High Throughput Screening High Throughput Screening Technologies for developing Technologies for developing

purification processes of proteinspurification processes of proteins

Michel Eppink, Synthon BV, Nijmegen

Overview

• Introduction

• From small to large processes

• New protein purification technologies– Resin Screening Studies– Resin Scouting Studies

• Case Studies I + II

• Conclusions

Introduction

Why Purification of Proteins?

• Development of robust purification processes (DSP) for therapeutic proteins is essential

• “Time to market” for DSP development saves money (DSP is a costly process)

• Reduce the amount of column/filtration steps and increase the overall yield

• Diminish the amount of impurities such as host cell related proteins

• Insulin (first recombinant product commercialized 1983)

• Granulocyte Colony Stimulating Factor (GCSF)

• Gonadotrophins (HCG, FSH)

• Interferon α/β

• Interleukins

• Growth Hormone

• Erythropoetin (EPO)

First Recombinant Proteins

Monoclonal Antibodies

• Large biomolecules (150 kD)

• Contain both heavy (50 kD) and light (25 kD) chain

• Expression mainly in eucaryotic cell lines (NS0, PerC6, CHO, HEK or other cell lines)

• Mainly glycosylation at the CH chains

• Activity determined by Fab region

Principle of a Chromatographic Step

Sample

Flow through

WashElution

Column with Resin

Ligand:-Ionic-Hydrophobic-Affinity

Equilibration

Chromatogram

BKA0023 IMAC003 :10_ UV1_ 280n m BKA002 3 IMAC003 :10_ Logb ook

0

10 00

20 00

30 00

40 00

mAU

30 0 40 0 50 0 600 m l

sam

ple

inj

ect

ion

Sta

rt w

ash

ing

Wa

sh 2

Wa

sh 3

Sta

rt e

lutio

n

stri

p

1M

Na

OH

Flow through

Wash

Elution

Development DSP processesin early years

Batch wise resin screening in small tubes

Process Development with “in house” equipment

From Small to Large Processes

Exp

erim

enta

l spa

ce

Small → Large

From small to large

0.01 – 0.2 ml

200 - 300 ml

Miniaturization

Scouting Studies

Research Scale

Development Scale

Large Scale

0.05 - 10 ml

1-10 % (1-10 liter)

100% (10-500 liter)

New Purification Technologies

Robotic Handling System

• Small scale purification in microtiterplate and/or minicolumn format

• Automatic handling of the samples

• (Fast) assay analysis (EIA, HPLC, UV, Protein, etc.)

• Parallel testing of different purification conditions (pH, salt, additives, organic solvents, resins, etc.)

• Resin screening/scouting studies can be performed in a reproducible way

Freedom EVO Protein Chromatography Workstation

Tecan Readeroptional, for result analysis

Te-Chrom

Te-Stackoptional, for collection of fractions

Freedom EVO ≥ 100LiHa / RoMa

Hotels/ carriers sample and buffer preparation

Resin Screening Studies

Resin Screening Studies I

Essential part of the screening studies is the

pipetting robot

Pipet chromatographic resin with high

reproducibility from a 12-well plate

Into a 96-well filter plate (in the near

future maybe a 386-well plate would

probably be feasible?)

Analysis with UV/VIS Reader

Resin Screening Studies II

Robot Liquid Handling Batchwise

chromatography

(96 experiments /day)

•Titer (ELISA/UPLC)

•HCP (ELISA)

•Protein

•UV

•CE

•MS (MALDI/ESI)

•SDS-PAGE

map

Centrifugation/Vacuum filtration

� Screening of Resins� Low/medium resolution� Determination of DBC� No flow/bedheight properties

Case Study I

Case Study I

• Cell supernatant of a CHO cell line contains a glycoprotein with a molecular mass of approx. 30-40 kD (heterodimer)

• Development intermediate step with HIC resins (screening/scouting)

• HTS occurs with robotic system

• Detection is performed by UV, protein and/or product specific tests

Case Study I

Elution profile’s at different salt concentrationsResins screened with different (NH4)2SO4 concentrations

Resin Scouting Studies

Atoll RoboColumns

Pre-packed with the resin of choice per each row

Bed heights: 2.5, 5, 10, 30 mm; inner diameter: 5 mm

Column volumes: 50, 100, 200, 600 µl

Freedom EVO Protein Chromatography Workstation

Sample loading Collection of fractions

Sample preparation/ clarification

Elute with Buffer X

Elute with Buffer Y

Column equilibration

Collect Fractions

Analysis (Tecan Reader)

Workflow in High Throughput Chromatography

Sample Loading

Collect Fractions

Analysis (TECAN Reader)

Preparation of Elution Buffers

Case Study II

• Comparison of chromatogramsCommon LC instrumentation

vs.

RoboColumns processed on Freedom EVO

• Example study: Separation of 2 proteinsSeparation of lysozyme and cytochrome c on a cation exchange column, using a step gradient of 0,1 M or 1 M NaCl respectively

Case Study II

Chromatograms from a common LC instrumentation and Freedom EVO® are identical

Extremely high reproducibility from column to column

Case Study II

0 5 10 15

Time (min)

0

1

2

mAU

xE+3

Time [min]

OD

280

nm

[mA

U]

Chromatograms for the separation of two proteins (lysozyme, cytochrome c (each 1 mg/ml) on cation exchangers packed in 200µl Atoll columns for 8 RoboColumns using 0.1 M NaCl or 1 M NaCl respectively, processed on a common LC instrumentation or Freedom EVO®

respectively

Shift due to lower

dead volume

Data kindly provided by TimSchroeder, Atoll, Germany

Platform Technology for DSP processes

Pipetting robot studies

pH, buffer, salt, organic solution screening

Robot/Column Scouting studies

Selected resin(s)

Parameter screening (Experimental Design)

pH, buffer, salt, organic solution screening

Selected resin

Robot/Column optimization studies

M.H.M. Eppink (2007). Biopharm International, 20, 3, 44-50.M.H.M. Eppink (2009). Biopharm International, Mar 2, Supplement

Conclusions

• Robot handling equipment important for Downstream Processing (DSP)

• Fast performance of purification processes

• Large screening window

• Small amount of product needed

Acknowledgement• TU DelftMarcel Ottens

• SynthonGuy de RooKim BurgersXiaonan LiMeng Liu

• TECANMarc BrusDennis BertensDirk de Regt

• AtollJurgen FriedleTim Schroeder

• Kalsruhe Institute of TechnologyJuergen Hubbuch