Streamlining Process Monitoring in Cell Culture Analysis & Spectrometry

Streamlining Process
Monitoring in Cell Culture
Analysis & Spectrometry
Daniel Merriman
Strategic Marketing Manager
Thermo Fisher Scientific
Femi Egbebi, PhD
Senior Global Product Manager
Media Development
Services/Omics Capabilities
Krzysztof Inglot, PhD
Technical Sales Specialist

Application and technical experts
present an overview of process
monitoring systems in the biopharma
field.
Streamlining Process
Monitoring in Cell Culture
Analysis & Spectrometry

Streamlining Process Monitoring
with TruBio
SUT Technical Sales Specialist, CEE & Middle East
Proprietary & Confidential | 25-August-2021
3

Quality Scale-Up is Critical
• Time to market
• Manual data entry
What are the challenges?
What is most critical?
• Consistent/predictable product quality
• System validation
• Data integrity
• GMP manufacturing facility regulatory requirements
• Risk mitigation
• Speed to market
• Efficient, traceable data transfer
• Quality of data maintained throughout the
manufacturing process
4

Industry Challenge
• Integration of existing equipment causing automation islands
• Highly engineered, product-specific PLC based facilities
• Unique and nonstandard data output
Facility flexibility
Mobius CellReady
EMD Millipore
BioBLU
Eppendorf
UniVessel Glass
Sartorius
ez2-Control
Applikon
Allegro STR
Pall
Xcellerex XDR
GE Life Sciences
Xcellerex XDR 10
GE Life Sciences
BIOSTAT STR
Sartorius
HyPerforma S.U.B.
Mobius Single-Use Bioreactor
EMD Millipore
5

Our Solution
A highly configurable, open-architecture system from R&D to production
TruBio Software
Research/PD Clinical to production
6

Our Solution
One consistent solution from R&D to production
Thermo Scientific™
TruBio™ Software
Clinical
Production
TruBio Software
DCS/SCADA consistent data flow
Research &
Process Development
7

DeltaV Network
Remote Access
Redundant DeltaV Network
Plant LAN
Operator Station Professional Plus Application Station/Historian
Scales, pumps, G3Lab Universal Controller for
Small Scale Vessels
OPC
SERVER
Plant Messenger
OPC compliant third party bioreactor controllers, Analyzers or other devices
G3Pro Universal controller for highest vessel flexibility as single or dual systems
G3Lite controller for one vessel
Terminal Server
DeltaV Controller
Centrifugation
Mixers
Clarification Chromatography
Virus Removal
Ultrafiltration Sterile filtration
Filtration
OPC
OPC
Operator Station PCs or mobile devices with browser (TS)
8

Introduction to TruBio
9 Proprietary & Confidential | 25-August-2021
• Software application with inbuilt control
strategies specifically for bioprocesses.
• Commercial off the shelf (COTS), FDA 21 CFR
part 11 compliant.
• Easy to plan, modify and implement new
process requirements using the standard
TruBio functions and features.
• Based on Emerson’s DeltaV with easy scale-
up and -down between R&D, PD and
manufacturing.
• TruBio 5.0 requires DeltaV 12.3 or higher
What is TruBio?

Introduction to TruBio
What are Control Strategies
• Control Strategies are a predefined function
which assist the operators achieve their process
requirements.
• TruBio contains the following functions as
standard (e.g.):
• pH Control
• DO (Dissolved Oxygen) Control
• Temperature Control
• Weight Control
• Dosing
• Continuous
• pH Base and pH Acid
• Pump Totals
• Multi Feed Dosing
• The following functions come as standard in
TruBio to assist in easy setup and process
manipulation:
• Calibration of IO and calibration records (e.g. pH,
DO, pumps)
• Save/load files: easy exchanging control
strategies between sites
• Vessel selection – Move from one vessel
independent of size to another.
• Use the same IO tower for running different vessels.
TruBio Functions

TruBio and Vessel Integration

TruBio
TruBio and Vessel Integration
• Seamless integration with other manufacture vessels and rockers e.g.
• Integration with other manufacture equipment e.g..

