purification of next · cell and gene therapy atmp are here to stay. gene therapy products • over...
TRANSCRIPT
Purification of next generation medicines
ChairDr Richard Turner
Director of PurificationProcess Sciences
MedImmune
Dr John LiddellSenior Scientific
AdvisorCPI NBMC
Dr Oliver HardickChief Executive Officer
Puridify
Dr Kajsa Stridsberg-FridenSenior Project Manager, Downstream Processing
GE Healthcare Life Sciences
Purification of Next Generation Medicines
Richard TurnerDirector Biopharmaceutical Development. MedImmune. 29th November 2017
4
Purification - Progress
Purification of therapeutic drugs - current and future
5
Automation + DOE
Analytics Continuous
Development
strategies Scale, COG,
FTP
New
Chromatography AI
New
Modalities
Speakers
Daniel Smith – Rapid Process Development of Viral Vectors
John Liddell – Supporting the changing bioprocessing landscape
Oliver Hardick – New Primary capture technology
Kajsa Stridsberg-Friden – Next Generation Protein A Chromatography
6
Rapid process development of viral vector-based products for cell and gene therapy
applications - challenges and opportunities
Dr Daniel SmithChief Scientific OfficerCobra Biologics
Rapid Process Development of Viral Vector-based Products for Cell & Gene Therapy Applications: Challenges & Opportunities
Dr Daniel Smith,
CSO, Cobra Biologics
14th Annual BioProcessUK Conference, Cardiff, Nov 2017
Cell and Gene Therapy ATMP are here to stay.
Gene Therapy Products
• Over 650+ products in development/market
• 75% of products in Phase I & II development
• 9 approved therapies (4* EU; 3* US; 3 ROW)
• Adeno-, Lenti- and AAV-based delivery contribute to ca.
60% of the vectors used.
• A number of C> products have received Fast-track
Approval, Orphan Disease and/or Break-through status.Data Source:
Gene Therapy Market 2015-2025, Roots Analysis Report (2015)
Alliance for Regenerative Medicine Data Report (2016)
Gene Therapy Works
• Gene Therapy is saving the lives of patents today
• Focused on Oncology & Monogenetic Disorders
Overview of Adeno-Associated Virus
AAV is rapidly becoming the delivery vehicle of choice for many gene therapy indications
• In 2015, 25% of all viral vectors in development where AAV-based and used in 60% (80 out of 133) of Gene
Therapy trials for monogenetic diseases [Roots Anal. 2015]
• Small non-enveloped virus, containing single stranded DNA
• Infects both dividing and non-dividing cells
• Replication defective
• Very low immunogenicity due to size (~20nm)
• Not associated with any human disease
• Can be used for in vivo gene deliveries
(source: http://goo.gl/YRqjvB)
A ‘Classical’ Approach to recombinant AAV Production & Purification
TRANSGENE
ITRITR
REP2 CAP (X) E2A E4 VARNA
Helper Virus
E1-Expressing cell
AAV-vectors
Upstream Variations on a Theme Downstream relies on non-scalable process steps
Cell Pellet (+/- SN)
Cell Lysis
Nuclease Treatment
Gradient (Iodixanol)
Ultra-centrifugation
Gradient
Ultra-centrifugation
Harvest AAV bands
+ Analysis (qPCR)
Harvest AAV bands
+ Analysis (qPCR)
Buffer Exchange
Formulation
Empty
Full
OR
AAV Vector production via a scalable platform based approach
Genetic Engineering News, (2017); Vol. 37, No.13 ‘Manufacturing of AAV Vectors for Gene Therapy’
Requirements for a platform
• Standard approach
• Rapid Fit evaluation
• Cost of goods
• Speed
• Reliability
• Flexibility
• Scalable
• USP/DSP & Analytics
AAV vectors come in a variety of flavours
• 12+ naturally occurring human & nonhuman primate serotypes
• Numerous recombinant forms including hybrids and synthetics AAVs
• Sequence identities among the capsid range from ∼55 to 60% (AAV4 & AAV5) to >99% (AAV1 & AAV6)
• Capsid surface topology is conserved in all serotypes
rAAV1 rAAV2 rAAV3 rAAV4 rAAV5 rAAV6 rAAV7 rAAV8 rAAV9 Hybrids Synthetic
█ ▼ ? ▌▐█▼ █▼█ ?Primary
Receptor
? V
‡▼
? ‡█ █?Secondary
Receptor‡
‡▼
Variety adds complexity
Considerations for the development of AAV production & Purification
