Ingrid Maes

1
AT improving manufacturing performance and
developing manufacturing excellence, by using PAT
in vaccine manufacturing
Paving the way to process understanding
P

• Ingrid Maes
• Competence Centre Pharma

2
Outlook

• Improving manufacturing performance and
  developing manufacturing excellence, by using PAT
  in vaccine manufacturing
• PAT the definition and the benefits
• PAT and vaccines
• Report on experiences with PAT implementation
  (Case study)How PAT leads to increased process
  understanding
• Conclusions

3
Process Analytical Technology

• Process control through new technologies, focus
  on manufacturing science
• System for designing (process development),
  analyzing and controlling manufacturing
  processes,based on timely measurements of
  critical Q performance attributesof
  raw-materials, in-process materials and processes
  with the goal of ensuring final product Q.
• Processes to assure acceptable end-product Q at
  the completion of the process (quality by design)

FDA, Guidance for Industry PAT A framework for
innovative Pharmaceutical Manufacturing and
quality assurance, September 2004
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PAT Framework
0

• PAT Understanding controlling the process
• A process is well understood when
• All critical sources of variability are
  identified and explained
• Variability is managed by the process
• Product quality attributes can be accurately and
  reliably predicted
• Process Understanding is inversely proportional
  to risk

Dr. C. Watts, FDA, CDER 2005
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What is the true gain of PAT?

• Time-to-market
• Fast process development
• Fast product release(real-time product
  release)
• Supply Chain improvement
• Improved market responsiveness
• Reduction of production cycle times
• Decrease warehousing

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Case Bordetella pertussis cultivation
Joint development project, between 3 partners
Competence Centre PharmaceuticsSupplier of
process control, analytical solutions, IT
infrastructure

• Governmental institute, Department of Public
  Health
• Provide vaccines for the Dutch market
• Research for new or improved vaccines
• Advisor to the minister of Public Health
• Key role in calamities involving infectious
  diseases

Innovation initiative of Ministry of Economic
Affairs of The Netherlands
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PAT implementation methodology (Roadmap), during
process development
Methodology leading to increased process
understanding

• Preparation
• Assessments
• Risk
• Product
• Process
• Analyze
• Control
• Release Philosophy
• Optimization

Valida-tionPlan
Change Mana-gement
New technology introduction
Assessments
Time
Risk assess-ment
Process assessment
Knowledge manage-ment
Analyze
Analyzer assessmentDoEData collectionData
mining / MVDA
Control
Control strategy developmenRelease philosophyt
Optimisation
Continuous optimisation improvement
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The Product The Whooping Cough Vaccine

• Whole cell vaccine against B. pertussis infection
• Inactivated cells are the actual product
• Outer membrane proteins are presented to immune
  system
• Outer membrane composition is crucial for vaccine
  efficacy(virulence)
• Virulence activated genes (VAGs) expression
  depends on extra cellular conditions
• VAGs code for proteins is crucial for vaccine
  efficacy
• Classical (animal) tests do not provide enough
  discriminative power for assessment of CQAs

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Measuring VAG expression
Micro array
Protein analysis

• Protein analysis (Western Blotting, ELISA)
• Supernatant analysis (with 1H and 13C NMR)
• Protein profiling(nano LC-MS)

• DNA microarrays for expression profiling

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Search for Critical To Quality (CTQ) process steps
Critical process steps, in terms of critical to
end-product efficacy
Primary process
Secondary process
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The Process Batch Cultivation

• Input materials are thoroughly controlled
• Master / Working seedlots
• SOPs
• Defined medium
• Batch process control only at basal level
• pH
• DO (dissolved oxygen)
• T

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Monitoring Critical Attributes
Analyze

• pH, DO and T are timely but insufficient
• mRNA and protein analysis are sufficient but not
  timely
• Online monitoring of critical attributes is
  necessary

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Process NIR spectroscopy (as PAT tool)
In situ measurement sterilizable

• 4L working volume
• DO, pH, T control

Process output
Process feed
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Bioreactor/cultivation trajectory monitoring

• NIR spectra are measured every 30-60s
• No quantification of analytes, but the overall
  process behavior is assessed
• Output is a process trajectory representing the
  behavior of the batch process (as a fingerprint)

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Multivariate process trajectory
Multivariate process trajectory

• Multivariate scores summarize normal variation in
  process parameters
• The trajectory of the scores tracks the status
  and evolution of a batch
• Control limits on these summary variables
  distinguish in-control vs out of control
  operation

becomes
(PCA)
Many variables of one batch are plotted.
All the variables of same batch plotted But now
as the first SUMMARY t1.
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Cultivation monitoring during batch progress
In-situ / real-time NIR data
Essential process data NIR fingerprint
Multivariate process trajectory
Development of process specifications
Multivariate Data Analysis(PCA)
Other process data (pH, DO, T)
Correlation between end-product quality
parameters and key process parameters
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Batch-to-Batch comparison

• First the process NIR measurements are assessed
  with all additional testing in place, resulting
  in a process trajectory
• When process trajectory correlate with additional
  tests, a tunnel can be defined within which
  product quality is assured
• Validation of this tunnel can lead to real time
  QA
• Runs that fall outside tunnel still need
  additional tests

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Experimental resultsCorrelation NIR and
microarray data

• For 8 consecutive batch cultivations
• Process monitoring (NIR, pH, DO, T) throughout
  process process trajectory monitoring
• Microarray analysis at end point
• Comparison between measurements
• no difference process trajectory no difference
  in array??
• disturbance in process trajectory disturbance
  in array??