TruBio
TruBio Third-party interfacing examples
TRUBIO DeltaV system
Eppendorf
Incubators
GE Chrom Skids Parker NFF Skids Parker TFF Skids
Satorius Scales
Hamilton Probes
Various
Manufacture
TCUs
Remote IO
Panels
Profibus Modbus Ethernet IP
Traditional IO
OPC OPC OPC
Modbus
Traditional IO/ Modbus/
Profibus

Standard TruBio – Process Configuration without
Programming
14

TruBio 5.0 + G3Lab Universal controller ≤ 250L
HyPerforma Glass Bioreactor HyPerforma Rocker
Benchtop Single-Use Bioreactor HyPerforma SUB
15

Customized TruBio – Mapping the Process
16

Customized TruBio – Mapping the Process
17

Facilitie examples
18

Chromatography skid

Eppendorf incubators
WFI – Water For Injection
TruBio adaptation

Same control software throughout: TruBio Software
Data Integrity Maintained Through Tech Transfer
Scale: Commercial
Vessel size: Up to 5,000 L
Scale: Clinical
Vessel size: 30 L–2,000 L
Scale: Research/PD
Vessel size:1 L–50 L
Superior connectivity:
• Secure
• Repeatable
• Traceable
21

The Data Flow
Data integrity and precise management to the top
Enterprise management level
(Planning, logistics, finance)
Plant management level
(Scheduling, analysis)
Production process control
(DCS, SCADA)
Plant floor equipment, control,
instrumentation, sensors, I/O
TruBio Software
Provides inherent connectivity
to DCS, consistent from R&D to production
22

TruBio
DreamReport
Provide Data
Generate and Execute
Design and Configure
Real-Time
Data and Alarms
HMIs, PLCs, DCS, SCADA, …
Connect to Data Sources
HISTORICAL DATA
• ODBC & OLE
• OPC HDA
• CSV files
• Native drivers for
proprietary archives
REAL TIME
Data and alarms
• OPC DA-AE
• Native Drivers
Dream Report logger can archive
real time data into any open
database
• SQL Server
• MySQL
• Oracle
• Access
• …
Provide Data
Provide Data
Provide Reports
• Design reports in easy environment
• Use Page Templates
• Use Report Templates
Define and periodically execute
any type of calculations:
Min, Max, Average, Current,
Integral, Duration, Counter, SQL
commands, free Mathematical
Calculation…
Automatically:
❑ On Schedule
❑ On Event
Manually:
❑ Select Report
❑ Change period or select batch
❑ Generate new report
Automatically Publish and Distribute
Generate
Reports
Design
Reports
Configure Data
Aggregation
Access existing
3rd-party archives
Data
Logger
Web Portal File Server Email Printers Mobile
FTP Server

Femi Egbebi, PhD
Multi-Omics for Cell Culture
Medium and Process
Development
24

Why multi-omics for medium and process development?
Untargeted towards targeted

Traditional vs. multi-omics media development workflow
Traditional workflow: multiple full-factorial DOEs
< 50 molecules: amino acids, ~10 metabolites
Multi-omics workflow: metabolic pathway analysis
8000 molecules: 1000 metabolites, 7000 proteins
Tech
transfer
Media
mixture
DOE
Medium
optimization
DOE
Process
optimization
Non-GMP /
dry powder
Feed
optimization
DOE
Panel screening
(Metabolic baseline
establishment)
Multi-omics data
interrogation
(Acquisition, analysis,
interpretation)
Media & feed
design DOE
Media, feed &
process optimization
Non-GMP /
dry powder
Multi-omics provides more information, better insights into nutritional requirements
Titer
Titer

Introduction: multi-omics driven medium design
Metabolic
Baseline
• Adapt to a panel of media
• Perform growth performance assay (GPA)
• Multi-omics sample collection
Multi-Omics
Analysis
• Automated sample preparation
• Sate-of-the-art multi-omics analysis
• Multi-Omics integration and interpretation
Omics
Driven DOE
• Identify pathways and networks influencing GPA features
• Design and validate DOE factors of interest in AMBR
• QC of mAbs and medium formulation
Biological function-oriented cell culture media development

Establishing a metabolic baseline
Characterizing the cell culture process and fed-batch media
➢ Robust and scalable: Pursues the small-scale
model; is relevant and predictive to the large-scale
process.
➢ Fed-batch cultivation: Utilizes run data from a
panel evolution to identify which conditions to analyze.
▪ Low VCD vs High VCD
▪ Low Titer vs High Titer
▪ Low Viability vs High Viability
➢ Spent media analysis: Metabolic run data
(Glucose, Lactate, Glutamine, Ammonia etc.) can guide
initial interpretation of Multi-Omics data sets.
➢ Quality attributes of mAbs: Glycosylation
pattern, charge variants, aggregates, and low-
molecular-weight species.
Media diversity & feature integration
29