Inherent Complexity and Variation
Vandenberghe LH, et.al. (2010), Hum. Gene Therapy 21:1251-1257
Variation in the ratio of Full- and Pseudo-Vector particlesSerotype-dependent particle distribution in vector
production
Full nullallogeneicsyngeneic
“Empty”
• Empty : Full vector ratios can vary
widely (3:1 – 30:1) – Serotype & Process
• Some Clinical-grade vectors have had
>90% pseudo content
• EM analysis is the ‘Gold Standard’ for
analysis
• Not suitable for rapid PD
• Full / Empty ratio usually determined by ELISA & qPCR
• Total Particle analysis is performed using a conformational
specific antibody (A20) in an ELISA
• Genomic Particle analysis is performed by qPCR with
primers sets usually directed at amplicon regions in the
ITR sequences
Challenges for Viral Vector Process Development
The Importance Analytics• Analytics also require development and optimisation
for use as in-process assays
• DNA template for standard
curve requires optimisation
• Linear Plasmid vs Circular
• Free ITR ends for standard
curve
• 3x variation in titre of
reference material observed
• Requires alignment to
transgene-specific qPCR
D’Costa S, et. al., (2016) Mol Ther
Methods Clin Dev, 5:16019
Challenges for Viral Vector Process Development
10:15:1
4:1
18:116:1
20:1
2:1
Optimisation of AAV2::eGFP production
Balancing Quality vs Quantity in USP
• Process development requires the combination of
optimised analytical approaches
Improving Vector Quality (Empty/Full Capsids)
• Media Supplementation supports enhanced vector
quality
Reducing the burden on purification by using Serum & Protein
Free Media & enhancing the number of ‘Full’ capsids.
Locking the USP early in Process Development
T25 Flask (25cm2) HyperFlask (1700cm2)
68-fold scale up
Depth Filtration
Cell Lysis
Nuclease Treatment
TFF
Filtration
Salt Addition
Optimised USP
Scalable Approach: Scale Down & Scale Up Consistency
AAV vectors are prone to aggregation
• AAV-vectors have a tendency to aggregate at
certain concentrations (1x1013 particles/mL)
• Aggregation seems to be promoted by
residual DNA, host proteins and HSPGs
• The combination of ELISA and qPCR based
analytics can indicate gross aggregation
• Affinity ligand based on camelid single domain antibody
• AAV bind at Neutral pH and is eluted at Low pH (2-5)
• High yields of AAV
• No separation of E/F
Purification of AAV-vectors; Initial Capture of Vector
Capturing AAV with Affinity Chromatography
• Different affinities for different serotypes
• Need to minimize exposure to low pH during elution
VP1
VP2
VP3
LWFE
Image adapted from Data file 28-9207-54 AB,
AVB Sepharose™ High Performance, GEHC
Screening for AAV capture
Resin Screening
Binding Assessment
Elution Screening
Dynamic Binding Studies
DoE Optimisation
Design Space Confirmation
IEX Capture Screen
Data generated by CPI as part of an Innovate UK-funded project with Cobra
Purification of AAV-vectors; Separation of Full & Empty Capsids
Enriching for Full capsids with IEX Chromatography
• Separation based on small charge difference between Full &
Empty capsids
• Elution relies on very shallow duel linear gradients (pH &
conductivity) to effect the separation
Data Sourced from ‘Chromatographic separation of full
and empty AAV8 capsids, BIA separations
Challenges:
• Variations in AAV vector serotype
• Variations in genomic packaging content
• Will separation be sufficient for larger clinical and even commercial
volumes
• High degree of optimisation will be required
Mingozzi F & High KA., (2013),
Blood, 122:23-36
Model of the relationship between capsid dose
and outcome of gene transfer
Purification Challenges: Understanding AAV vector manufacturability
Structure – Function Relationships
• AAV vectors and small and stable
• Relatively pH and thermo-stable
• All serotypes have a similar tertiary structure
Heat induces exposure of the N-
terminal stretches of VP1 AAV2
capsids.
