Aseptic Upstream by uri703

Aseptic Upstream Bioprocessing 2022 Presented by Beth Zielinski-Habershaw, PhD

9:00am-9:30am

Introductions

What will this this course provide? Knowledge in methodologies and principles that underlie mammalian cell culture and aseptic upstream processing. Comprehensive education in • • • •

principles of clean room function and ISO standards proper aseptic technique in ISO 5 cell culture hoods inoculation of genetically modified cells in the aseptic suite cell scale up methods for the ultimate transfer of therapeutic material to downstream personnel

Understanding and integration of cell culture methodologies and techniques with performance indicators in order to optimize individual proficiencies.

After this course you will Be knowledgeable in the FDA guidance for clean room aseptic processing Have clear understandings of and technical acumen for aseptic upstream processing and scale up. Be prepared to work effectively in aseptic upstream bioprocessing suites which includes monitoring of clean room environments while culturing mammalian cells.

Table of Contents Cells used for industrial production FDA guidance The clean room environment Biosafety cabinets Monitoring for viable and nonviable particles Cell culture contamination Basic equipment and media Inoculation into spinner flasks Scale up

9:30am-11:00am Cells used for industrial production FDA guidance The clean room environment

CELLS USED FOR INDUSTRIAL PURPOSES

The big picture…..

Microbial Biomolecules: Properties, Relevance, and their Translational Applications 2020, Pages 69-89

Flow of aseptic upstream bioprocessing

Inoculum

Harvest

Spinner Flasks( 100ml-500ml)

Precipitation

Bioreactor (3-20K L)

Centrifugation

Filtration

What are CHO (Chinese Hamster Ovary) cells?

Alcibiades -

Jamiri

Are these both CHO cell lines?

Creation of several CHO cell lines •

Mammalian systems are often preferred over other hosts, such as bacteria, plants, and yeast.

•

Capability for proper protein folding, assembly and posttranslational modification.

•

CHO systems have the added advantages • Can be grown in suspension at scales required to meet market demands • Genetically engineered to express high amounts of recombinant protein Grown in serum-free and chemically defined media • Less risks of propagating human viruses Wurm FM (2013) CHO quasispecies—implications for manufacturing processes. Processes 1:296–311

How are CHO cells genetically manipulated? from nucleofection to clone development Stable cell lines • High expression level of the gene of interest plasmids must integrate into transcriptionally active chromosomal regions. • Vector promoter elements drive the expression of recombinant protein and are derived from viruses, mice, and human

Biopharmaceutical Processing

What are the CHO cells producing? The cells that you are inoculating and maintaining have been genetically modified to produce a therapeutic monoclonal antibody A therapeutic monoclonal antibody is a protein that is capable of binding, in a specific manner, to another protein called a ligand. Once administered to the body, the therapeutic monoclonal antibody binds its complementary ligand and changes cell and gene function in order to combat disease

Monoclonal antibodies 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) Common production molecules

Ligand

Exquisite specificity with fewer off-target interactions and improved safety profiles! Cancer Research, UK

Are CHO cell lines always stable?

https://www.genscript.com/gsimages/news/why-are-cho-cells-critical-in-bioprocessing-1.jpg?=02

Screening

Please watch at leisure: https://vimeo.com/501897392

FOOD AND DRUG ADMINISTRATION (FDA)

FDA: Guidance for drug and biopharmaceutical development Formed June 30, 1906 The FDA is a governmental body that provides guidance for bringing a drug, biologic or medical device to market The FDA enforces rules governing the process for bringing a drug, biologic or medical device to market Offices in Maryland and throughout the country

What is FDA 21 CFR - Code of Federal Regulations? “The Code of Federal Regulations (CFR) is a codification of the general and permanent rules published in the Federal Register by the Executive departments and agencies of the Federal Government. Title 21 of the CFR is reserved for rules of the Food and Drug Administration. Each title (or volume) of the CFR is revised once each calendar year. A revised Title 21 is issued on approximately April 1st of each year.” FDA.gov

Food and Drug Administration 21 CFR The regulations enable a common understanding of the regulatory process by describing the requirements to be followed by drug manufacturers, applicants, and FDA. 21 CFR Part 210. Current Good Manufacturing Practice in Manufacturing Processing, packing, or Holding of Drugs. 21 CFR Part 211. Current Good Manufacturing Practice for Finished Pharmaceuticals. 21 CFR Part 212. Current Good Manufacturing Practice for Positron Emission Tomography Drugs. 21 CFR Part 314 For FDA approval to market a new drug. 21 CFR Part 600. Biological Products: General. FDA.gov