Multi-omics analysis workflow
Enable large-scale sample analysis and data interpretation
Analytical sensitivity/reproducibility & decision accuracy
30

Small molecule identification & quantification
• Orbitrap ID-X Tribid MS system
• 3 mass analyzers: Quadrupole; LIT; Orbitrap
• Flexibility (Non-targeted & targeted assays)
• Tandem in time
• Quantification by Compound Discoverer
software
High accuracy, precision, resolution and sensitivity
Identify more endogenous metabolites
31

Identification & quantification of large molecules
32
• Orbitrap Fusion Lumos Tribid MS
• Optimized HPLC-MS method
Identify / quantify more proteins in less time and with high reproducibility
Proprietary & Confidential | 20-Apr-2021
< 0.5 min RT shift across 100 runs < 20 % intensity drop across 100 runs

Case study: Application of multi-omics approach in the
development of a CHO production medium
33
Experimental approach
• Goal: Perform a proof-of-concept experiment to demonstrate the ability to design/improve cell
culture medium from multi-omics data sets
• Background:
• CHO-S cell line produces ~40% more IgG in Medium 1 vs. Medium 2
• Medium 1 and Medium 2 are chemically-defined CHO media
• Target:
• Improve Medium 2 to exceed Medium 1 performance by 50% using traditional and omics
approaches in parallel

IgG titer of CHO cell line in Medium 1 and Medium 2 in a simple fed-batch process in 1L bioreactor
CHO S cell line demonstrated 40% higher titer in Medium 1 as compared to Medium 2
Simple fed-batch
process sampled for
metabolomics (daily)
and proteomics
(arrows)

35
Mass spec omics analysis
Titer influencing pathway
identification
Omics guided DOE
Medium 2
Medium 1
Omics approach prototype
Traditional approach prototype
>1500
changes
7000 proteins + 1000 metabolites
identified
Pathways highlighted
Successfully met project goal utilizing omics approach in medium development

Multi factorial DOE based on multi-omics analysis
Titer improvement is impressive - competitive enough against other improvement options
Amonia Influencers TCA Intermediates Lactate Influencers Critical Amino Acids Anti-Oxidants
1 -1 1 -1 -1 -1
2 -1 -1 1 -1 -1
3 1 1 -1 1 -1
4 -1 -1 -1 -1 1
5 -1 -1 -1 1 -1
6 -1 1 -1 1 1
7 1 -1 1 -1 1
8 1 1 -1 -1 1
9 1 1 1 -1 -1
10 1 -1 -1 1 1
11 1 1 1 1 1
12 -1 1 1 1 -1
13 -1 -1 1 1 1
14 1 -1 1 1 -1
15 1 -1 -1 -1 -1
16 -1 1 1 -1 1
Final optimization resulted in a 3x increase in titer for customer

Why choose Thermo Fisher for media development?
37
Experience and expertise Collaborative
Driving performance through collaboration
• 50 years of cell line / media development
experience
• 150 projects completed since 2006
• Decades of tech transfer expertise
• Worked with >100 client PD groups
• 35% of projects from repeat customers
• Dedicated business team, support team
and program managers
Consistency
• World-class processes and facilities
• Assurance of supply to meet demands
• Scalability to GMP
• Rapid Prototyping Service

Daniel Merriman
Real-time Monitoring of
Fermentation & Cell Culture
Senior Manager, Strategic Marketing
38

What the FDA says about PAT
• PAT provides an opportunity to move from the current “testing to document quality” paradigm to a
“Continuous Quality Assurance” paradigm that can improve our ability to ensure quality was “built-in”
or was “by design” - ultimate realization of the true spirit of cGMP!
• Greater insight and understating of processes
• At/On/In-line measurement of “performance” attributes
• Real-time or rapid feedback controls (focus on prevention)
• Potential for significant reduction in production and development cycle time
• Minimize risks of poor process quality and reduce regulatory concerns
• Ajaz S. Hussain, Ph.D.
• Deputy Director, Office of Pharmaceutical Science, CDER, FDA
• ‘PAT Initiative: Next Steps’, 2004
39