A: Full B: Empty
Bennett A., et.al. (2017) Mol. Therapy: Meth. & Clin. Dev. 6;171-182
AAV serotypes show variation in thermal stability
AAV capsids can undergo conformational changes
Analysis of capsid conformation and VP1 N-terminus exposure.
Kronenberg S., et al. (2005) J. Vir.
79:5296-5303
Bleker S., et al. (2005) J. Vir. 79:2528-40
Can the Bioprocess conditions modify Vector Conformation and
influence processing?
pH; flow; salt
Conclusion
Hitchcock T., et al. (2017) BioProcess International, 19 Sep ‘Development Approaches to AAV production’
Challenges:
• Gene Therapy & AAV vectors are here to stay
• AAV Variety adds Complexity for scalable manufacture
• Major challenges in achieving rapid process development
• Most bioprocess solutions are imported from ‘classical
biologics’ technologies
• Analytical technology lags behind process technology
Opportunities:
• Important to develop fast screening approaches to provide a
handle for rapid process development
• Fundamental to better understand capsid structure-function
relationships and how these relate to the proposed bioprocess
AAV Vector heterogeneity at the physical & functional level
Supporting the changing bioprocessing landscape
Dr John LiddellSenior Scientific AdvisorCPI NBMC
Copyright © 2017 Centre for Process Innovation Limited. All Rights Reserved.
14th Annual bioProcess UK Conference (29 November 2017)
Supporting the changing
bioprocessing landscape
John M Liddell PhDSenior Scientific Advisor
at CPI’s National Biologics Manufacturing Centre
CPI’s National Biologics Manufacturing Centre
Outline
The Evolving Bioprocess Landscape
Case Study Examples
Summary
Who are CPI?
CPI is a UK technology innovation centre and the process element of the Government’s
High Value Manufacturing Catapult.
We use applied knowledge in science and engineering combined with state of the art facilities
to enable our clients to develop, prove, prototype and scale-up the next generation of
products and processes.
NATIONAL
FORMULATION CENTRE
OPENING 2018
NATIONAL INDUSTRIAL
BIOTECHNOLOGY FACILITY
NATIONAL PRINTABLE
ELECTRONICS CENTRE
NATIONAL BIOLOGICS
MANUFACTURING CENTRE
CPI’s Biologics Network – Oct 2017
Changing Healthcare Landscape
Monday, 19 March 2018
Aging Population
Patient Centric
Delivery
Cost Pressure
Pay for results
Biosimilar Substitution
Precision Medicine
Right treatment for right patient
Emerging Markets
Treatment vs Cure
COMPLEXITY
LA
CK
OF
MA
NU
FA
CT
UR
ING
PL
AT
FO
RM
Legacy
microbial
products
Fab/ Dab
pDNA
Extended
half life
mAb
Conjugate
vaccines
ADC
NA therapeutics
Viral
vaccine
Bispecific
VLP
Viral gene
therapy
Oncolytic
vaccines
MRT
Stem cell
therapies
Somatic cell
editing
Germ cell
editing
Exosome
therapies
Collaborative R&D Project Examples
INNOVATIVE PROCESSES AND
PROCESS TECHNOLOGIES
Novel CHO platforms
Smart formulation in DSP
Continuous, integrated DSP
Nano-template assisted membrane
crystallisation for DSP
Predictive technology to de-risk and
streamline drug development
Cell and bioprocess engineering for rapid
and intensive continuous production
NEW AND IMPROVED
ANALYTICAL TECHNOLOGIES
CD for higher order structure
HDX-MS for higher order structure
PAT for lyophilisation
Microfluidic approach to test for CQAs of
lentivirus vectors (EngD)
PROCESS DEVELOPMENT FOR NOVEL
PRODUCTS AND FORMULATION
Industrial manufacturing platform for AAV
Novel glycosylated drugs and HT analytics
Improved VLP production platform (EngD)
Process development for gold core, glycan
coated nanomedicines
Platform for aggregation profiling
Drug development and manufacturing
economics evaluation (EngD)
Improved Downstream Process (DSP) Operation through
Formulation Innovation.
