Examples?  9CFR

 Qualifying vendors who can provide the appropriate documentation.  Appropriate validated manufacturing and test methodologies  Substituting plant proteins

Source: www.bestlifeonline.com

 During Manufacturing  Low pH buffers  Temperature exposure  Solvent/detergent

 During facility/equipment cleaning Source: www.agric.wa.gov.au

218

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Viral Inactivation and Removal  Specific steps for viral inactivation  Heat (Primatone)  Acid (low pH)

Very effective

 Nanofiltration (similar to FBS production process)  Inactivation can be incorporated into multiple positions of the manufacturing process  Typically towards the end of the process

219

Viral Removal (Filtration)

Source: BipPharm International Volume 19, Issue 10

220

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Viral Removal (Filtration)

Source: Planova filters: virus removal for biotherapeutic products

221

Viral Removal (Filtration)

Source: Planova filters: virus removal for biotherapeutic products

222

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Viral Removal (Filter Integrity)

The Ashai Gold Particle Test System: • Automated • Validated • Minimizes operator error • Provides assurance of post use filet integrity

Source: Planova filters: virus removal for biotherapeutic products

223

Viral Validation Studies

Source: BipPharm International Volume 19, Issue 10

224

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UF/DF Objective: Successfully concentrate the therapeutic to its final formulation and exchange the buffer to one that is preferred for storage stability and ease of administration. 225

225

What is Ultrafiltration/Diafiltration(UF/DF)? • The UF step is a concentration that reduces the amount of volume of the product by separating the molecules based on the membrane pore size or molecular weight cutoff. • The DF is a diafiltration step that exchanges one buffer solution with another (final formulation buffer).  UF/DF can include Tangential Flow Filtration (TFF).

https://www.google.com/url?sa=i&url=htt ps%3A%2F%2Fwww.bpesys.com%2FPDF% 2Fultrafiltration‐diafiltration‐tff‐fact‐ sheet.pdf&psig=AOvVaw2g9oVguDAXGrs8 eqfihn7A&ust=1616676132094000&sourc e=images&cd=vfe&ved=2ahUKEwiUxOqF‐ sjvAhXDDt8KHZM‐D3EQr4kDegUIARCFAg

https://www.emdmillipore.com/Web‐US‐Site/en_CA/‐/USD/ShowDocument‐Pronet?id=201306.5278

226

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What Tangential flow filtration (TFF)?  TFF is a cross‐flow filtration process used for separation of protein and particles.  The fluid passes parallel to the membrane filters, rather than being pushed through a membrane perpendicularly.  Particles that pass through the membranes flows to what is called the permeate side then to drain.  The rest of the molecules (protein) flow across the membranes and are recycled back into a feed tank. https://www.logcheck.com/wp‐ content/uploads/2018/05/Slide1.jpg

 Retain the Retentate! 227

227

Ultrafiltration/Diafiltration (UF/DF)  Process parameters (examples)  Load Factor  Flow Rates  TMP  Number of Cycles  Diafiltration Buffer (pH, Osmolality)  Performance indicators (examples)  Bioburden  Endotoxin  pH  Osmolality  SE‐HPLC

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Drug Substance Release Testing Objective: successfully test your product for important attributes during the manufacturing process to ensure quality. 229

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Drug Substance Testing  Appearance: Color, Clarity  Identity: ELISA  Purity: cIEF, SE‐HPLC, rCE‐SDS,  Adventitious Agents: Bacterial Endotoxins, Bioburden  Potency: Bioassay  Quantity: protein concentration  pH  Osmolality  Polysorbate 20

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Vial Filling Objective: successfully package your protein for storage and distribution. This function is performed outside of RI (Puerto Rico) 231

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Vial Filling  Aseptic Process (always validated)  Automated process (isolator and/or strict ISO classifications)

Source: www.ivtnetwork.com

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Vial Filling Steps and Equipment  Vial Wash and Depyrogenation  Filling, Stoppering, Lyophilization, Crimping, Visual Inspection

Source: https://www.youtube.com/watch?app=desktop&v=tnaefPcN3mE

Source: https://www.emergentbiosolutions.com

Source: https://www.contractpharma.com

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Drug Product Release Testing Objective: successfully test your final product for important attributes prior to release to ensure quality. 234

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Drug Product Testing  Appearance: Color, Clarity  Identity: ELISA  Purity: SE‐HPLC, CE‐SDS, RP‐HPLC, SDS‐PAGE  Adventitious Agents: Bacterial Endotoxins, Sterility  Potency: Bioassay  Quantity: Protein Concentration  pH  Osmolality  Polysorbate 20  Particulates

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Break

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Equipment Cleaning

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What are we removing?

