The BioeconomyBioproducts

Development, Production and Analysis Education and Training

Skilled Technicians Needed

Sonia Wallman, PhDNBC2 and c3bcBiomanufacturing ConsultantBio-Link Summer FellowsBerkeley, CAJune 8, 2016

History of Northeast Biomanufacturing Collaborative and Center

Since 2003, working with biomanufacturing employers to define the skills, knowledge and abilities (SKAs) needed by technicians to develop, produce and analyze bioproducts. Starting with biopharmaceutical production and analysis in 2003, 2007 and 2013; with biofuels in 2013 and finishing up with industrial biotechnology in 2015 (and 2016), we will synthesized these inputs from local (and global) employers into a single set of SKAs to educate and train technicians to support the development and growth of the bioeconomy. The synthesis of these inputs on what biotechnicians need to know and do is driving the production of hands-on and online curriculum and instructional materials, including laboratory manuals, textbooks and virtual industrial modules to facilitate deep understanding of what is required by technicians to biomanufacture bioproducts, creating an awareness of the many career pathways in biomanufacturing.

Biofuels Workforce Summit May 23, 2013 in Honolulu, HI

Bioproducts Upstream ProcessesBIOPHARMACEUTICALS, INDUSTRIAL

ENZYMES, GREEN CHEMICALS, BIOFUELS

• DEFROST CRYOVIAL OF CELLS FROM WORKING CELL BANK

• INOCULATE MEDIA WITH CRYOVIAL OF CELLS

• SCALE-UP• HARVEST• CENTRIFUGATION• FILTER MEDIA (DEPTH

FILTRATION AND STERILE FILTRATION)

• HOLD

BIODIESEL FEEDSTOCKS: WASTE AND AG

• COLLECT OIL PRODUCTS SUCH AS WASTE FRY OIL AND PLANT OILS

• TRANSPORT• STORE• HEAT• FILTER• HOLD

Bioproducts Downstream ProcessesBIOPHARMACEUTICALS, INDUSTRIAL

ENZYMES, GREEN CHEMICALS, BIOENERGY

• DEPTH FILTRATION• LIQUID COLUMN

CHROMATOGRAPHY (ION EXCHANGE, AFFINITY, HYDROPHOBIC INTERACTION=HIC, SIZE EXCLUSION=GEL FILTRATION)

• TANGENTIAL FLOW FILTRATION• STERILE FILTRATION• COLLECT BATCH• FORMULATE, FILL AND FINISH (for biopharmaceuticals)• DISTRIBUTE

BIODIESEL FEEDSTOCKS: WASTE AND AG

• TRANSESTERIFY OIL WITH ALKALINE CATALYST AND METHANOL INTO FAME (FATTY ACID METHYL ESTERS=BIODIESEL)

• SEPARATE INTO FAME-MeOH AND GLYCEROL-MeOH LAYERS

• DISTILL MeOH FROM BOTH LAYERS – RECYCLE MeOH

• WATER WASH FAME LAYER AND FILTER OR DISTILL AT HIGH VACUUM

• DISTRIBUTE BIODIESEL• SELL GLYCEROL CRUDE OR PURIFIED

BY HIGH VACUUM DISTILLATION

Pacific Biodiesel (Big Island Biodiesel)5M gallon/year Fry Oil to Biodiesel Facility

Collected fry oil into heated tanks, oil strained, placed into funnel shaped tanks where sodium hydroxide cleaves glycerol from triglyceride and methanol is used to add a methyl group to the free fatty acids to produce FAME or fatty acid methyl esters or biodiesel. Glycerol to bottom of funnel and biodiesel is drawn off and dewatered. BIB follows this by distillation, producing crystal clear biodiesel.

Vessels and Pumps

Vessels store/contain liquids, solids, or gasses involved in bioproducts production, such as bioreactors, tanks, drums, cylinders, and bins.