• Apply Arecor formulation technologies to screen and evaluate
smart DSP formulations to improve product stability, reduce
viscosity, expand design space and increase yield
• Create a new development route for difficult to manufacture
biologics products
Speed up DSP development, transfer
to manufacture and operability.
Platform applied to a wide variety of
new and challenging products. DSP
improved with in-process smart
formulations is likely to offer a cost-
effective, scalable alternative to
traditional processes enabling
improved yields and product quality.
DSP activities (high capacity
chromatography, low pH hold, UF/DF)
can cause product destabilisation
resulting in degradation and aggregation,
compromising product quality, safety and
process productivity
INDUSTRIAL CHALLENGE IMPACT? !
FUNDED BY
CASE STUDY 1
Arecor Project
PARTNERS
Transfer and Scale-up of FDB USP, DSP
and Analytical mAb Platform Processes at CPI
Example of comparative quality
data for final mAb product quality
(HCP & SEC) of FDB001 mAb
purified by mAb platform process
produced at the different
locations at 10L scale (FDB
&CPI) and scaled to 200L at CPI
ANALYSIS
Appearance CHO HCP
A280/A600 Protein A
SEC uplc MFI
cIEF DLS
Reduced CE DSC
Non reduced CE
USP: 10L followed by scale-up to non-GMP 200L Sartorius Biostat™ STR SU bioreactor DSP: Scale-up and consistency
Step Yield (%)
DSP step FDB 10L CPI 10L CPI 200L
Protein A 97.5 84.3 104.4
VI filtrate 82.4 84 99.8
CIEX 92.7 91.8 90.4
AIEX 93.7 93.8 93.7
DSP intermediates of consistent quantity retained for formulation studies
Testing Downstream Processing Formulation Impact
Two rounds of assessment of the impact of the elution buffer formulations were
tested using the high throughput automated liquid handling Perkin Elmer JANUS
BioTx platform at CPI.
This was coupled with application of multiple high resolution analytical methods
to determine the impact of the formulation buffers on step performance and
product quality.
The second round (refined formulations) achieved elution profiles comparable to
the control, important for process yield.
Significant stabilisation of mAb through downstream processing was achieved
using Arecor chromatographic elution formulations optimised in this second
round evaluation.
Integrate technologies to increase number of high quality
candidates entering development, develop better platforms for
manufacture and improved methods to select candidates with
best chance of being safe, easy to manufacture and formulate
Harnessing UK innovation to
streamline the biologics supply chain
from molecule to medicine
Strong UK consortium, including major
companies and SME technology
suppliers
Current technologies not optimised for
next-generation molecules.
High failure rate during drug
development significant contributor to
costs.
Assessment methodologies to select
best candidates are not well developed
INDUSTRIAL CHALLENGE IMPACT? !
FUNDED BY
CASE STUDY 2
BioStreamline
PARTNERS
BioStreamline Components
LONZA, UCB, CPI
Data generation through experimental studies (USP/DSP/analytical)
used to develop algorithms to predict molecule developability risk from
molecular features.
SPHERE FLUIDICS
Developing an automated platform with UCB to give more than a 10-
fold productivity increase in antibody number through pipelines.
HORIZON DISCOVERY
Applying gene editing technology (CRISPR) to develop flexible
efficient cellular systems to support the future needs of biologics
manufacture.
ALCYOMICS LTD
Novel immunogenicity tool to used to predict safety problems before
clinical trials.