Mammalian cells Used for protein production

Contamination Source: https://gut.bmj.com

Contamination

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Contamination Control  Aseptic technique is critically important during the entire biomanufacturing process.  Cleaning and sterilization of equipment must be performed on a regular basis  Potential contamination sources of product include bacterial, fungal and viral from many different sources. Source: www.americanpharmaceuticalreview.com

Contamination at any stage of the biomanufacturing process is extremely expensive to mitigate; especially viral contamination!! 239

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Types of Equipment Cleaning  Clean‐in‐Place (CIP)‐without assembly (automated chemical cleaning system)  Large pieces of equipment…    

Bioreactors Media and Buffer tanks Strong Acid and Base WFI rinses and monitored by pH

 Clean out of Place (COP)  Small pieces of equipment

Similar Challenges  Complete coverage  Complete removal of raw materials and cleaning agents  Validation

 Fixtures, clamps, fittings, etc.  Washer system (Lancer)

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CIP Process  Pre‐rinse with WFI (water for injection) or PW (purified water)

 wets the interior surface of the tank and remove residue  provides a non‐chemical pressure test of the CIP flow path.

 Caustic solution single pass flush through the vessel to drain. Caustic is the main cleaning solution  Caustic solution re‐circulation through the vessel  Intermediate WFI or PW rinse  Acid solution wash – used to remove mineral precipitates and protein residues.  Final rinse with WFI or PW – rinses to flush out residual cleaning agents  Final air blow – used to remove moisture remaining after CIP cycle Source: www.pharmtech.com

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Cleaning Validation is a must……  Smaller items cleaned in a glass washer can be challenged using organic carbon (i.e. sucrose) and rinses can be submitted for TOC analysis.  Vessels can be challenged with riboflavin and visually inspected  Riboflavin fluorescence under UV light and can easily be detected. Source: Sani‐Matic, Inc.

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Sterilization

Objective: Absence of viable organisms

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Sterilization  Steam in Place (SIP)‐without assembly  Large pieces of equipment…  Bioreactors, media and buffer tanks, process piping  Maintain a temperature of 121.1˚C for desired time  Requires validated

 Autoclaves  Smaller pieces of equipment     

Fixtures, clamps, fittings, etc. Generally double wrapped Established load patterns Established sterility shelf life (TBD) Maintain a temperature of 121.1˚C for desired time  Requires validated

Similar Challenges  Complete sterilization  Validation

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Autoclave: Sterilization out of place  Purge Phase

 Steam flows through the sterilizer beginning the process of displacing the air; temperature and pressure ramp slightly to a continuous flow purge.

 Exposure (Sterilization) Phase

 During this phase, the exhaust valve closes causing the interior temperature and pressure to ramp up to the setpoint. The program then maintains the desired temperature (dwells) until the desired time is reached.

 Exhaust Phase

 The pressure is released from the chamber through an exhaust valve and the interior is restored to ambient pressure, although contents remain relatively hot.

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GMP Autoclave  Clean steam supply (sampling port)  Instrument air  316L ss construction  Controller  Thermocouple located in drain (cold spot)  Dirty and clean sides (single vs. double door configurations)

Source: www.aoghmedical.com

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Steaming Validation is a must……  For autoclaves, a load pattern is established and validated using thermocouples and biological indicators.  For bioreactors, media and buffer tanks, and process piping a similar approach is used.  Typically B. Stearothermpohilus is used as an indicator. Growth at 55°C‐ 60°C is monitored. Must be negative for growth.  TC/BIs are snaked into autoclave pouches and process piping  TC/BI are distributed in autoclave chamber and vessels  Must maintain 121.1 °C for exposure period  Typically 3 consecutive runs are performed (both for load patterns and reactors)

Source: https://iopscience.iop.org

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Aseptic Processing Runs……  Once load pattern and/or process parameters are established, equipment is sterilized and then tested using TSB (tryptic soy broth)  Typically performed as 3 consecutive runs  Samples are collected for sterility testing  Added assurance

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Alternative Sterilization Methods Objective: Absence of viable organisms

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Irradiation  Methods  Gamma  Used in the syringe manufacturing process (also, needles, tubing, bags)  Validated with sublethal dose  Dose audits conducted quarterly

 E‐beam (Electron beam)  Used in medical device manufacturing (attention to adhesives and polymers)  Validated with sublethal dose  Dose audits conducted quarterly

 UV (ultraviolet)  Used for surface treatments (BSC)/isolators

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Facility Cleaning Objective: successfully minimize adventitious agent contamination through the use of appropriate disinfectants. 251

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Facility Cleaning  Levels of cleaning  Disinfection‐destroys microbes from surfaces (typical disinfection agents include Lysol (phenolic), quaternary agents (ammonium compounds), bleach, peroxide)  Almost always effective against spore forming bacteria (but not always sporicidal‐example Sporicidin)

 Sanitization‐reduces the number of microbes. Typically performed with IPA (70%).  Not very effective against spores  Some viricidal properties

 Sterilization‐process of full destruction of microbes including those that are spore forming

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Facility Cleaning‐Cleaning Agents  Vesphene, LpH, Vestasyde     

Kills broad range of microorganisms Typically applied daily Contact time with little residue following rinsing Quick destruction of microorganisms Historically rotated on monthly basis (philosophy is changing)

 Frequency  Critical areas  Flooring and horizontal surfaces are cleaned daily  Walls and ceilings are cleaned monthly  Sporklenz cleaning is preformed quarterly (ceilings, walls, and floors)

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Assessment

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Break

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EM‐Microbiology Objective: become familiar with different microbes and the importance of gram status. 256

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What are we looking for?