What does 21 CFR 211 include?

https://www.plianced.com/compliance-wiki/wp-content/uploads/2019/11/FDA-CFR-1024x576.jpg

GUIDANCE DOCUMENT: Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing Practice. Guidance for Industry. OCTOBER 2004. Content current as of 05/04/2020

“This guidance is intended to help manufacturers meet the requirements in the Agency's current good manufacturing practice (CGMP) regulations (2l CFR parts 210 and 211) when manufacturing sterile drug and biological products using aseptic processing. This guidance replaces the 1987 Industry Guideline on Sterile Drug Products Produced by Aseptic Processing (Aseptic Processing Guideline). This revision updates and clarifies the 1987 guidance.” FDA.gov

Litigation: Sanofi, Genzyme 'negligently allowed a virus contamination in the manufacturing facility' By Eric Palmer Mar 12, 2012 06:56pm

“An Idaho widow has sued Genzyme and parent Sanofi ($SNY), saying the viral contamination at a plant led to a shortage of Fabrazyme. The lawsuit, which can be accessed via a link from a Pharmalot story, says that in June 2009, the defendants "negligently allowed a virus contamination in the manufacturing facility where Genzyme makes Fabrazyme [leading] to a shortage of the drug, which resulted in Genzyme not meeting demand of Fabrazyme to Fabry disease patients." The situation was made worse in November 2009 after the FDA found more contamination in Fabrazyme, resulting in a further reduction in the drug's availability. "Due to this shortage, Genyzme began arbitrarily rationing Fabrazyme toward the end of 2009. The problems at the plant led Genzyme to sign a consent decree with the FDA, and it has been working to resolve the issues. ”

Viral contamination in biologic manufacture and implications for emerging therapies Nature. Published: 27 April 2020

Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB)—a biopharmaceutical industry consortium including more than 20 biotechnology companies housed at the Massachusetts Institute of Technology’s Center for Biomedical Innovation—collected comprehensive data on virus contaminations in cell culture operations from CAACB member companies36.

Viral contamination in biologic manufacture and implications for emerging therapies Nature. Published: 27 April 2020

1.Contaminating virus and host cell line are indicated where known. Note: some contamination events were reported publicly and in more detail to the CAACB. 2.CVV, Cache Valley virus; EHDV, epizootic hemorrhagic disease virus; MVM, minute virus of mice; PCV-1, porcine circovirus type 1; Reo3, reovirus type 3.

Factors influencing microbial contamination of manufacturing environment and therapeutic product Figure 2. Major factors that influence the microbiological contamination risk of biopharmaceuticals. The human factor might influence and dominate the other factors.

THE CLEAN ROOM ENVIRONMENT

A few facts about aseptic processing standards Food and Drug Administration Title 21 CFR 211: Guidelines for aseptic processing in pharmaceuticals and biopharmaceuticals Specialized HVAC equipment Strictly regulated air pressure and humidity control for appropriate anteroom and clean-room environments that are low in particulate matter Clean-rooms constructed of nonporous materials ISO 7 standard

Aseptic processing is performed in enclosed environments (hoods) that maintain ISO 5 conditions

Aseptic Processing Areas: Big Picture

Easily cleanable floors, wall, and ceilings of smooth, hard surfaces

Temperature and humidity controls

Bioprocessinternational.org

High Efficiency Particulate Air Filters (HEPA)

Aseptic Processing Area: Clean Room An isolated environment, strictly controlled with respect to: Airborne particles of viable and nonviable nature Temperature Humidity Air pressure Air flow Air motion Lighting

ISO classifications

https://www.blue-line.com/cleanroom-fda-and-eu-classifications

ISO classifications The particulate matter in the corridors outside the aseptic processing area must be no greater than ISO Class 8 At the next level of stringency, particulate matter contained in room air within an aseptic processing area should be no more than the ISO Class 7 standard allowance Biosafety cabinets (BSCs) are located in an ISO 7 clean room suite The air within the BSC must be ISO Class 5

https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.prnewswire.com%2Fnews-releases%2Fsignaturebiologics