Better candidate selection will
lower risk of developability
failure and increase the
efficiency of the supply chain
High drug clinical failure rates
a major contributor to
development costs
Developability tool data generation
200 mAb sequences evaluated experimentally and in silico for more detailed study
Short list of 50 mAb variants with different developability features selected
Cell lines expressing the molecules produced
Developability risks data collected (CPI / Lonza). A wide range of biochemical, physical
methods used to assess developability challenges for each molecule
Evaluation of different chemometric approaches (statistical, machine learning,
clustering etc.) to describe data and generate decisional and predictive tools
BIOCHEMICAL DATA
Charged variants
Glycoforms
Aggregation
Fragmentation
Impurity quantitation
DEGRADATION PATHWAYS
Isomerisation
Oxidation
Accelerated & real time stability
pH stability
BIOPHYSICAL DATA
Aggregation onset
Hydrophobicity
Protein / protein interaction
Rheology
Higher order structure
Replace multi-step chromatography with innovative Continuous
Template-Assisted Membrane Crystallization as main
purification step for Downstream processing
Highly innovative, high risk, high
reward project that needs a large
multidisciplinary consortium to make
progress in
DSP (batch) ≈ 66% mAb production cost
mAbs ≈ $125 billion, > 50%biopharma
market
Reuse & storage challenges
High buffer requirements
Large footprint
INDUSTRIAL CHALLENGE IMPACT? !
FUNDED BY
CASE STUDY 1
Arecor Project
PARTNERS
Horizon 2020
www.amecrys-project.euProject Amecrys Basic Approach
Permeable membrane
containing silica
nanotemplates to
achieve super-
saturation and rapid
crystal growth
DEVELOPMENT
APPROACH
Pure protein
Intermediate
purification
Bioreactor filtrate
PRE-TREATMENT
CONDITIONING
Pre treatment and
conditioning
Inline concentration
In line diafiltration
Potential for a truly
continuous protein
process
Amecrys Components
Cell line and USP process
Conventional purification method
Analytical methods
Generation of baseline data and intermediates
Crystallisation protocols development
Nanotemplate and crystallisation
Silica based nanotemplates
Surface modification – hydrophobicity/ hydrophilicity
Control of pore size distribution
Nanotemplate impact on crystallisation rates
Modelling support
Summary
Evolving
biopharmaceutical
landscape presenting
new development
challenges
New approaches to
development in both
USP and DSP
necessary
An integral part of
the UK’s strategy to
maintain its position
as a world leader in
biologics
manufacture
NATIONAL BIOLOGICS
MANUFACTURING
CENTRE OPENED IN
SEPT. 2015
Expertise in translation of
development to manufacture
High throughput
development platforms
GROWING NETWORK
OF PARTNERS AND
COLLABORATIONS
Progressing on multiple
projects
Strong forward pipeline of
activities
Primary capture purification offeringnew single-use bioprocessing opportunities
Dr Oliver HardickChief Executive OfficerPuridify
Development of the next generationprotein A chromatography resin
Dr Kajsa Stridsberg-FridenSenior Project Manager, Downstream ProcessingGE Healthcare Life Sciences
Development of the next generation Protein A chromatography resin
Kajsa Stridsberg Fridén, PhDSenior Project Manager, GE Healthcare
• Opportunities and challenges with Protein A chromatography
• Design of affinity ligand for improved alkaline stability
• Design of solid support for increased dynamic binding capacity
• Optimization of solid support in combination with affinity ligand
• mAb applications
• Purification performance
• Life time study
• Summary and conclusions
Outline
KA610250917PP I Nov 2017
Opportunities and challenges with Protein A chromatography
Protein A: the key for efficient mAb manufacturing
Protein A is well-established as the key purification step in mAb manufacturing processes.
Key success factors:• Natural affinity is the result of millions of years of
evolution and offers high purity and yield in one step.
• Engineered variants of Protein A offer significant advantages for industrial manufacturing.
• Proven and regulatory-accepted technology for fast and predictable development to manufacturing scale.
KA610250917PP I Nov 2017
Increased time and cost pressure on development and manufacturing of mAbs:
• Increasing upstream titers can make the Protein A chromatography process step a bottleneck.
• The combination of high nutrient load and low alkaline stability of the Protein A ligand leads to increased bioburden risk and costly process deviations.
Challenges with Protein A chromatography
KA610250917PP I Nov 2017
First challenge: Protein A resin capacity constraints
• Long manufacturing lead times and required Protein A resin volumes higher than necessary.
• Lack of prepacked disposable chromatography column solutions for the high-titer processes.