PAT steps
Source: Process Analytical Technologies for Pharmaceuticals: Global Markets. January 2018. bcc Research.
• Design phase: The critical quality
attributes (CQAs) influenced during
the process are studied and critical
process parameters (CPPs) are
determined
• Analysis phase: Analysis of the
appropriate CQAs studied in the first
phase
• Monitoring phase: monitor the CPPs
• Control phase: Controlling CPPs
based on understanding the process,
utilise real-time data, effect consistent
performance and product quality
PAT
Process
Design Analysis Monitor Control
40

Implementation of gas analysis at the lab
Outlet Gas
DCS, PLC or
SCADA
Inlet Gas
Oxygen, Carbon Dioxide, Nitrogen, Argon
Outlet Gas
Oxygen, Carbon Dioxide, Nitrogen, Argon
Derived Values (RQ, CER, OUR)
Methanol, Ethanol, Water, Hydrogen, Ammonia, Hydrogen
Sulfide …
Prima BT
Inlet Gas
Vent
41

The value of respiratory gas analysis
Oxygen Uptake Rate can be used to trigger nutrient additions to maintain viability
Glutamine depletion
42

Bacterial & microbial fermentation
E. coli fed batch fermentation over 60 hours
MS measures O2, CO2, N2 & Ar in inlet and outlet and calculates RQ
Reagent introduced at temperature To
to induce cells to change from multiplying to
expressing desired product
S. cerevisiae fed batch fermentation over 60 hours
MS measures O2, CO2, EtOH, N2 & Ar in inlet and outlet and calculates RQ
Ethanol is initial carbon source, then switch over to glucose to achieve RQ of 1 and
desired product
43

Process control by RQ – fed batch
Fed batch process with hexose carbon source
Aim to maintain RQ at 1
Nutrient is added in increments as fermentation progresses
When RQ drops to 0.8, add glucose and biomass continues to grow
Second & third triggers for glucose addition set at 0.85
This degree of control can only be provided with very precise RQ measurement
No other technique can match this level of precision
44

Cell culture – early experiment (VCC and off-gas)
Significantly higher VCC in Bio 1 that Bio 2
Significantly more CO2 evolved and O2 consumed in Bio 1 than Bio 2
Changes in feed gas composition produce significant noise levels in feed
concentrations
Source: Applications of off-gas mass spectrometry in fed-batch mammalian cell culture. Goh Hai-Yuan et al., 2019
45

Cell culture – later experiment (VCC and off gas)
Significantly higher VCC in Bio 1 that Bio 2
Significantly more CO2 evolved and O2 consumed in Bio 1 than Bio 2
Changes in feed gas composition produce significant noise levels in feed
concentrations
46

Sparge gas O2 and VCC correlation
47

RQ determination – cell culture
The GasWorks software can calculate and report automatically and in real-time
the Oxygen Uptake Rate (OUR) and Carbon Dioxide Evolution Rate (CER) in
terms of percentages of inlet flow (CDC and OXC), and also the Respiratory
Quotient (RQ).
CDC and OXC reached plateau just after 100 hours and
subsequently diverged suggesting a switch in cellular metabolism.
In the lag-phase, the RQ values obtained are not significant
as very little respiratory activity is occurring.
RQ values was between 1.0-0.8 during the logarithmic growth and
stationary phase stages, matching theoretical values of 0.9 based on
glutamine and glucose oxidation (Fitzpatrick et al., 1993).
Source: Online characterization of mammalian cell culture off-gas composition by magnetic sector MS for real-time bioprocess monitoring
Floris et al., 2020
48

Types of biotech customers
Pharmaceuticals
Antibiotics, Proteins, Enzymes, Hormones, Vaccines,
Vitamins
Food Ingredients
Monosodium Glutamate, Riboflavin
Agricultural Products
•Plant Nutrients, Animal Food Supplements
Biomaterials
•Collagen, Biodegradable Plastics
Bio-fuels
•Ethanol, Propanol, Butanol, Hydrogen for Fuel Cells
49

Resources
Web resources – video, specific PAT pages, e-books, product pages etc…
50

Thank you

Thank you for participating!
CLICK HERE to learn more and
watch the webinar
Daniel Merriman
Strategic Marketing Manager
Femi Egbebi, PhD
Media Development
Services/Omics Capabilities
Technical Sales Specialist

Streamlining Process Monitoring in Cell Culture Analysis & Spectrometry

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