Aseptic Processing Areas: Clean Room Airflow Unidirectional or Laminar Airflow (FDA Guidance definition) An airflow moving in a single direction, in a robust and uniform manner, and at sufficient speed to reproducibly sweep particles away from the critical processing or testing area 9/2004 Guidance for Industry-Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing Practice

https://lh3.googleusercontent.com/proxy/ zrcilm7Tk7oFDkcBGakBpc9McuNsjHaxKTGR0bDoLlM5JNsSPvkJsw_tplOoJRs5plBTNBUJO9ZIux-IygWmidF2XkzmYrbxgSxB9uKskKbhxZyx6PxWs6Ch0Wq-EBuN2914x1-k6wb_2bVHctVRzeMJg

Turbulent/Nonunidirectional Airflow Airflow which does not meet the definition of unidirectional airflow Edges, solid flat surfaces, person or equipment Moving

Air changes and flow patterns Air flow over critical areas should be uni-directional (laminar flow) at a velocity sufficient to sweep particles away from filling/closing area Non-viable air particles are monitored on a daily basis

ISO Class ISO 5 ISO 6 ISO 7 ISO 8 Conventi onal building

Average number of air changes per hour 240–360 air changes per hour (unidirectional airflow) 90–180 air changes per hour 30–60 air changes per hour 10–25 air changes per hour 2–4 air changes per hour Mecartcleanrooms.com

Aseptic processing areas: pressure differentials = prevent contaminations Positive air pressure used to prevent the ingress of contaminants from less clean areas Cleanroom person and materials enter from adjacent clean corridor or clean area Biopharmaceuticals

Negative air pressure effective in containing or preventing dispersion of sensitive or highly toxic materials Pharmaceuticals

https://www.achrnews.com/NEWS/Home/Images/ Alnor-Airflow%20Testing%20and%20Cert%201.jpg

ASSESSMENT QUESTIONS 37

11:00am-11:10am

Break 38

11:10am-12:10pm Biosafety cabinets Monitoring for viable and nonviable particles Cell culture contamination

BIOSAFETY CABINETS

ISO classifications The particulate matter in the corridors outside the aseptic processing area must be no greater than ISO Class 8 At the next level of stringency, particulate matter contained in room air within an aseptic processing area should be no more than the ISO Class 7 standard allowance BSCs are located in an ISO 7 clean room suite The air within the BSC must be ISO Class 5

https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.prnewswire.com%2Fnews-releases%2Fsignaturebiologics

Laminar flow hoods

(Image Source: http://edgeqld.org.au/fringes/diy-laminar-flow-hood/) (Image Source: http://edgeqld.org.au/fringes/diy-laminar-flow-hood/)

Biosafety cabinet (BSC) Special hood for preparing hazardous compounds such as antineoplastic drugs and other chemotherapy medications Worker stands or sits behind the shield and places the hands into the cabinet to manipulate the sterile compound https://encrypted-tbn0.gstatic.com/images?q=tbn:ANd9GcRueyjZZ4diX3ftHbdvLJT54-D7rWZhARb46Q&usqp=CAU

BSC Classification Class 1 Cabinets Provides personal and environment protection. Used when working with low to moderate risk biological agents. Biosafety levels: 1, 2 and 3 Class 2 Cabinets Provides personnel, environment and product protection. Used when working with low to moderate risk biological agents. Biosafety Levels: 1, 2 and 3 Class 3 Cabinets A highly specialized laboratory “glovebox”. A Class 3 cabinet provides the same protection as a Class 2 but is designed for working with Biosafety Level 4 highly infectious agents and provides the highest level of protection for the environment, product and user. Used when you are working with very high risk biological agents. Biosafety Level: 4

Labogene.com

Biosafety cabinets Class I

(Image Source:https://www.unr.edu/ehs/policies-manuals/biosafetymanual/chapter-8)

Class II

Class III

(Image source:https://www.unr.edu/ehs/policies-manuals/biosafetymanual/chapter-8)

(Image Source: http://www.antechscientific.com/biosafety-cabinet-class-iii.html)

Starting Up Turn off the ultraviolet sterilizer (if so equipped) as soon as you enter the room. (Turn on all blowers) and cabinet illumination lights. Allow 5 minutes of operation to purge the system; check the flow alarm system and visual alarm function (if so equipped). Decontaminate readily accessible interior surfaces with a disinfectant appropriate for the agents or suspected agents present. (70% IPA) https://www.totalcleanair.com/wp-content/uploads/2020/06/Biological-Safety-Cabinet-5.jpeg

Basic rules for working in BSC Maintain vigilance about sanitizing items handed to you from outside the BSC Do not cross your arms or objects over open containers Minimize touching anything that is handed to you from outside the BSC and be sure to sanitize with 70% isopropyl alcohol (IPA) Work at least 4 inches from the inside edge of the front vent.