Upstream titer development Consequences of limited capacity
5–50 mg/L
50–1000 mg/L
1000–5000 mg/L
1982–1985
2005–2015
5000–10000 mg/L
2015–2025
KA610250917PP I Nov 2017
2005–2015
Second challenge: bioburden and cross-contamination risks
• Protein A capture step exposed to crudest feed and highest concentrations of nutrient load.
• Current Protein A ligand solutions have lower resistance to the commonly used cleaning and sanitization solution 1 M NaOH.
• Less efficient cleaning, with potential cross-contamination risk and fouling, impacts dynamic binding capacity.
• Elevated bioburden risk.
Elevated risk for bioburden at the Protein A step Consequences of limited alkaline stability
mAb capture step:Elevated bioburden andCross-contamination risk
Upstream Downstream
KA610250917PP I Nov 2017
Ligand Base matrix
• Point mutation
• Length of ligand
Base matrix + ligand
Development of affinity chromatography resin
• Bead size
• Pore size
• Matrix volume
• Coupling chemistry
• Length of ligand
• Ligand density
KA610250917PP I Nov 2017
• Next-generation Protein A chromatography resin.
• Resin and ligand design developed from well-established MabSelect SuRe™ ligand and high-flow agarose base matrix.
Built on the heritage and proven performance of the MabSelect family of resins
Introducing MabSelect™ PrismA
New picture required
KA610250917PP I Nov 2017
Design of affinity ligand for improved alkaline stability
• Selection of stable single domain.
• Theoretical and empirical identification of alkaline-sensitive amino acids.
• Point mutation of sensitive amino acids.
• Multimerization into alkaline stabilized ligand.
• Thorough functional evaluation.
• More than 400 constructs were screened using the Biacore™ SPR system.
Protein engineering towards increased alkaline stability
SPR = surface plasmon resonance
KA610250917PP I Nov 2017
Evaluation of alkaline stability of ligand using Biacore™ SPR system
Cleaning with 0.5 M NaOH in 100 cycles using polyclonal IgG as sample.
Contact time for 0.5 M NaOH: 10 min per cycle.
Cycle 100Cycle 2Cycle 1
KA610250917PP I Nov 2017
Alkaline stability screening
Biacore™ results
Cycle
Rem
ain
ing
rela
tive
cap
acit
y (
%)
Tetramer ligands compared
KA610250917PP I Nov 2017
Alkaline stability study performed in columns
• Using Protein A chromatography resin with the final hexamer ligand construct.
• > 95% of DBC retained after 150 cycles with 0.5 M NaOH.
• Stable DBC enables higher load volumes over the resin lifetime.
Cycling with buffer only (no protein sample). Cleaning with 0.5 M NaOH,15 min contact time per cycle. High stability in 0.5 M NaOH over repeated cleaning cycles.
DBC = dynamic binding capacity
KA610250917PP I Nov 2017
Alkaline stability study performed in columns
• Using Protein A chromatography resin with the final hexamer ligand construct.
• > 90% of DBC retained after 150 cycles with 1 M NaOH (compared with ~ 50% retained DBC for MabSelect SuRe™ LX).
• Save footprint by aligning your CIP solutions throughout chromatography steps.
Cycling with buffers but no sample. Cleaning with 1.0 M NaOH 15 min contact time per cycle.
Re-engineered protein A ligand enables repeated cleaning with 1.0 M NaOH.
DBC = dynamic binding capacityCIP = cleaning-in-place
KA610250917PP I Nov 2017
Design of solid support for increased dynamic binding capacity
Pore size versus matrix volume
• Pore size optimized for the chosen ligand.
• Pore size and matrix volume optimized with respect to pressure-flow properties.
• Development of new matrix synthesis methods to meet the requirements on the new solid support.
