Biopharmaceutical manufacturing processes are complex, challenging and utilize living organisms to produce safe and efficacious biopharmaceuticals. These molecules themselves have high molecular weights and complex structures that will exhibit heterogeneity such that at any given vial contains not one active ingredient but a population of biologically active molecules which must have maximal benefit to the patient with minimal deleterious effects. The necessity for controlling variation in processes, and hence product, is self-evident when we consider how our actions affect the lives of the patients our products are developed for. This presentation focuses on understanding the various origins of process variation and examines strategies for reducing their impact or eliminating them all together. http://parker.com/dh

1. July 28, 2016
Understanding and Controlling
Bioprocess Variation

2. Aims and Objectives
1. Link between biopharm product and
bioprocess variation.
2. Sources of bioprocess variation.
3. Use process development to minimize
bioprocess variation.
4. Best operating practices to minimize
bioprocess variation.
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3. Biopharmaceuticals
Biological Medicines
• Recombinant Proteins
• Vaccines
• Gene Therapies
• Cellular Therapies
Produced with
Live Expression Systems
• Mammalian Cells (eg CHO)
• Bacteria (eg Escherichia coli)
• Yeast (eg Pichia Pastoris)
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4. Structure of a Biopharmaceutical is
Critical to Safety & Efficacy
Biopharmaceutical Characteristics
• 1000x larger than aspirin with complex
structures
• Function highly dependent on structure
• Heterogeneous mix of molecular structure
in the final drug product
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A range of modifications can be made to the
protein once the cell has translated the gene

5. Structure of a Biopharmaceutical is
Critical to Safety & Efficacy
Examples of post-translational
modifications
• N-terminal pyroglutamines
• Oxidations
• Deamidations
• Glycosylations
• Glycations
• C-terminal lysine processing
1. Product profile to match that
verified in clinical trials
2. Manufactured using a
process consistent with that
used in clinical trials
3. “Process is the product”
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Recreated from “Application of Quality by
Design Paradigm to the Manufacture of
Protein Therapeutics” by del Val et al.
http://dx.doi.org/10.5772/50261

6. Process Validation: General
Principles and Practices (FDA)
Manufacturers should:
• Understand variation and the sources of variation
• Detect the presence and degree of variation
• Understand the impact of variation on the process and ultimately on
the Critical Quality Attributes (eg post-translational modifications,
glycosylation)
• Control the variation in a manner commensurate with the risk it
represents to the process and product
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7. Process Validation: General
Principles and Practices (FDA
Establishing scientific evidence that a process is
capable of consistently delivering quality product
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Process
Design
i. Building & Capturing
Process Knowledge &
Understanding
ii. Establishing a strategy
for Process Control
Process
Qualification
i. Design of a facility &
qualification of utilities
and equipment
ii. Process Performance
Qualification
Continued
Process
Verification

8. Quality by Design: ICH Q8 (R2)
Traditional Approach to Process Development:
• Regulatory filings described the manufacturing
process like a recipe
• Didn’t include the description of why the process
was designed the way it is
Quality by Design to Process Development:
• More concerned with CQAs than how they
are derived
• Multidimensional design spaces created
during development
• These are descriptions of how the process
can be operated to derive product of the
appropriate quality
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9. Quality by Design: ICH Q8 (R2)
• Risk management tools eg:
FMEA can been used to
identify possible critical process
parameters
• Identified process parameters
can be studied using DOE to
generate design spaces
• UMETRICS explain the need to
account for uncertainty in
regression model so that the
risk of failure is acceptably low
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10. Understanding the Sources of Variation
During Single-use Bioprocessing
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1. Biological
2. Raw Materials & Consumables
3. Operational Inputs (measurement,
method, personnel & machine)
4. The Environment

11. 1. Biological Variation
Vaccine candidate with protein
disulfide isomerase (PDI)
over-expression
• Under normal expression conditions in
Pichia, 6 different product variants were
detected
• Significant reduction in O-linked
mannosylation which can trigger rapid
clearance from blood stream
• PDI is chaperone protein that promotes
disulfide bond formation & correct folding
• Vaccine candidate has 11 disulphide
bonds
• Significant improvement in product
quality achieved
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Malaria protein
alone
(kDa)
Co-expression
with PDI
(kDa)
20.2
20.4 20.4
20.6 20.6
20.8
20.9
21.1
Electron spray ionization
mass spectrometry
Initial peak at 20.4kDa contains no
O-mannosylation and is followed by 5 peaks
containing 162 Da adducts of mannose
Molecular Design of Recombinant Malaria Vaccines Expressed By Pichia pastoris
(David Narum in Quality by Design for Biopharmaceuticals Edited by Rathore & Mhatre)

12. 1. Controlling Biological Variation
Through Automating Bioreactor Feeds
Cell Culture Process:
• 3 x 10L 18 day cell cultures
• 4L starting volume with 10% bolus daily
feeds from Day 5
• Day 5 Cell Viabilities for each bioreactor
was >98% demonstrating a consistent
starting point for comparison
Manual Feed Method:
• Each bolus prepared daily and delivered
under gravity
MabTec® Automated Feed Method:
• 1 batch of media prepared for automated
feeding over 18 days
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SciLog MabTec® a bioreactor
feed control system from
Parker domnick hunter

