modern vaccine and biologics manufacturing strategy
TRANSCRIPT
Prepared for AndyJuly 08, 2021
Modern Vaccine and Biologics Manufacturing Strategy
Cytiva 2
Precision medicine for cancer
Cytiva 3
新冠病毒疫苗，主要關注抗原依然是 S抗原
February 19,2020
減毒病毒疫苗
滅活病毒疫苗
減毒細菌疫苗
滅活細菌疫苗
蛋白疫苗
多糖疫苗
多糖蛋白結合疫苗
病毒樣顆粒(VLP)
病毒載體疫苗
DNA疫苗
mRNA疫苗
Covid-19 vaccine development
Cytiva 4
Immune cellsare
collected by a
procedure called
apheresis
107 T cells
50-100mL
Cells are tested for safety and
potency and packaged for
injection back into thepatient
Cells are
reinfused
into the
patient at
clinical
center
108 T cells
50-100mL
107 T cells
50-100mL
Biopharma, Cell therapy and Vaccine
Cytiva工藝開發流程
工藝放大流程
Biopharma, Cell therapy and Vaccine tall tale Solution
Introduction to protein purification
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Sample purification and preparation
TherapeuticsDiagnostics
Functional Studies
Structural Studies
Basic Research
PurifiedProtein
TherapeuticsAntibodyVaccine
AntibioticHormoneCytokinesEnzyme
InhibitorsTest reagentsBiomaterial
Sample purification
UV
Column Gel media
Crude sample Buffer
Monitors
Collection
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Chromatography
Chromatography principle
29160894AA | June 2015 10
Medium
Sample
Target protein
Buffer
Other molecules
Protein properties that matter for protein purification
Hydrophobicity
Net charge
Size
Ion exchange chromatography (IEX)
Affinity chromatography (AC)
Hydrophobic interaction chromatography (HIC)
Size exclusion chromatography (SEC)or gel filtration
Biospecific affinity(e.g., a tag)
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The principles of chromatography techniques
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Affinity Chromatography(AC)
Ion exchange Chromatography(IEX)
Hydrophobic interaction Chromatography(HIC)
Size exclusionChromatography(SEC)
• Bind – elute principle
• Requires specific elution conditions
• Concentrating effect
• Diffusion – no binding
• Any elution conditions
• Diluting effect
Purification strategy
Purification protocol development - step by step
Define the purification objectives
Consider what’s
known about the target
protein
Develop the individual
purification steps
Combine techniques
Develop analytical
assays
Introduction to CiPP purification strategy
Purity
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Stage
Sample prep
Capture
Isolate, concentrate and stabilize
intermediate
Purification
Remove bulk impurities
PolishingRemove finalcontaminants
Yield (%)
Number of purification steps
Protein recovery plotted against the number of purification steps
Purification strategy combining multiple steps
How to combine purification techniques
AC
SEC
AC
IEX
AC
HIC IEX
IEX
SEC SEC
HIC
Use orthogonal techniques and minimize sample handling
IEX
Capture
Intermediate purification
Polishing
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ÄKTA™ system provides efficient purification of your target protein
Cytiva 18Ref.: https://m.match.net.tw/pc/life/health/20210612/5921991
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Ref.: https://www.nature.com/articles/s41563-020-0746-0
Different types of vaccine
Cytiva
1Sub-unit vaccine
• Additional content, if needed
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Recombinant protein
Plasmid Construction
Protein expression
clarification and
filtration
Protein purification
Functional Analysis
To produce large quantities of the protein of interest
cDNA
Plasmid
2HN
COOH
2HN
COOH
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There are many ways to improve protein purity and yield
• Sample conditions
• Optimize binding conditions
• Optimize elution conditions
• Multi-step purificationNo imidazole in the binding buffer Imidazole in the binding buffer
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Impact of multi-step purification on protein purity
97 000
66 000
45 000
30 000
20 100
14 400
Mr
97 000
66 000
45 000
30 000
20 100
14 400
Mr
97 000
66 000
45 000
30 000
20 100
14 400
Mr
1 3 4 5 6 7 8 92
LMW Start FT wash eluted 1 2 3
HisTrap Superdex
F
Desalting
AffinityGST His Ab
Gel filtration
Size exclution
Cytiva 24Viral fusion core
The function of Spike glycolprotein
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Vaccine based on S protein
• Full-length S protein
• RBD/FC fusion protein
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Different Tag technologies
Fc domain-ProteinA Nickel-Histidine
Cytiva
Fc fusion protein Purification Toolboxes
Two-step process
Capto S ImpAct
Capto Q /
ReadyToProcess™
Adsorber Q
Three-step process
MabSelect™ PrismA MabSelect PrismA
Capto™ adhere (FT)
Capto adhere ImpRes (FT)
Capto MMC
Capto adhere (FT)
Capto adhere ImpRes (FT)
Capto adhere ImpRes (B/E)
Three-step process
MabSelect PrismA
B/E = bind and elute, FT = flow-through
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His-Tagged protein Purification Toolboxes
Main characteristics:
