The Process of Transgenic TFF Development

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The Process of Transgenic TFF Development
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Presentation Agenda

• Overview
• Tangential Flow Filtration Systems
• What is Tangential Flow Filtration
• Microfiltration/Clarification
• Parameters
• Optimization Protocol
• Scale-Up
• Smart Tips
• Ultrafiltration
• Parameters
• Optimization Protocol
• Scale-Up
• Smart Tips
• Summary

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Overview

• Transgenic expression systems require unique
  capabilities and different processes than other
  protein production systems
• The diverse nature of transgenic systems (milk to
  plants to duck weed) require additional attention
• Consequently, developing a Transgenic Tangential
  Flow Filtration ( Transgenic TFF) process
  requires special attention to process
  optimization and operating conditions

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Proteins From Cell Culture Typical Downstream
Processing Flow Diagram
Product Source Material
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Prime Stages For Process OptimizationMicrofiltra
tion
Product Source Material
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Prime Stages For Process OptimizationUltrafiltra
tion
Product Source Material
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Prime Stages For Process OptimizationConcentrati
on
Product Source Material
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Prime Stages For Process Optimization Final
Concentration / Buffer
Product Source Material
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Overview What is Tangential Flow Filtration?
Permeate
Pores
Membrane
Shear
Gel Layer
Retentate Channel
Retentate Flow
Permeate
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Microfiltration/Clarification Parameters -
Overview

• Situation Analysis
• Large distribution of particle sizes
• Complex particle interaction
• Casein proteins present
• Fats and lipids
• Plant matter
• Low purity
• Objective
• Achieve high purity, in a low solids, low
  viscosity solution quickly and repeatable

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Clarification Parameters to Optimize

• Retentate Channel Height
• Mathematically or Empirically
• Objective is to minimize while still
• Sufficiently high to allow passage of clumps
  (plant matter)
• Prevent excessive Pressure Drop
• Provide proper angle of attack

Typical Starting point for Milk based 0.5mm
to 0.875mm Typical Starting point for Plant
based 0.75mm to 1.5mm
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Clarification Parameters to Optimize Retentate
Velocity/Shear Rate
VELOCITY
SHEAR

• Optimization Requirements to Minimize Gel
  Layer and Fouling
• Right velocity for protein passage
• Higher permeate rate does not represent
  higher total passage

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Flux Versus Recovery
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Clarification Parameters to Optimize Retentate
Velocity/Shear Rate
VELOCITY
SHEAR

• Starting point for Milk and Plant Based
• 0.7 to 1.2 m/s and 5,000 to 10,000 sec-1

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Microfiltration/Clarification Parameters
Membrane Chemistry
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Microfiltration/Clarification Parameters
Membrane Chemistry
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Microfiltration/Clarification Parameters
Membrane Chemistry
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Microfiltration/Clarification Parameters
Membrane Chemistry
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Microfiltration/Clarification Parameters
Membrane Chemistry
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Microfiltration/Clarification Parameters
Membrane Chemistry
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Clarification Parameters to Optimize Membrane
Pore Size Selection

• Primary Objective
• Select the tightest membrane which passes
  the target substance
• Selection Criteria
• Start with a membrane which is 5 to 10X larger
  than the target
  substance
• Adjust according to assay results

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Microfiltration/Clarification Parameters
Membrane Structure
Traditional MF
Asymmetric MF
Highly Asymmetric MF
Ultrafiltration UF
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Transmembrane Pressure (TMP)
Microfiltration/Clarification Parameters

• _ Inlet Outlet Permeate
• 2
• Higher TMP More Gel layer Fouling

Typical Starting Point for Milk and Plant Based
2 to 6 psig (0.1 to 0.4
bar)
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Microfiltration/Clarification Parameters

• Temperature
• Understand molecular sensitivities
• Look for interaction at different temperatures,
  especially interaction with casein

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Clarification Optimization Protocol

• Define the process needs
• Select Channel Height 0.75mm
• Select initial membrane material and pore size
• Select sample size membrane area and temperature
  based on future scale-up plans and protein
  compatibility
• Concentrate two (2) samples, 10 fold at two shear
  rates
• 5,000 sec-1
• 10,000 sec-1
• Zero Backpressure
• Remove the backpressure valve
• Enlarge the retentate line
• Transmembrane Pressure 2 to 4 psig (0.1 to 0.3
  bar)

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Clarification Optimization Protocol

• Collect retentate and permeate samples
  simultaneously
• Start, 2X, 4X, 8X and 10X
• Total 10 samples
• Assay the samples as pairs
• Graph the Percent Passage Results
• How did concentration affect passage?

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Clarification Optimization Protocol

• If the transmission of the target substance
  decreases with concentration
• - Typically diafilter the solution
  prior to the percent transmission
  dropping below 70
  percent.

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Clarification Optimization Protocol

• If the transmission of the target substance
  remains the same
• - concentrate 10X and diafilter the
  concentrate 3X

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Clarification Optimization Protocol

• If the transmission of the target
  substance
  increases and then decreases
  
  with concentration
• - diafilter the solution
  
  at the maximum percent
  transmission achieved

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Clarification Optimization Protocol

• Repeat this process for preferred
• membranes, TMP and channel heights

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Critical Factors Clarification Scale Up

• Membrane Area / Process Volume
  Should Remain Constant
• Maintain Critical Parameters from Optimization
• Production System Flow Dynamics

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Velocity Effects on Protein Passage and Permeate
Rate
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Temperature Effects on Protein Passage and
Permeate Rate
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Microporous Membrane FiltrationMembrane Area /
Process Volume
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Key Clarification Issues Transgenic Expression
Systems

• Presence of Casein
• High Debris Level (Plant Matter)
• Fats and Lipids
• Proper Clarification Reduces Chromatography Costs
• All Make Optimization Critical!