13. 1. Controlling Biological Variation
Through Automating Bioreactor Feeds
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14. 1. Controlling Biological Variation
Through Automating Bioreactor Feeds
Benefits:
• Eliminates variability derived from rates of
addition
• Reduced opportunity for error & failures
• Process documented with Parker domnick
hunter’s SciDoc Software
• Easier to control between batches
• Operator time freed up for other operations
Automation Eliminated:
• 30 manual operations
• 15 autoclave cycles
• 15 feed preparations
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15. 2. Variation from Raw Materials
& Consumables
ICH Q7
1. Procedures in place for accepting or rejecting received
materials
2. System for evaluating suppliers of critical materials
3. Materials purchased against an agreed specification
4. Identification of the manufacturer if not the supplier
5. Changing of materials covered under the Change
Control process
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16. Biopharmaceutical Manufacturing
with Single-use Technologies
• Single-use options are
available for most if not all
steps in the bioprocess
• Reduced utilities
requirements
• Reduced risk of product cross
contamination
• Delay to CAPEX during
product development
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Parker domnick hunter
automated single-use
sterile filtration system

17. 2. Variation from Raw Materials
& Consumables
• Supplier provided tubing from
the upper and lower limits of the
tubing ø specification
• Two distributions were being
supplied
• Overmolding process established
on the high ø tubing not
appropriate for low ø tubing
leading to variable quality
• Worked with supplier to provide
a single-distribution of tubing ø
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Supplied tubing diameters

18. FDA Guidance Document: PAT – A framework
for Innovative Pharmaceutical Development,
Manufacturing & Quality Assurance
A system for designing, analyzing & controlling
manufacturing through…
• Timely measurements of critical quality and performance
attributes of raw, in-process materials and processes
• Goal of ensuring final product quality
PAT Tools
• Multivariate tools for design, data
acquisition & analysis
• Process analyzers
• Process control tools
• Continuous improvement & knowledge
management tools18

19. 3. Application of PAT to Enabling
CQAs to be met
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Virus Reduction Filtration
SciFlex® NFF
Final Ultrafiltration
SciPure® TFF
Final Bulk Sterilization
SciFlex® TFF

20. 3. Application of PAT to Enable
CQAs to be met
Virus Reduction Filtration
SciFlex® NFF
• Opportunity to provide
feedback control to divert
process or via heat
exchanger
• Documented evidence
validated temperature limits
were not exceeded
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SciTemp®
pre-calibrated
single-use
temperature
sensor

21. 3. Application of PAT to Enable
CQAs to be met
Final Ultrafiltration
SciPure® TFF
• Control to ensure
conductivity specification
of Drug Substance has
been met
• Documented evidence of
process reproducibility
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SciCon®
pre-calibrated
single-use
conductivity
sensor

22. 3. Application of PAT to Enable
CQAs to be met
Final Bulk Sterilization
SciFlex® TFF
• Control to ensure validated
pressure limits are not
exceeded
• Documented evidence of
process reproducibility
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SciPres®
pre-calibrated
single-use
pressure
sensor

23. MVA for Making Sense of Data
• Multivariate data analysis can be
used to compress data from multiple
sensors
• Data from all relevant process
parameters are concentrated to a
few highly informative graphs
• Simplifies overview, analysis and
interpretation
• Enable use of data by increasing ease of use
• Transfer of compressed information
back to raw data for analysis
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24. 3. Reducing Variability in Operations
R/P Stat Method is a patented process control
method linked to process analysers
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25. 3. Reducing Variability in Operations
Case Study: Final Bulk Filtration & Dispense
• Automated system for
performing final bulk filtration
& dispensing
• Up to 80L of final drug
substance can be dispensed
into bags or bottles in aliquots
of 125 mL to 2L
• SciPres® pressure sensors &
R/P Stat Method incorporated
to ensure validated pressure
limits not exceeded of critical
sterilizing-grade filtration
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26. 3. Reducing Variability in Operations
Case Study: Final Bulk Filtration & Dispense
• Automation of a
ten step process
• Standardization of the
filtration & dispense
operation between
operators, batches
and campaigns
• Increased dispense
accuracy
• Reduced opportunity
for error
1. Filter flush
2. Equilibrate
3. Air purge
4. Filtration
5. Air purge
6. Filter integrity test
7. Dispense set-up
8. Prime
9. Dispense
10. Product recovery
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27. 4. Controlling Variation derived from
the Environment
Case Study: Final Bulk Filtration & Dispense
• Process previously performed
using a vertical laminar
flow hood
• Required considerable
maintenance and aseptic skill
from two operators
• Closed system for filtration
and dispense of bulk
• Significantly reduces the
risk of environmental
contaminations.27

28. Conclusions
The effective control of bioprocesses
is required to ensure patient safety.
Sources of process variation:
1. Biological
2. Raw Materials & Consumables
3. Operational Inputs (measurement,
method, personnel & machine)
4. The Environment
Process Development and
Manufacturing Strategies can reduce
the impact of variation
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29. Contact us
North America: +1 877 784 2234
ROW: +44 (0)191 410 5121
dhprocessinfo@parker.com
www.parker.com/dh-bioprocessing
https://www.linkedin.com/company/
bioprocessing-and-pharmaceuticals
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