designing affinity ligands for downstream process intensification

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Prof. Dr. H. Henning von HorstenHTW-Berlin University of Applied Sciences

3rd Biologics & Biosimilars Congress, March 6th-7th, 2017, Berlin, Germany

Designing Affinity Ligands for Downstream Process Intensification

Prof. Dr. Hans Henning von Horsten

HTW-Berlin University of Applied Scienceswww.htw-berlin.de

Prof. Dr. Hans Henning von Horsten


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Key drivers of process intensification

FlexibilityProductivityCost effectiveness Consistent Product Quality

Conventional Batch Unit Operations

Continuous Unit Operations

n cumulative cycles

ProcessIntensification

Consistent Product Quality

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ICHQ8 (R2)

Critical quality attribute (CQA): A physical, chemical, biological, ormicrobiological property or characteristic that should be within anappropriate limit, range, or distribution to ensure the desired productquality( ).

CDR-integrity

N-Glycan Profile

K-Clipping

Deamidation

OxidationDisulfideShuffling

Glycation

Aggregation

Adventitious viruscontamination

AdventitiousTSE

Endotoxin

Residual DNA

Residual HCP

Column Leachables/Protein A

IsoaspartateRacemization

Critical Quality Attributes

Bioburden

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ContinuousBioreactor /

Cell Retention

NF

Pool

PCC-cycle 1

Low pH-EluateHold

Protein A AffinityCapture

PCC-cycle 2

PCC-cycle 3

IEX-1 IEX-2

UF/DF

Intensified Process: Semi-Continuous Process Operation

BulkDS

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Load Elute

ColumnSwitch

Load

Periodic Countercurrent Chromatography

ReequilibrationCycle

I II II I

�� Intensified use of

capture capacity

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Periodic Countercurrent Chromatography

I II II I

Surface induced conformational changes

High on-resin protein concentrations matching resin capacity

Denaturing elution at low pH

Aggregates/HMM-species

PCC��Cumulative entrapment of aggregation seeds after multiple runs

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Protein A-induced product aggregation

Fig. 2a: SEC Profiles illustrating increased vulnerability of protein A-purifed IgG by exposure to pH 3.0

Gagnon et al., J. Chromatogr., A 1395 (May 2015): 136–142.

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Protein A-induced product aggregation

Fig. 2b: Semi-log plot of monomer decay rate, R0, against pH for solution-only

experiments (filled triangles) and experiments including a prior protein A

chromatography step (open circles).

Mazzer et al., J. of Chrom A, Vol 1415, 9 Oct 2015, pp 83–90

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Alternative Affinity Ligand Design

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Prerequisites:

Reasonable selectivity

Mild nondenaturing elution

High capacity binding

Non-immunogenic leachables

Alternative Affinity Ligand Design

Possible Solution:

� multivalent ligand binding

� competitive elution

� LMM-Ligand: enhanced ligand density

� Carbohydrate-Ligands

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Alternative Affinity Ligands

I. Arylboronic acids

*

**

Dennis Hall, 2005* Boron Science New Technologies and Applications Narayan S. Hosmane (Ed.) Crc Pr Inc; 3. Oktober 2011** Smoum et al., Magn. Reson. Chem. 2003; 41: 1015–1020

X

II. Coordinated transition metal ions

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Alternative Affinity Ligand Concept

Dennis Hall, 2005

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Dennis Hall, 2005

Binding Mediators

I. Aryldiboronic Acids

II. Transition Metal Ions

X

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Arylboronic acid functionalized ligands: Bivalent Ligand Concept

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+

Binding


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+

Competitive Elution:(Cis-diols/

alpha-hydroxycarboxylates)


R

R

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Lactate - no longer a problem

Yuk et al., 2014

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Investigation of Affinity Ligand Performance

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[Source: PALL/Fortebio]

Biolayer Interferometry

time

nm

shift

Baseline Association Dissociation

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www.htw-berlin.de

3rd Biologics & Biosimars Congress ThankYou!

Prof. Dr. Hans Henning von

Horsten

[email protected]

+49-30-5019-3636

Life Science Engineering

HTW-Berlin University of Applied Sciences

Wilhelminenhofstraße 75A,

12459 Berlin