Introduction to Biopharmaceutical Manufacturing Laboratory Exercises
June, 2021 1
Laboratory Exercises • Aseptic Processing • Cell Viability
• Environmental Monitoring • Mixing Study • Introduction to bioreactors (review of parts/functions)
• Chromatography 2
Aseptic Processing- Cell Thaw • Equipment and Raw Materials • Gowning
• Trained Staff • Cell Thaw • Personnel Monitoring
• Environmental Monitoring of BSC/LFU 3
Equipment and Raw Materials Examples: Equipment ID BSC -001 BSC-002
Certification Date 09/06/2020 08/13/2020
Certification Due Date 09/30/2021 08/31/2021
Minihelic Reading 0.25” 0.25”
Initial/Date CM 5/3/2021
Media Type Touch Plates
Manufacturer Remel
Media Lot Number 9280464
Date of Expiration 05/05/2020
Initial/Date CM 5/3/2021
Cell Culture Media IMX
Manufacturer
Media Lot Number
Date of Expiration
Initial/Date
Amgen
9280464
08/05/2021
CM 5/3/2021
What do you think it takes to use these?
100% Team Sport
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Typical Gowning Scenarios ISO 7 ISO 5
ISO 8
https://www.lifescienceleader.com
https://industry24h.com https://www.criticalenvironmentsolutions.co.uk
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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)
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Recording (Aseptic Cell Thaw)
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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
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Recording (Cell Viability)
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Image (Cell Viability)
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Calculation (Cell Viability) • Quick Calculation • From the hemocytometer count XX live cells and XX dead cells • Determine Viability 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑉𝑖𝑎𝑏𝑙𝑒 𝐶𝑒𝑙𝑙𝑠 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑉𝑖𝑎𝑏𝑙𝑒 𝐶𝑒𝑙𝑙𝑠+𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑁𝑜𝑛𝑣𝑖𝑎𝑙𝑏𝑒 𝐶𝑒𝑙𝑙𝑠
x 100%
• The cell preparation is XX % viable
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Calculation (Cell Viability) • Quick Calculation • From the hemocytometer we counted 20 live cells and 2 dead cells • Determine Viability 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑉𝑖𝑎𝑏𝑙𝑒 𝐶𝑒𝑙𝑙𝑠 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑉𝑖𝑎𝑏𝑙𝑒 𝐶𝑒𝑙𝑙𝑠+𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑁𝑜𝑛𝑣𝑖𝑎𝑙𝑏𝑒 𝐶𝑒𝑙𝑙𝑠
20 x 20+2
x 100%
100%= 91%
• The cell preparation is 91% viable
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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 𝑉𝑖𝑎𝑏𝑙𝑒 𝐶𝑒𝑙𝑙𝑠 # 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑
20 x 4
x 104 x dilution factor
104 x 10 = 5.0 x 105
𝑐𝑒𝑙𝑙𝑠 𝑚𝐿
Now multiply by the total cell suspension volume (i.e., 20 mL) to get total cell number:
5.0 x 105
𝑐𝑒𝑙𝑙𝑠 𝑚𝐿
x 20 mL= 10 x 106 cells 13
Environmental Monitoring • •
• • •
Monitoring is performed on a routine basis Looking for both non-viable particulates and viable Established ISO standards for non-viable particulates Establish Alert and Action Levels Trending data
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Non-Viable vs. Viable
Source: http://cutestockfootage.com
Source: http://faculty.fiu.edu/ 15
Environmental Monitoring- Non-Viable
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Common Bacteria
E. coli https://www.researchgate.net/
B. subtillus https://www.gettyimages.com/
S. epidermis https://http://faculty.weber.edu/
S. aureus https://en.wikipedia.org/
P. aeruginosa https://en.wikipedia.org/
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Environment Monitoring – Non-Viable (video)
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Environment Monitoring –Viable (video)
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Environment Monitoring – Surface (Floor video)
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Mixing • Importance of Mixing • Media/Cell Suspension/Buffers/Product Pools • Vessel Shape • Impellor Type • Speed • Cell Shear • Foam Generation • Vortex
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Impellers:
Gentle Marine Blade Impellers Pitched Blade Impellers
Source: BioProcess International; January, 2009
Rushton Impellers
What do you think the difference is? 22
Recording (Mixing Study)
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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 24
Bioreactors • Key Features: • Materials of Construction • Probes • Temperature • pH • Dissolved oxygen (DO)
• Agitation • Antifoam addition • Sampling ports
Source: https://www.labcompare.com
• SIP system-closed • Nutrients (glucose, protein, etc.)
Source: https://www. www.b2bcentral.co.za
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Recording (Cell Bioreactors)
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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
96 93.5 91 88.5 86 83.5 81 78.5 76 73.5 71 68.5 66 63.5 61 58.5 56 53.5 51 48.5 46 43.5 41 38.5 36 33.5 31 28.5 26 23.5 21 18.5 16 13.5 11 8.5 6 3.5 1 Time (hours)
Process Value Setpoint
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7.2
pH
Recent BioFlo Run (1001 Data)
BioFlo Run 1001- pH 8
7.8
7.6
7.4
7
6.8
6.6
6.4
6.2
6
96 93.5 91 88.5 86 83.5 81 78.5 76 73.5 71 68.5 66 63.5 61 58.5 56 53.5 51 48.5 46 43.5 41 38.5 36 33.5 31 28.5 26 23.5 21 18.5 16 13.5 11 8.5 6 3.5 1
Process Value Setpoint
Time (hours)
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35
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Dissolved O2 (%)
Recent BioFlo Run (1001 Data)
BioFlo Run 1001 Dissolved Oxygen 38
37
36
33
32
96 93.5 91 88.5 86 83.5 81 78.5 76 73.5 71 68.5 66 63.5 61 58.5 56 53.5 51 48.5 46 43.5 41 38.5 36 33.5 31 28.5 26 23.5 21 18.5 16 13.5 11 8.5 6 3.5 1 Time (hours)
Process Value Setpoint
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Protein Purification
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Purification (Recording)
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Closeout/Evaluation
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GFP Purification Demo
GFP Purification on Anion IEX LC Cartridge GFP Load
Equilibrate with Load buffer
➢ ➢ ➢ ➢
Wash with Load buffer
Load buffer – 50 Mm Tris HCl pH 8.5, Cellufine MAX Q-h anion IEX cartridge (1 mL) Elution buffer – Load + 1.0 M NaCl GFP in load buffer
Elute with 1.0 M NaCl in Load buffer
High salt wash
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Troubleshooting GFP Load
Wash NO binding of GFP to the cartridge?
At pH 8.5 the GFP has a net –ve charge and will not bind to a MAX S-h cation exchange resin.
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