Pumps are used to move liquids through the system, such as into or out of a vessel. There are many different types of pumps, but they all use either rotational or centrifugal force to induce flow in liquids.

Tank holding Water For Injection (WFI) Peristaltic Pump

Compressors, Fans and Motors

Piping and ValvesPiping is cylindrical tubes made of varying lengths and materials such as 316L stainless steel, plastics, and iron that carry materials (usually in liquid or gas form) from one location to another. Valves are devices that are attached to piping or equipment to control the flow of liquids or gases. There are a wide variety of valve types that can start/stop or throttle (open or close in increments) the flow. Valves are vital to process control. They can be remotely opened or closed by computers to control variables such as flow or level.Piping and valves are the most common pieces of equipment found in a biomanufacturing facility.

Piping & Instrumentation Diagrams

P&IDA drawing or diagram which shows the piping of the process flow together with the installed equipment and instrumentation, including computer process controllers.

Biomanufacturing is Process ControlledBig Island Biodiesel 5M Gallon/Year

Quality Control LaboratoryBIOPHARMACEUTICALS,

INDUSTRIAL ENZYMES, GREEN CHEMICALS, BIOFUELS

BIODIESEL

TriState Biodiesel in Bridgeport, CTBuilt Using Recycled Equipment

Lonza Biopharmaceuticals in Portsmouth, NH

Quality Control TestingBIOPHARMACEUTICALS,

INDUSTRIAL ENZYMES, GREEN CHEMICALS, BIOFUELS

BIODIESEL

• Cell Count (growth rate)• Analyate Analysis (metabolism)• ELISA (protein ID, amount, and

activity)• SDS PAGE (protein ID and amount)• TLC (thin layer chromatography)• HPLC plus detector (protein, lipid,

carbohydrate IDs and amount of each)

• Stability Testing• Genome Sequencing• PCR Identification

Scale-Up Using Aseptic Technique

Shake Flask Inoculation using Biosafety Cabinet Class 100

Upstream Processing Count Cells

Scale-Up to Stainless Steel Biopharmaceutical Production Bioreactors

Inoculation of Microalgae Seed Bioreactor

Scale-Up of Microalgae toOpen Pond Production Solar Bioreactors

SludgeContinuous Centrifuge

Media OutCells + Media In

Harvest Protein Rich Media by Continuous Centrifugation

Harvest by Batch Centrifugation (1000 liters of Chlorella vulgaris culture)

Process controlled centrifuge to separate cells (microalgae) from media

Centrifuge drum and fins for separation of microalgae from media

Cellular paste is collected below

the blades of the centrifuge

Microalgae are harvested; media is

reprocessed into useable water

Depth Filtration to Remove Cells

Depth Filtration Housings, Piping, Valves

Tangential Flow Filtration (TFF) forSeparation and Concentration

Using TFF with the right cut off filters, the biologic of interest can be separated from other thing

For instance HSA has a molecular weight of 69KD. To make sure that the protein of interest is retained, a 10KD cut-off filter is used.

After ultrafiltration, we can diafilter, adding the phosphate buffer at pH 7.1 that we will also use to equilibrate our affinity column to prepare it for affinity chromatography of HSA.

Tangential Flow FiltrationSeparates and Concentrates

TFF for Biopharmaceutical Protein Concentration or Buffer Exchange

TFF for Microalgae Concentration

Photo taken at the ATP3 Large Scale Algal Cultivation, Harvesting and Downstream Processing Hands-On Training at AZCATi (Arizona Center for Algal Technology and Innovation) in Mesa, AZ

From Open PondTo Centrifuge

Chromatography for Purification and Quality Control Chemistry of the Protein, Lipid or

Carbohydrate of Interest

Separation Science Used for Downstream Processes and for Quality Control Chemistry Monitoring of both