1. High binding capacity: ~ 40 mg/mL
2. High flow rate: 150 ~200 cm/hr
3. BioProcess resin: Regulatory suppot file (RSF)
4. RSF containing: Product Quality, Product stability, Excretable compound, Manufacturing description..etc.
Ni Sepharose 6 Fast Flow
IMAC Sepharose 6 Fast Flow
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Process workflow
Upstream Production
Clarification
Concentration and Buffer exchange
Capture: MabselectPrismA
Polishing: Capto adhere
Capture: IMAC 6 FF
Polishing1: Capto MMC
Polishing 2: Capto Q
Virus Filtration
Formulation
Sterile Filtration
Case Sharing
• RBD/FC fusion protein • Full-length S protein
Cytiva
2Virus vector vaccine
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Cytiva
The sizes of viral vectors
Adenovirus (AdV)
~ 90 nm
Lentivirus
~ 80–120 nm
Adeno-associated
virus (AAV)
~ 25 nm
Antibody
~ 5 nm
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Cytiva
Recombinant virus vectors for cell- and gene therapy
Preventive vaccines
Rota (80 nm)
Polio (30 nm)
HAV (30 nm)
Hep B
AAV (25 nm) Adeno
(70–90 nm)
Flu (90–120
nm) Rabies (75 × 180 nm)
Mumps (200 nm)
Measles (100 × 300 nm)
Pox (200 × 300 nm)
Lenti (80–120)
JE
Yellow Fever
VLP
(40–60 nm)
(40–60 nm)
(40–60 nm)
(42 nm)
11/30/202132
Sizes of common viruses
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Adenovirus vector platform
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5 µm inactive shell
85 µm particle size
N+
Octylamine - Multimodal ligandVery strong protein binding
Viral particle
Host cell proteins (HCP)DNA fragmentsEndotoxinsDetergentsBenzonase™Etc…
Concept
Capto™ Core 700
Cytiva
Features and benefits
High purity and yield with high load
Purity and yield
Capto™ Core 700 achieves the same HCP removal and virus recovery as gel filtration
Load and dilution
Capto Core 700 allows this purity and yield to be obtained with significantly higher load and without any dilution to the feed
High productivity without compromising purity or yield!
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Benefits of Capto Core
• Saving Column volume • More sample volume
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Adenovirus vector platform
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Cytiva 39
Adenovirus vector platform
Cytiva
3mRNA vaccine• Additional content, if needed
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Ref.: https://theconversation.com/what-is-mrna-the-messenger-molecule-thats-been-in-every-living-cell-for-billions-of-years-is-the-key-ingredient-in-some-covid-19-vaccines-158511
How mRNA vaccine works?
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Transcription with RNA polymerase (IVT, In-vitro transcription)
mRNA purificationGet plasmid DNA Encapsulated mRNA
Strategy for producing mRNA vaccine
Cytiva
Plasmid and mRNA manufacturing process
Cell expansion and production
Cell concentration
Lysis Clarification ConcentrationGroup
separationCapture Polishing
Formulation and sterile filtration
Bulk fill
Synthesis from
pDNA template
Optional conditioning
mRNA capture
Formulation Bulk fillEncapsulation
and LNP formation
Polishing purification OR
filtration
Formulation and fill
CappingOptional
condition
1. Plasmid manufacturing
3. mRNA encapsulation2. mRNA manufacturing
LNP, lipid nanoparticle.