Matrix volume
Pore size
KA610250917PP I Nov 2017
Dynamic binding capacity versus pressure-flow properties
DBC Flow rate
Porosity
Particle size
Trade off Effect of particle size and porosity on DBC and flow rate
Particle size
Porosity
DBC
Flow rate
DBC- dynamic binding capacity
KA610250917PP I Nov 2017
Base matrix optimized for high flow throughput
Flow properties of MabSelect™ PrismA AxiChrom™ 300, 20 cm bed height, temperature 20°C
High-flow agarose base matrix provides excellent pressure-flow performance | Verified in large-scale BioProcess™ columns
KA610250917PP I Nov 2017
Optimization of solid support in combination with ligand
Variation of ligand length
Pore surface
Hexamer construct
Tetramer construct
Example: different ligand length with maintained ligand density
KA610250917PP I Nov 2017
Base matrix and ligand design for optimal binding capacity
Varying ligand length on base matrix A (larger pores) Varying ligand length on base matrix B (smaller pores)
Combination of base matrix and ligand design required for optimal binding capacity.Large pores allow for larger ligand densities, even with longer ligand constructs.
KA610250917PP I Nov 2017
QB10 = dynamic binding capacity at 10% breakthroughRT = residence time
• Up to 40% increased DBC compared with MabSelect SuRe LX at 2.4 min RT.
• Up to 30% increased DBC compared with MabSelect SuRe LX at 4 min RT.
• Up to 25% increased DBC compared with MabSelect SuRe LX at 6 min RT.
Significantly increased DBC
Comparison of DBC
Polyclonal IgG results
QB10 = dynamic binding capacity at 10% breakthroughRT = residence time
KA610250917PP I Nov 2017
mAb applications
Monoclonal antibody binding capacities
Dynamic binding capacity mAb1 Dynamic binding capacity mAb2
MabSelect PrismA 100 mg/mLMabSelect SuRe LX 90 mg/mLMabSelect SuRe 66 mg/mL
Equilibrium capacity mAb1
KA610250917PP I Nov 2017
QB10 = dynamic binding capacity at 10% breakthrough
Capture of monoclonal antibody using different Protein A resins
Protein A chromatography resins:
• MabSelect™ PrismA
• MabSelect SuRe™
• MabSelect SuRe LX
Load: 80% of QB10 (6 min residence time)
Load mAb1 (g/L resin) Load mAb2 (g/L resin)
MabSelect PrismA 58 63
MabSelect SuRe 39 36
MabSelect SuRe LX 46 43
Purification using standard protocol Load
QB10 = dynamic binding capacity at 10% breakthrough
KA610250917PP I Nov 2017
Recovery (mAb1)
HCP (mAb1)* HCP (mAb2)**
Performance benchmarking: recovery and removal of host cell protein (HCP)
* Start HCP 1.4 x 105 ppm** Start HCP 5.7 x 105 ppm
Recovery (mAb2)
KA610250917PP I Nov 2017
Aggregates (mAb1)* Aggregates (mAb2)*
hcDNA (mAb1)**
Performance benchmarking: aggregate and host cell DNA (hcDNA) removal
hcDNA (mAb2)***
** Start hcDNA 8037 ppm*** Start hcDNA 6785 ppm
* MabSelect PrismA: aggregate level associated with higher load.
KA610250917PP I Nov 2017
Pool volume (mAb1) Pool volume (mAb2)
Leached Protein A (mAb1)* Leached Protein A (mAb2)*
Performance benchmarking: pool volume and leached Protein A
* MabSelect PrismA leached protein A level associated with higher ligand density and ligand length. CV = column volume
KA610250917PP I Nov 2017
Lifetime study with mAb-containing cell culture supernatant
• More than 90% remaining DBC using mAb-containing feed and 0.5 M NaOH as cleaning agent in between cycles (15 min contact time per cycle).
• Higher alkaline stability enables longer lifetime and improves process economy.
Resin comparision Alkaline stability with protein
MabSelect™ PrismA 91%
MabSelect SuRe™ LX 83%
MabSelect SuRe 61%
DBC = dynamic binding capacity
KA610250917PP I Nov 2017
Conclusions
Conclusions
• New optimized ligand with better alkaline stability.
• New base matrix with optimized bead size and porosity.
• The combination of new base matrix and a longer ligand increases the dynamic binding capacity at all residence times tested.
• Performance in terms of recovery and purity similar to reference Protein A resins.
• The improved alkaline stability enables efficient cleaning of the resin using 0.5–1 M NaOH over many purification cycles.
• The use of 1 M NaOH for sanitization enables increased bioburden control.
KA610250917PP I Nov 2017
KA610250917PP I Nov 2017
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