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Key Clarification Issues Transgenic Expression
Systems

• Rationale for Optimization
  Recommendations
• Higher channel height
• Asymmetric membrane pore structure
• Low TMP

Tech Tip For Proper Membrane Selection
Difficult to clean internal surfaces of a sponge
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Process OptimizationConcentration Parameters

• Retentate Channel Height
• Mathematically or Empirically
• Lower channel reduces pump flow while increasing
  pressure drop. Look to balance
• Typical Starting point for 0.375mm to 0.75mm
• Higher the final desired protein concentration
  the higher the retentate channel
• Reach gt30 protein concentration with 0.875mm

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Process Optimization Concentration Parameters
Concentration vs. Retentate Channel Height
(Albumin)
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Process Optimization Concentration Parameters

• Retentate Velocity / Shear Rate
• Optimal is
• Minimize Gel Layer and Fouling
• Balance Higher Flux Rate Against Larger Pump and
  Energy Cost
• Starting Point is 1.0 to 1.5 m/s and 10,000 to
  16,000 sec-1

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Process Optimization Concentration Parameters
Membrane Chemistry
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Process Optimization Concentration Parameters

• Membrane Pore Size Selection
• Goal Select the loosest membrane which retains
  the target substance
• Good starting point is pore with 1/3 to 1/2 the
  molecular weight
  of the target substance
• Neutrality of C membranes causes them to act
  larger than
  the same pore size PES membrane
• The higher the concentration desired
  
  the lower the molecular weight membrane
  you should select
• Tighter membrane restricts passage of lower
  molecular weight contaminates

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Process Optimization Concentration Parameters

• Transmembrane Pressure
• Higher TMP More Gel Layer and Fouling
• Typical ranges
• 10KD membrane 40 80 psig (2.7 5.4 bar)
• 30KD membrane 30 60 psig (2.0 4.0 bar)
• 50KD membrane 25 55 psig (1.7 3.7 bar)
• Temperature
• The higher the temperaturethe higher the
  permeate rate

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Process Optimization Concentration Protocol

• Define the process needs
• Select Channel Height 0.5mm
• Select initial membrane material and pore size
• Select sample size membrane area, temperature and
  concentration based on future scale-up plans and
  protein compatibility
• Conduct initial optimization by placing system in
  full recirculation mode

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Process Optimization Concentration Protocol

• Optimize Velocity/Shear Rate
• Start at a high shear rate (21,000 sec-1 , 2m/s
  velocity) and slowly turn down the pump in steps
  of 2,500 sec-1 shear rate, every 5 minutes
• Set initial TMP to middle of recommended range
  for given membrane
• Be sure to maintain constant TMP during run
• Graph Permeate Rate vs. Shear Rate
• Look for Steps or changes in the slope of the
  line in graph of Permeate Rate vs. Shear Rate

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Process Optimization Concentration Protocol
Optimize TMP

• Operate System at Optimized Shear Rate / Velocity
• Begin at the Low Range of the Initial TMP,
  Measure Permeate Rate
• Increase Transmembrane Pressure 3 psig (0.2 Bar)
  and Measure the Permeate Rate
• Wait 5 Minutes, Then Measure the Permeate Rate
• If Permeate Rate Increases Then Increase the
  Transmembrane Pressure Again
• Continue to Increase the Pressure Until the
  Permeate Rate gtgt
  No Longer Increases During Each Step or
  
  gtgt Does Not Hold That Increase for Five Minutes

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Process Optimization Concentration Protocol
Key Points

• The OPTIMUM MATHEMATICAL Transmembrane Pressure
  is the Last Pressure Reading Where the Increase
  in Pressure Resulted in an Increase in Permeate
  Rate
• The MOST STABLE Transmembrane Pressure is the
  Next to Last Pressure Reading
  Where the Increase in Pressure
  Resulted in an Increase in Permeate Rate

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Process Optimization Concentration Protocol
Final Concentration

• Concentrate Sample to Desired Final Concentration
  at Optimized Shear/Velocity and TMP
• Repeat With Alternate Membranes and Channel
  Heights for Complete Comparison

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Process Optimization Concentration Scale-Up

• Most Critical Factors in Ultrafiltration
  Scale-Up are
• Maintain Critical Parameters From Optimization
• Production System Flow Dynamics

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Discussion Key Concentration Issues

• Passage of Lower Molecular Weight Contaminates
  
• Reduces Chromatography Cost
• Desired Final Concentration
• Cleaning
• Membrane Life

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Summary

• With Proper Optimization Transgenic TFF Allows
  for Low Cost Downstream Processing
• In Addition to Traditional Considerations of
• membrane selection
• tangential velocity
• trans-membrane pressure

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Summary

• Specialized Challenges Presented In Transgenic
  TFF Processing Also Require Consideration of
• Shear
• Channel Height Selection
• Temperature
• Casein
• Fats and Lipids
• Expression System