Technique

Thermo Fisher Scientific

Technique

MONITORING VIABLE AND NONVIABLE PARTICLES

https://pediaa.com/wp-content/uploads/2018/06/DifferenceBetween-Viable-and-Nonviable-Particles-Comparison52 Summary.jpg

Viable microbes travel on non-viable particulates Monitoring of non-viable particulates and viable microbes is performed daily to ensure aseptic environmental conditions

https://static01.nyt.com/images/2015/08/28/science/01obs-dust/01obs-dust-articleLarge.jpg? quality=75&auto=webp&disable=upscale

Non-viable (Particulate) monitoring In order to monitor air flow efficiency in regards to maintaining aseptic conditions in clean rooms, viable and nonviable particulate samples are taken daily. ISO 14644-1/2:2015; EU GMP, Annex 1; PIC/S, GMP Annex 1 (2017); FDA Aseptic processing cGMP:2004 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 and BSC

Met One (Beckman Coulter) non-viable particle counter

Viable particulates: microbes

Common microbes

Low-temperature electron micrograph of a cluster of E. coli bacteria, magnified 10,000 times. Each individual bacterium is oblong shaped. Photo by Eric Erbe, digital colorization by Christopher Pooley, both of USDA, ARS, EMU.

Scanning Electron Micrograph of Staphylococcus epidermidis Photo Credit: Janice Carr Content Providers(s): CDC/ Segrid McAllister

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

https://abbkineau.files.wordpress.com/2016/11/image-34316.jpg

Viable particle monitoring Viable particle counting is performed in order to confirm that the microbial load or bioburden is negligible and aseptic conditions are maintained Testing is performed multiple times/day Combination of active air sampling and passive monitoring Active monitoring indicates how many microbes are in a m3 of air SAS microbial air sampling Settling plates indicate how many microbes could settle onto a surface Touch plates indicate how many microbes are in approximately 25cm2 area

2030lab.com

Americanpharmaceuticalreview.org

Touch plates for viable particle counting

SAS microbial air sampler for viable particle counting

Environmental monitoring data 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.

Trending data Sensitivity Sensitivity of a bioburden tests used to detect the presence of microbes decreases with test volume size and decreases in bioburden density

Trends Charting trends overtime allows for visualization of the overall efficiency of the inspected process. Trends should show stability in the system; ie low bioburden levels. Consistency and compliance with cGMP guidelines

CELL CULTURE CONTAMINATION

Culture contamination with bacteria

Eppendorf

Culture contamination with bacteria

Eppendorf

Culture contamination with bacteria

Eppendorf

Culture contamination with mycoplasma

David M Phillips/ Science Source/ Getty Images

The presence of mycoplasma must be assayed for.

Detection of mycoplasma

Direct agar culture Indirect Hoechst DNA staining PCR-based methods

Over 190 mycoplasma species are known, but only eight are responsible for ~95% of cell culture contamination events. • Mycoplasma arginini • Mycoplasma fermentans • Mycoplasma hominis • Mycoplasma hyorhinis • Mycoplasma orale • Mycoplasma pirum • Mycoplasma salivarium • Acholeplasma laidlawii ATCC.ORG

Culture contamination with fungus

Eppendorf

Culture contamination with yeast

Eppendorf

ASSESSMENT QUESTIONS 74

12:10pm-1:10pm

LUNCH 75

1:10pm-2:10pm

Basic equipment and media Inoculation into spinner flasks

BASIC EQUIPMENT AND MEDIA

BASIC EQUIPMENT

Equipment for Large-Scale Mammalian Cell Culture (MassBiologics of the University of Massachussets Medical SchoolMattapanUSA)

Early-stage cell culture

Fig. 2 Equipment used for seed train expansion: a T-flasks, b shaker flask, c roller bottles, d spinner flask, and e WAVE bioreactor on a rocking platform

Equipment for Large-Scale Mammalian Cell Culture (MassBiologics of the University of Massachussets Medical SchoolMattapanUSA)

Late-stage cell culture Bioreactors Fig. 7 A stainless-steel bioreactor with instrumentation and piping for sterilization, gas flow, and fluid transfer

Equipment for Large-Scale Mammalian Cell Culture (MassBiologics of the University of Massachussets Medical SchoolMattapanUSA)

Bioreactors Fig. 8 Disposable bioreactors from different vendors. The bag used in the stirred-tank disposable bioreactors contains an impeller and a sparger. The probes are either integrated as part of the bag or they are inserted using an aseptic method after the bag is in place

