Vaccine Downstream processing –an overview...Title or Job Number | XX Month 201X 21 ~90 µm...

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Imagination at work Vaccine Downstream processing –an overview Mia Bennemo May, 2015

Transcript of Vaccine Downstream processing –an overview...Title or Job Number | XX Month 201X 21 ~90 µm...

Page 1: Vaccine Downstream processing –an overview...Title or Job Number | XX Month 201X 21 ~90 µm chromatography bead 100 nm influenza virus 200 x 500 nm Pox virus 1-7 nm proteins Diffusion

Imagination at work

Vaccine Downstream processing –an overview Mia Bennemo May, 2015

Page 2: Vaccine Downstream processing –an overview...Title or Job Number | XX Month 201X 21 ~90 µm chromatography bead 100 nm influenza virus 200 x 500 nm Pox virus 1-7 nm proteins Diffusion

See tutorial regarding confidentiality disclosures. Delete if not needed.

Overview

• Vaccines overview

• Demands on vaccine purification

• Common techniques for vaccine purification

• Example of a purification process

• Summary

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Vaccine Overview

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Cell culture / Fermentation

Purification

Fill and Finish

Analysis (QC/QA)

The

Vacc

ines

Virus based

Protein based

The

Man

ufac

turi

ng p

roce

ss

Polysaccharide based

DNA based

Bacteria based

How Vaccines are manufactured

Number and order of the different steps depends on the specific vaccine production

Vaccine Downstream processing – an overview May 2015

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Demands on vaccine purification

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Safety and quality is priority

Regulatory requirements

• Safe vaccine with no or minimal adverse effects

• Effective dose

• Stability

• Process control

• Reproducable process

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Downstream purification of vaccines

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Downstream processing of viruses Available technologies

Harvest • Lytic virus • Non-lytic virus

• Detergent

• Mechanical disruption / Homogenization

• Osmotic shock

• Freeze-thaw

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Cell culture

Harvest

Clarification

Primary purification

Secondary Purification

Formulation

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Impurity challenges after lysis

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Cell lysis

Organelles/cell membrane /lipids

Antigen (e.g. virus)

chemicals

DNA/RNA

proteins

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Goal with purification

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Cell lysis

Antigen (e.g. virus)

Purification

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Downstream processing of viruses Available technologies

Clarification • Filtration

– Normal flow – Tangential flow

• Centrifugation

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Cell culture

Harvest

Clarification

Primary purification

Secondary Purification

Formulation

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Normal flow filtration

• Removal of cell debris and larger particulates

• Porosities from 0.2 - 20 µm

• Scalable

• Single-use

• Straight forward process set up

• Not recommended for harvest with high particulate content

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Feed Flow

Filtrate Flow

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Downstream processing of viruses Available technologies

Primary purification • Tangential flow filtration (TFF) • Density gradient centrifugation • Precipitation • Chromatography

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Cell culture

Harvest

Clarification

Primary purification

Secondary Purification

Formulation

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Tangential flow filtration

Cross flow

Permeate flow

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• Sweeping effect clean filter surface • Allow greater throughput on smaller surface area

Feed pressure

RETENTATE PRESSURE

Permeate, (Filtrate)

BACKPRESSURE VALVE

RECIRCULATION PUMP

Feed Reservoir

( concentrate )

UF or MF Device

Dilute Feed Material

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• Hollow fiber cartridge consists of tubular fibers • Concentration/ diafiltration • Microfiltration • Suitable for shear sensitive material • Possible handle high particle loads (ex cell harvest) • Defined pore sizes • Re-usable • Scalable

Tangential flow filtration

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• Cassettes consists of sheet membranes • Concentration/ diafiltration • Defined pore sizes • Re-usable • Scalable

Hollow fiber filters Flat sheet cassettes

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Downstream processing of viruses Available technologies

Secondary purification • Density gradient

centrifugation • Selective precipitation • Chromatography

– IEX, MM, AC, HIC, SEC – Bead format (Packed bed) – Membrane format (Capsule)

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Cell culture

Harvest

Clarification

Primary purification

Secondary purification

Formulation

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Ion exchange chromatography

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Surf

ace

net c

harg

e Cation

Anion

pH

Anion exchange chromatography

• (-) Negatively charged molecules binds to (+) positively charged ligands

Cation exchange chromatography

• (+) Positively charged molecules binds to (-) negatively charged ligands

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Hydrophobic interaction chromatography

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Separation by hydrophobicity

• Hydrophobic surfaces of proteins interact with the ligand in precence of salts

• High salt content enhance and low salt weakens the interaction

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Size exclusion chromatography

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Exluded from pores

Enter a fraction of the pores

Enter all pores

Abso

rban

ce Sample injection

High molecular

weight

Intermediate molecular

weight Low molecular

weight

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Affinity chromatography

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Specific binding

Few affinity resins available for vaccines

• Agarose based affinity resin for adeno associated virus

• Pseudo affinity resins for influenza – sulphated cellulose

– sulphated dextrane

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Chromatograpic purification of large molecules can be challenging

Title or Job Number | XX Month 201X

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~90 µm chromatography bead

100 nm influenza virus

200 x 500 nm Pox virus

1-7 nm proteins

Diff

usio

n co

nsta

nt

25 nm polio virus

Flow through chromatography recomended

Bind-Elute chromatography possible

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Pores

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• Host cell proteins and DNA fragments bind to the core and viruses stay in the void.

Core bead chromatography

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Process example

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Polio IPV

Seed N-2 Cell

expansion

Seed N-1 Cell

expansion

Production bioreactor

Virus propagation

Clarification NFF

Removal of cell debris and large particles

TFF Conc of

polio virus

SEC Separation

of polio virus from

small molecular

compounds

AIEX (FT) DNA

removal. Polio virus

in flow through

Virus inactivation

formaldehyde

Formulation Sterile

filtration, mixing with

other strains

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Summary

• Robust downstream process can ensure consistent high quality

• Most vaccines have unique purification processes

• Preferably use scalable techniques when developing new processes

• Purification of particles in binding mode can be difficult with classic chromatography

• Core bead chromatography suitable for purification of particles of sufficient size

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Thank you

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