Upstream and Downstream Processes

Stationary Media and Moving Buffer System Result in Competition and Separation

Liquid Column Chromatography

Large Scale Chromatography Column Packed with Beads (Media) for Separation

Chromatography System Components

• Chromatography Column and Media (Beads)

• Peristaltic Pump, Pipes and Valves

• Mixer for Buffers• (UV) Detector to identify

the product of interest

Liquid Column Chromatography ProcessPURGE Air from Column using Equilibration BufferPACK Column with Media (e.g. ion exchange, HIC,

affinity or gel filtration media)EQUILIBRATE Column with Equilibration BufferLOAD Column with Protein of Interest in

Equilibration Buffer WASH Column with Equilibration BufferELUTE Protein of Interest from Column with

Elution Buffer of high or low Salt or pHREGENERATE Column or Clean and Store (NaOH)

Chromatogram

WASH=Flow Through ELUTE=Eluate

Skills, Knowledge and Attributes for Biomanufacturing Technicians

Specific SKAs for Biomanufacturing Time Spent/Importance of EachProduction and Processing 22Operation and Control 7.6Quality Control Analysis 5.6Computers and Electronics 3.9Operation Monitoring 3.2Equipment Selection 2.3Equipment Maintenance 1.3Repairing 1.1Engineering and Technology 0.7Design 0.7Systems Analysis 0.6Technology Design 0.1Installation 0.1

Lab Science, Medical Devices and Biomanufacturing Common Core Critical Work Functions

• Maintain a Safe and Productive Work Environment• Provide Routine Facility Support• Perform Measurements/Tests/Assays• Comply with Applicable Regulations and Standards• Manage and Communicate Information• Perform Mathematical Manipulations

Online Biomanufacturing Resourcewww.biomanufacturing.org/

CHAPTER 1 INTRODUCTION ENGINEERING UNIT OVERVIEW CHAPTER 2 FACILITIES CHAPTER 3 METROLOGY CHAPTER 4 VALIDATION CHAPTER 5 ENVIRONMENTAL, HEALTH,AND SAFETY (EHS)CHAPTER 6 OPERATIONAL EXCELLENCEQUALITY UNIT OVERVIEW CHAPTER 7 QUALITY ASSURANCE CHAPTER 8 MICROBIOLOGICAL CONTROLCHAPTER 9 QUALITY CONTROL BIOCHEMISTRYPRODUCTION UNIT OVERVIEW CHAPTER 10 UPSTREAM PROCESSING CHAPTER 11 DOWNSTREAM PROCESSING CHAPTER 12 PROCESS DEVELOPMENT APPENDIX A MASTER GLOSSARY

Michael Cicio - Today’s Biologics: From Bench Top to Bottle.Tags: Keynote Speeches, Bioman 2006

Virtual Industrial Biomanufacturing

Virtual Downstream ProcessingMonoclonal antibody proteins (or mAbs) are the single largest class of recombinant biological drugs to date and represent about a third of the total biopharmaceutical market. The recent success of monoclonal antibodies for a wide range of disease therapies has led to the development of industrial production operations that manufacture pharmaceutical-grade mAbs both efficiently and safely. The following modules introduce a typical mAb bioprocessing workflow, detailing the equipment and processes used in biomanufacturing within a regulated environment. http://faculty.mc3.edu/downstreamprocessing/story.html

NBC2 Biofuels Curriculum – Textbook

NBC2 Biofuels Curriculum – Laboratory Manual

Career Pathway Data Collection:Job Titles of Some Program Graduates

• Environmental Monitoring Technician• Laboratory Automation Scientist• Laboratory Technician• Manufacturing Associate I• Manufacturing Tech I• Manufacturing Tech 2• Process Improvement Leader• Quality Assurance Associate• Quality Analyst• Quality Control Analyst• Quality Operations Product Release Coordinator l• Research Associate• Research Scientist• Research Technician• Senior Laboratory Technician• Senior Scientist • Technical Writer• Technical Writer/Scientist III