Mammalian cells Used for protein production

Contamination Source: https://gut.bmj.com

Contamination

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Different Bacterial Shapes  EM is your report card  Bacteria come in various shapes:    

Cocci or round/spherical Bacillus or rod‐shaped Spiral or twisted Variable

Source: www.imagesofbacteria.blogspot.com

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The Gram Stain  Developed by the bacteriologist Hans Christian Gram in 1884  Gram staining differentiates bacteria by the chemical and physical properties of their cell walls by detecting the presence of the peptidoglycan, which is present in the cell wall of Gram‐positive bacteria.  Consists of 4 steps: Application of

Reagent

Primary Dye Trapping Agent Decolorizer Counterstain

Crystal violet Iodine Alcohol Safarin/carbol fuschin

Color Gram Positive Gram Negative purple purple purple purple purple colorless purple pink

Be careful with this step

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Bacterial Wall Structure

Why is gram status so important?

Source: https://www.researchgate.net

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The Gram Stain

Source: https://www.onlinebiologynotes.com

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Common Bacteria

E. coli

B. subtillus

S. aureus

Source: https://www.researchgate.net

Source: https://www.gettyimages.com

Source: https://en.wikipedia.org

S. epidermis Source: https://http://faculty.weber.edu

P. aeruginosa Source: https://en.wikipedia.org

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Endotoxin  Gram negative microbes shed endotoxin  Endotoxins are large molecules consisting of a lipid and a polysaccharide composed of O‐antigen, outer core and inner core found in the outer membrane of Gram negative bacteria.  Effects include:  Inflammation (TASS)  Altered gene transcription

 Detectable  Gel clot method  Chromogenic method  Turbidimetric method Source: www.abbkine.com

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Environmental Monitoring Objectives: demonstrate that your choice of cleaners is robust and that your cleanrooms do not harbor high bacterial counts 264

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Environmental Monitoring  Monitoring is performed on a routine basis  Looking for both viable and non‐viable particulates  Established ISO standards for non‐viable particulates  Establish Alert and Action Levels  Trending data

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Environmental Monitoring

Source: https://www.gotopac.com/art‐cr‐iso‐cleanroom‐classifications

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Environmental Monitoring  Non‐Viable (Particulate) Monitoring  Active Sampling  Active monitoring indicates how many particles are in a m3 of air  Measures different sized particles at .1 µm to 5.0 µm  Performed in cleanrooms, BSCs, and LFUs

Source: https://www.climet.com

Source: https://encrypted‐tbn0.gstatic.com

Source: https://media.beckman.com

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Lab Environmental Monitoring

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Environment Monitoring – Non‐Viable (video)

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Environmental Monitoring  Viable EM requires QC personnel to qualify the media  Typically challenged with ≤ 100 cfu of various microbes including:     

Aspergillus brasiliensis ATCC™ 16404 Bacillus subtilis ATCC™ 6633 Candida albicans ATCC™ 10231 Pseudomonas aeruginosa ATCC™ 9027 Staphylococcus aureus subsp. aureus ATCC™ 6538

 Growth after 72 hours demonstrates the media can support microbial growth and can be used for EM monitoring activities.

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Environmental Monitoring  Viable Monitoring  Combination of active air sampling and passive monitoring  Active monitoring indicates how many microbes are in a m3 of air  Settling plates indicate how many microbes could settle onto a surface  Touch plates indicate how many microbes are in approximately 25cm2 area

Source: https://www.rapidmicrobiology.com

Source: https://www.emdmillipore.com

Source: https://eagleanalytical.com

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Recommended limits for microbial contamination (EU) Recommended limits for microbial contamination Grade

Air sample cfu/m3

Settling plates (diam. 9 Contact plates (diameter Glove print 0mm cfu/4 hours) 55 mm) cfu/plate 5 fingers cfu/glove

100

‐

200

100

‐ Source: https://www.pda.org/docs

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Lab Environmental Monitoring

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Environment Monitoring – Air and Surface (Videos)

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Assessment

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Thank You!  Contact Information:  Cahil McGovern, Ph.D. (email: cahilmcgovern@uri.edu)  Beth Zielinski‐Habershaw, Ph.D. (email: bzielinski@uri.edu)  Malcolm Pluskal, Ph.D. (email: m.pluskal@verizon.net)  Sharon McGuire, MS, (email: smcguire@uri.edu)

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Final Day Wrap Up and Evaluation

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