Vaccine downstream processing—an overview

DCVMN 10 March 2017

Outline

Vaccines overview

Demands on vaccine purification

Common techniques for vaccine purification

Example of a purification process

Summary

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Vaccine overview

Vaccines and production

Vaccines The manufacturing process

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Bacteria based

Virus based

Protein based

Polysaccharide based

Cell culture/fermentation

Purification

Fill and finish

Analysis (QA/QC)

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E. Coli Bacteria from NIH Image LibraryInfluenza virus by Kat Masback.

Demands on vaccine purification

• Regulatory requirements

• Vaccine with no or minimal adverse effects

• Effective dose

• Stability

• Process control

• Reproducible process

Safety and quality is priority

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Vaccine image by www.torange-pt.com.

Available technologies for downstream purification of vaccines

Lytic virus

Non-lytic virus

• Detergent

• Mechanical disruption/homogenization

• Osmotic shock

• Freeze-thaw

Yeast- and bacteria-based vaccines

Cell culture

Harvest

Clarification

Primary purification

Secondary purification

Formulation

Process flow

Release of target molecules

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Impurity challenges after lysis

Process chemicals

DNA/RNA

Proteins

Cell membranes/organelles/lipids

Antigen related impurities

After cell lysis Impurities

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Antigen (e.g. virus)

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Goal with purification

Purified sample

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Antigen (e.g. virus)

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Clarification

Cell culture

Harvest

Clarification

Primary purification

Secondary purification

Formulation

Filtration

• Normal flow (NFF)

• Tangential flow (TFF)

Centrifugation

Process flow Available techniques

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• Removal of cell debris and larger particulates

• Porosities from 0.2 to 20 µm

• Scalable

• Single-use technology

• Straightforward process set-up

• Not recommended for harvest with high particulate content

Normal flow filtration (NFF)

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Feed flow

Filtrate flow

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Purification

TFF

Density gradient centrifugation

Selective precipitation

Chromatography– IEX, MM, AC, HIC, SEC

– Bead format (packed bed)

– Membrane format (capsule)

Process flow Available techniques

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Cell culture

Harvest

Clarification

Primary purification

Secondary purification

Formulation

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Chromatography techniques:AC = affinity, HIC = hydrophobic interaction

IEX ion exchange, MM = multi modal, SEC = size exclusion

TFF

• Sweeping effect clean filter surface

• Allow greater throughput on smaller surface area

Basic principle Schematic set-up

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Cross flow

Permeate flow

Feed pressure

RETENTATE PRESSURE

Permeate(filtrate)

BACK PRESSURE VALVE

RECIRCULATIONPUMP

Feedreservoir

(concentrate)

UF or MF device

Dilute feed

material

MF = micro filtration UF = ultrafiltration

TFF

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Cassettes consists of sheet membranes

Concentration/diafiltration

Defined pore sizes

Reusable

Scalable

Hollow fiber cartridge consists of tubular fibers

Concentration/diafiltration

Microfiltration

Suitable for shear sensitive material

Possible to handle high particle loads (e.g., cell harvest)

Defined pore sizes

Reusable

Scalable

Flat sheet cassettes Hollow fiber filters

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Anion exchange chromatography Cation exchange chromatography

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

Principle

Ion exchange chromatography (IEX)

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

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Hydrophobic interaction chromatography (HIC)

Hydrophobic surfaces of proteins interact with the ligand in presence of salts

High salt content enhance and low salt weakens the interaction

Separation by hydrophobicity Principle

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Size exclusion chromatography (SEC)

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Exluded from pores

Enter a fraction of the pores

Enter all pores

Ab

sorb

an

ce

Sample injection

High molecular

weight

Intermediate

molecular weightLow

molecular weight

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Affinity chromatography

Few affinity resins available for vaccines

Agarose-based affinity resin for adeno associated virus

Pseudo affinity resins for influenza

• sulphated cellulose

• sulphated dextran

Specific binding Principle

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Chromatographic purification of large molecules can be challenging

Pore size ~ 40–150 nm

1–7 nm proteins

25 nm polio virus100 nm influenza virus

200 × 500 nm Pox virusFlow-through chromatography recomended

Bind-elute chromatography possible

Pore size ~ 40–150 nm Average pore size 3–5 µm

Chromatography beads~ 2–6 min residence time

Membrane adsorbers ~ 2–60 s residence time

Virus ~100 nm

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The pore size determines the properties

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Core bead chromatography: host cell proteins and DNA fragments bind to the core and viruses stay in the void

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Formulation

Buffer exchange

TFF

Chromatography

• SEC

Process flow Available techniques

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Cell culture

Harvest

Clarification

Primary purification

Secondary purification

Formulation

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Process example

Process example inactivated polio vaccine (IPV)

Seed N-2 Cell

expansion

Seed N-1 Cell

expansion

Production bioreactor

Virus propagation

Clarification NFF

Removal of cell debris and

large particles

TFFConc. of

polio virus

SEC Separation of

polio virus from small molecular

compounds

AIEX (FT) DNA

removal. Polio virus

in flow through

(FT)

Virus inactivation formaldehyde

Formulation Sterile

filtration, mixing with

other strains

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Summary: robust downstream process can ensure 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 > 700 kDa

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