Equipment for Large-Scale Mammalian Cell Culture (MassBiologics of the University of Massachussets Medical SchoolMattapanUSA)

Bioreactor gassing Table 5 Mixing and aeration systems used in homogeneous bioreactors

Bubble-column and airlift bioreactors do not use an impeller, but rely on moving bubbles for mixing

Reactor type

Mixing mechanism

Aeration mechanism

Stirred tank

Impeller

Direct or indirect sparging

Bubble column

Bubbles

Sparging

Airlift

Bubbles, draft tube

Sparging

WAVE, rocking platform

Rocking

Surface aeration 83

Equipment for Large-Scale Mammalian Cell Culture (MassBiologics of the University of Massachussets Medical SchoolMattapanUSA)

Bioreactor spargers

Fig. 5 Types of spargers used in cell culture bioreactors: a macrosparger (arranged as a ring sparger), and b microspargers

A little more on sparging and gasses

Equipment for Large-Scale Mammalian Cell Culture (MassBiologics of the University of Massachussets Medical SchoolMattapanUSA)

Bioreactor connectors

Fig. 10 a Pall KleenPak connector and b its use to insert conventional probes to a disposable bioreactor [25]

MEDIA

Cell media prep: Manufacture media for cell growth (inoculum through final bioreactor) 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

Thermo Fisher Scientific

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 • Ham's F12 • 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)

This production suite relies on stainless steel bioreactors but integrates single-use technology for cell culture media storage and key transfer lines.

Genetic Engineering and Biotechnology News

Does the type of media make a difference?

Biotechnological products and process engineering Open Access Published: 07 April 2015

a Cell and b antibody concentrations as well as c lactate and d ammonium levels obtained with commercial CHO cell culture media in batch mode

Amgen thaw and scale up media

Amgen thaw and scale up media process parameters

Amgen thaw and scale up media performance indicators

osmolarity is a measure considering the volume of a solution whereas osmolality is measured considering the mass of a solution

Osmolarity versus Osmolality

The concentration of a solution that shows osmotic activity. (measure in volume)

The unit for osmolality is osmol/kg. Osmolality gives the concentration of a solute in a solution in terms of the mass of the solution. (measure in mass)

https://pediaa.com/wp-content/uploads/2017/08/Difference-Between-Osmolarity-and-Osmolality-2.png

Amgen expansion bioreactor media

Amgen expansion media process parameters

Amgen expansion media performance indicators

Amgen N-1 feed media

Amgen N-1 media process parameters

Amgen N-1 media performance indicators

Amgen production bioreactor media

Amgen production bioreactor media process parameters

Amgen production bioreactor media performance indicators

Amgen production feed media

Amgen production feed media process parameters

Amgen production feed media performance indicators

INOCULATION AND EARLY SCALE UP

Cells Used for Industrial Purposes

Courtesy of Amgen

Inoculation

111

Inoculum Successfully initiate a culture- this is likely an “open system” and requires training.

Process parameters: Stage 1 (vial thaw into 250ml shake flask)

Courtesy of Amgen

Cell Thaw: Inoculum Open system (risk) Work is done in a Biological Safety Cabinet (ISO 5) or isolator

The BSC protects the user and the product in the cabinet from bacterial contamination. Note direction of airflow

High air changes/hour HEPA filtered air

Adherent

Protects operator and the product Use of flasks or other culture vessel (i.e. bottle, spinner) Suspension

Cell Thaw: Inoculum Thaw 1 ml microtube quickly in sterile water Freezing media contains a specialized antifreeze that prevents ice crystals from forming in the cell membranes during the freezing process. The freezing media must be diluted quickly in order to maintain optimal cell viability Immediately upon thawing the 1 ml of cells are transferred to a flask containing cell culture media.

Incubator

https://www.pipette.com/ GetContent.ashx? ctn=BrandImages/H3551-45POpen-WeRes.jpg

Cell count and viability Once cells have been transferred to sterile culture media, a sample is removed, and cell number is counted

Automated Cell Counter

Cell viability is then determined using an automated cell counter Cells are placed in an incubator that has gas, temperature and humidity levels set according to your company’s SOPs

Arrow points to dead cell stained with trypan blue stain. Living cells are clear

https://bitesizebio.s3.amazonaws.com /cellculture/files/2013/03/Figure-4.jpg

ASSESSMENT QUESTIONS 118

2:10pm-2:40pm

Lab 1: Thawing and inoculation

119

Inoculation: Lab video

2:40pm-2:50pm

Break 121

2:50pm-3:50pm

Scale up

122

Phases of normal cell growth

A: Lag Adaptation B: Log Exponential growth C: Plateau Rate of proliferation slows D: Death Rate of death exceeds proliferation rate