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YES!
golden batch trajectory
Control limits
Multivariate cultivation trajectories for 7
different batches

• Root cause analysis of these disturbances in two
  deviating batch trajectories
• Are these disturbances affecting vaccine efficacy?

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Product efficacy monitoring during batch
progress
In-situ / real-time NIR data
Essential process data NIR fingerprint
Multivariate process trajectory
Development of process specifications
Multivariate Data Analysis(PCA)
Other process data (pH, DO, T)
Correlation between end-product quality
parameters and key process parameters
End product quality data(microarray analysis)
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Micro array analysis
Micro array
Protein analysis

• Protein analysis (Western Blotting, ELISA)
• Supernatant analysis (with 1H and 13C NMR)
• Protein profiling(nano LC-MS)

• DNA microarrays for expression profiling

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Microarray results

• For each gene the relative expression level is
  known
• Expression analysis (process understanding)
• Which genes respond to which process conditions?
• Is there a connection between these responses?
• Expression profiling (process trajectory)
• Does the experimental profile match the reference
  profile?
• How many genes deviate?
• Which genes deviate?

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mRNA expression profile
Does the experimental profile match the reference
profile?Yes, Overall expression correlates well
Reference profile
R gt 0.99
Sample (X-axis) vs. Reference (Y-axis)
Experimental profile
R gt 0.99

• Can this micro array method also be used to pick
  up a disturbance?

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pH, DO and T trends
Oxygen limitation
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mRNA Expression profile for an oxygen limitation
R gt 0.99

• Oxygen limitation at start of culture
• RNA isolation at end of culture
• Ribosomal genes and genes involved in oxidative
  stress are disturbed

Rlt0.95
Yes, micro array method can be used to pick up an
oxygen limitation as a disturbance!
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Working Plan

• Correlation between NIR signal and off line
  analysis (does NIR pick up disturbances?) ?
  Done!
• Establishing the allowed process variation in
  regard of product quality (tunnel) ?
  Feasibility (1 and 2) done ! ? DoE execution
  Running!
• Online NIR data processing and batch to batch
  comparison ? Feasibility done ! ? Based on
  results from DoE execution Running!
• Process disturbance root cause analysis and
  correction strategies? Based on results from
  second DoE execution starts second half 2006
• Implementation of automated process control based
  on NIR measurements? basic controllers and batch
  control system implementation done ? New control
  strategies (APC) starts second half 2006

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Develop Future Control Philosophy
0
Lab
Process
Transform to quality data
Close loop control (physical / chemical
parameters only)
Temp., pH, pO2, pressure,
Temperature, pH, pO2pressure
hold release
Required Quality information
Process output
Process feed
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Develop Future Release Philosophy RTR
0
Lab
Process
Transform to quality data
Close loop control (physical / chemical
parameters only)
Temp., pH, pO2, pressure,
hold release
Temperature, pH, pO2pressure
Required Process information
Process output
Process feed
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Learning points

• Building process understanding requires
  appropriate tests. Animal tests usually are not
  discriminative enough
• Genomics and proteomics combined should show all
  there is to know about the cells(mechanistic
  understanding)
• Process NIR measurements can be used to monitor
  the progress of the batch cultivation process (as
  a process trajectory monitoring tool)
• A multivariate process trajectory can pick-up
  main process disturbance (oxygen limitation),
  leading to off spec vaccine efficacy

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Requirements
Requirement for a successful PAT approach for
vaccine process development a multidisciplinairy
approach
Team approach
Implementation methodology
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Benefits

• Application of PAT methodologies in vaccine
  development and manufacturing are allowing
• Fast process development and tech transfer
  (robust processes)
• Process understanding
• Right first time (RFT) quality
• Fast release of the batch reduced cycle time
• Quality system build-in by decision

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etting out for process excellence
S
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Questions?
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Contact
Ingrid Maes Consultant Advanced
Technologies Siemens AG Competence Centre
Pharmaceutics, Antwerp Office Naviga Business
Park Nieuwe weg 1 B-2070 Zwijndrecht,Belgium Pho
ne 32 3 735 06 08 Fax [phone]
E-mail ingrid.maes_at_siemens.com www.siemens.com
www.siemens.de/pharma