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Cytiva 45
Cytiva
Plasmid and mRNA manufacturing process
Cell expansion and production
Cell concentration
Lysis Clarification ConcentrationGroup
separationCapture Polishing
Formulation and sterile filtration
Bulk fill
Synthesis from
pDNA template
Optional conditioning
mRNA capture
Formulation Bulk fillEncapsulation
and LNP formation
Polishing purification OR
filtration
Formulation and fill
CappingOptional
condition
1. Plasmid manufacturing
3. mRNA encapsulation2. mRNA manufacturing
LNP, lipid nanoparticle.
Cytiva
mRNA manufacturing & purification
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IVTUF/DF
Desalting
Oligo dT, Affinity
Polishing/Conc.Capto Core 700/
Hollow FiberCapping Sterile Filtration
Synthesis from
pDNA template
mRNA capture
dsRNA remove
Sterile filtration
Capping
Fibro Oligo dT
Sera-Mag Oligo(dT)-Coated
Source 15Q
HiRes QCapto core 400
dsRNA remove
Cytiva 48
Adenovirusvector
Upstream Productio
n
Cell Lysis and DNA
fragmentation
Clarification
UF/DFCapto Q ImpRes
CaptoCore 700 Formulation
Sterile Filtration
Spike proteinUpstream
ProductionClarification UF/DF
His-tagged, Affinity
CaptoMMC
Capto Q
MabSelect PrismACapto
Adhere
FormulationVirus
FiltrationSterile
Filtration
Plasmid DNA Fermentation Harvest Lysis Clarification UF/DFSepharose
6 FF
CaptoPlasmidSelect
Capto Q ImpRes
Formulation
mRNA IVT UF/DFOligo dT, Affinity
Polishing/Conc.
EncapsulationCapto Core 700/
Hollow FiberFormulation
Sterile Filtration
Summarized the Modern Vaccines
Adenovirusvector
Upstream Production
Cell Lysis and DNA
fragmentationClarification UF/DF
Capto Q ImpRes
CaptoCore 700
FormulationSterile
Filtration
Spike protein Upstream Production
Clarification UF/DF
His-tagged, Affinity
CaptoMMC
Capto Q
MabSelect PrismACapto
Adhere
FormulationVirus
FiltrationSterile
Filtration
Cytiva
Ä KTA System
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Cytiva 50
Proven technologyContinues ÄKTA™ tradition
• Incorporates over 50 years of experience in protein research
• Backed by 30 years of experience in developing protein purification systems
• ÄKTA systems are used by 100,000 researchers globally
•2012—ÄKTA pure: protein purification your way
1982—FPLC™ system released, predecessor to ÄKTA systems
1996—ÄKTAexplorer™,
single platform for all chromatography techniques1959—Sephadex™, the world’s
first gel filtration medium
2015 upgrade
2019—ÄKTA™ go : provides efficient purification of your target protein
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Ä KTA System
ÄKTA™ pure
Functions & Price & Scale-up
ÄKTA™ avant
Flexibility in researchMatch most current and future purification challenges
Productivity in process development Fast and secure development of purification processes
ÄKTA pilot 600
Prepare for GMP environment. Smaller GMP batches for early clinical studies Scale-up and robustness testing
ÄKTA process
ÄKTAprocess is a versatile platform providing thousands of configuration possibilities. The system is available in three flow rate ranges that extend up to 2000 L/h.
AxiChrom
BPG
Hitrap
HiScreen
XK/Hiscale
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System pump P9 A/P9 B
ÄKTA pure25:
Flow rate: 0.001-25 ml/min
Max. flow rate: 50ml/min
Pressure: 0-20MPa
Rinsing outlet
Solution inlet
Solution outlet
Pump head rinsing system
Function：--Rinse piston；--Rinse out buffer salt；
Maintenance：20% EtOH
（change rinsing solution every two week）Rinsing inlet
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Air in Pump
Buffer inlet
Buffer outlet
• If air gets stuck in the pump, manual purge might be necessary
• For lower amount of air, use pump wash
Air bubble
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Ports and flow paths of the Injection valve
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Tailor ÄKTA™ pure to fit your needs… to full automation...