High yields obtained today result from the combination of improved host cells, expression vectors, screening methods, media optimization, and process development innovations

Modes of bioreactor operation: 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

Source: https://www.semanticscholar.org

Critical factors for scale up “Change for the sensitive mammalian cells leads to shear stress and foaming issues, but nowadays these are quite well controlled due to the addition of antifoam and cell surfactants (Xing, Kenty, Li, & Lee, 2009).” Slow and gentle mixing Gassing system Foam prevention Accurate feeding Sterile sampling

https://www.researchgate.net/profile/ShuichiTakayama/publication/51046304/figure/fig6/ AS:277549058936843@1443184260593/Schematics-ofan-undisturbed-cell-cells-under-shear-stress-pressureand-their-gradients.png

Mixing

Minimum lateral shear

Typically, stirrer speeds for cell cultures are much slower than for microbial cultures. Most cell lines are stirred at around 100 rpm. Some robust cell lines can survive higher speeds, but yield of biomass suffers Xing, Kenty, Li, & Lee, 2009

Impact of mixing

Gassing in the bioreactor headspace Gas transfer occurs at gas/liquid interface No bubbles to cause foam Accumulation of CO2 in the headspace caused by the metabolism of the cells is prevented

Headspace

Dissolved oxygen and dissolved Dissolved oxygen and dissolved carbon dioxide carbon dioxide

Impact of pH on cell culture t of pH on cell culture

Gassing via the sparger This produces a steady stream of bubbles which can rise through the medium and exchange gas for as long as possible. Gassing via the sparger is more efficient but could cause foaming in cultures that have high levels of protein in the medium. (Add antifoam) Sparger

Foam formation in the 2L bioreactor Dense foam can entrap cells and pull them apart. Foam is due to proteins in the liquid medium and these can come from either protein rich media or protein secretion by cultured cells

Peter Czermak

Antifoaming agents Antifoaming agents have high surface activity that can lower the surface tension of local film when its micro droplet contacts liquid film. This local area of low surface tension will extend all around, losing elasticity and self-restoring ability, thus causing the rupture of liquid film.

Figure 7.15.. Oil drops in antifoaming/defoaming agents and their interaction with an air bubble. A is air, W is aqueous phase, O is oil. (a) Plain oil drop; difficult to achieve wetting of the drop by air. (b) Drop contains solid particles that are oriented in the O-W interface; a protruding particle can pierce the film between O and A. (c) The drop contains crystals that form a solid network; a protruding crystal can pierce the film. Vance Bergeron, Pieter Walstra, in Fundamentals of Interface and Colloid Science, 2005

133

Sampling

INFORS HT Super Safe Sampler

134

ASSESSMENT QUESTIONS 135

Introduction to scale up equipment

3:50pm-4:20pm

Lab: Scale up

136

BREAK 137

Cells Used for Industrial Purposes

Courtesy of Amgen

Flasks to flask transfer

How are cells transferred between flasks? Tubing can be cut and soft welded in an aseptic fashion Tubing can also be cut and clamped in an aseptic fashion

Mycap® CCX Cell Culture Shake Flask | Sartorius

Process parameters

Courtesy of Amgen

Process parameters

Courtesy of Amgen

Performance indicators

Courtesy of Amgen

Vial thaw and shake flask expansion calculations

Courtesy of Amgen

Cells Used for Industrial Purposes

Courtesy of Amgen

Flasks to expansion bags

Culture bag process parameters

Culture bag performance indicators

Expansion bag calculations

Courtesy of Amgen

Cells Used for Industrial Purposes

Courtesy of Amgen

Expansion bags to bioreactor

Courtesy of Amgen 153

154

Bioreactor to bioreactor

Courtesy of Amgen

Single Use Systems (SUS) Pros: Smaller carbon footprint Less water and energy use Higher cell densities possible per volume/size of culture Recyclable plastic???

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

Single Use Systems (SUS)

https://bioprocessintl.com/wp-content/uploads/2015/04/Rentschler_13468_600x400.jpg

https://www.pall.com/content/dam/pall/biopharm/segments/segment-singleuse2.jpg

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

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

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

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

3:50pm-4:20pm

Lab 3: Transfer and scale up

4:20pm-4:40pm

Evaluations and Wrap up

QUESTIONS? THANK YOU THANK YOU