Automated Small Scale Protein Purification and ... · Purification and Characterization for Accelerated Development of Protein ... Batch mode ... good similarity with Akta elution

1 1 © 2009 PerkinElmer © 2009 PerkinElmer © 2009 PerkinElmer © 2009 PerkinElmer

Irina Mineyev, Ph.D

Sr. Application Scientist

Automated Small Scale Protein Purification and Characterization for Accelerated Development of Protein Therapeutics

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Therapeutic Protein Process Development

Target

Discovery

Process

Development Clinical

Manufacturing

Commercial

Manufacturing

Clone

Selection

Cell Line

Development

Protein

Purification

Bioprocess

Scale Up Formulation

Steps were characterization and optimization are desired

For research use only. Not intended for diagnostic procedures.

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The potential design space is vast, the relationships between process parameters

and critical quality attributes must be determined experimentally

High Throughput Process Development

Parameter Choices Process Outcome

Cell line

Media components

Feed strategy

Control settings for pH, T, O2, CO2

Chromatography resin

Binding and Elution Buffers

Residence Time

Growth

Titer

Purity

Potency

Glycosylation

Charge heterogeneity

Stability

High throughput analytics are needed to address multi-factorial DOE studies in

Upstream, Downstream and Formulations Development

DOE complicated matrices of conditions


4 4

Small Scale High Throughput Process Development

Process

Parameters

Purification &

Sample Prep

Determination of Critical

Quality Attributes

Informatics Process

Outcomes

Purity &

Fragmentation

N-Glycan

Profiling

Charge Variant

Recovery and

Yield

Alpha Technology

- Immunoassays


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PerkinElmer Analytical Solutions for Accelerated Process Development

LabChip DS

Microvolume UV-Vis Spectrophotometry

for Protein Analysis

High Throughput Microfluidic UV-VIS

Label Free Quantitation of Nucleic

Acids & Proteins

Low Sample Requirement: 2-3 ul

Broad Dynamic Range (0.1-200 O.D.)

Autoblank and Background Correction

Short Run Time – 96 samples in under

5 min

Flexibility – 1-96 samples

LabChip GXII Touch

Reducing time to result for protein

characterization

Automated electrophoretic separations of RNA, DNA, Proteins and Glycans

Capillary electrophoresis performed in microfluidic chips

Sample directly from 96- or 384-well microtiter plate

Comprehensive and quantitative analysis with LabChip GX software

Optional 21 CFR Part 11 compliance package


6 6

LabChip GX/GXII System Components

A complete solution

Instrument

Software

Chips

Reagents

Optional 21 CFR Part 11 compliance package

LabChip GXII Touch

System Components


7 7

Crude or purified protein sample (2L)

Add Sample Buffer

Heat denature ~15min

Prepare Gel/ Dye-Solution and Ladder

Add Reagents to chip

Place Ladder and Buffer on GXII

Reusable chip Up to 400 samples

~ 40 sec per sample ~ 75 min for 96well plate

Protein

Analysis

Protein Assay Workflow

How A Chip

Works.mp4


How A Chip Works.mp4





8 8

View individual sample

results by selecting wells

on the plate map

Annotated electropherogram

and virtual gel shown for

each well

Peak and well tables display

relevant sample data

The protein peaks identified in

electropherogram

Data Analysis Software


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Carbonic Anhydrase Dilution Series - Sensitivity

1.9 ng/ul

15.6 ng/ul

7.8 ng/ul

2000 to 1.9 ng/ul 31.25 to 1.9 ng/ul


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Optimal expression condition is easily

observed

Goal

Develop high throughput purification and

expression system and test effectiveness of

identifying factors influencing protein

production.

Methods

Utilize PerkinElmer LabChip to run full

factorial quantitative analyses of expression

experiments of two level combinations on

four factors.

Authors’ Conclusion:

“Finally, the process that we have described

reduces both experimentation cost and time

while increasing throughput, making

possible what–if experiments that, up to

now, had been either prohibitively

expensive or too complex to perform.” Swalley, S.E. et al., Quantitative Analysis of Protein

Expression, Anal. Biochem, 351 (2006) 122-127

Protein Expression Optimization using DOE


11 11 Chen, X. et al, Electrophoresis. 29, 2008, 4993-5002

LabChip Produced

Comparable Data Faster

“Microchip CE-SDS… provide

sufficient resolution and

sensitivity for this purpose but

on a time scale approximately

70 times faster (41 s versus 50

min per sample) than

conventional CE separation”

LabChip

PA800

Comparison of microchip and conventional CE-SDS. Antibody

samples were denatured, reduced and analyzed with LabChip

or ProteomeLab PA800.

Antibody Quality: Comparison of Chip CE-SDS & CE-SDS

Analytical & Formulation,

Thousand Oaks, Amgen Inc.

CA, USA


12 12 Chen, X. et al, Electrophoresis. 29, 2008, 4993-5002

Protein fragmentation analyses by

microchip and conventional reducing

CE-SDS

• LabChip and conventional CE-SDS

show comparable profiles

• LabChip profile was generated in

~17 seconds as opposed to ~15

minutes by conventional CE-SDS

Antibody Quality: Comparison of Chip CE-SDS & CE-SDS

Analytical & Formulation,

Thousand Oaks, Amgen Inc.

CA, USA


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LabChip DS Instrument

230-750nm spectral analysis

96 well read-out in 5 minutes

DropPlate & classic 96 well plate

Integration ready

LabChip DS Polychromatic DropPlate reader

2 µl sample

Various path lengths (0.1-0.7 mm)

OD range (10mm): 0.05-200

Evaporation protection

16 or 96 well SBS format

DropPlate 16/96 Microfluidic chips and plates

DropQuant Data analysis software

Predefined protocols

Database/LIMS compatible

Flexible analysis & exporting

Interface to robotic systems

Sample Well

Optical chamber

Pump Interface


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LabChip DS - Broad Dynamic Range for Protein Quantification

BSA dilution

Up to 200 mg/ml concentrations

<0.1% CV across triplicate samples

Low range: 0.15 mg/ml

<4% CV across triplicate samples


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Workstations for Automated Protein Purification

BioTx Pro:

12 deck positions for protein purification techniques

8-tip dispense arms with Varispan™ for multi-tipped

processing from tubes to vials to plates.

Tip options include fixed, washable tips, disposable tips,

or both

BioTx Pro Plus:

20 deck locations

8-tip dispense arms with Varispan™

MDT 96 tip head for high throughput applications

Gripper on MDT head

Vacuum manifold

JANUS BioTx Pro

JANUS BioTx Pro Plus


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Features

Application-specific graphical interface to help guide

customers

User “Selects” a protocol and answer questions in simple table format

providing flexibility to define experimental variables in a simple and

straight forward process For research use only. Not intended for diagnostic procedures.

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Features


customers

WinPREP® – Robust, flexible liquid-handling software


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Features


customers

WinPREP® – Robust, flexible liquid-handling software

plate::shuttle station that automates fraction collection

with rack for housing up to 96 separate small scale

chromatography columns


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• 5ml sample loop

• 8X higher sample loading

• Adjustable residence times of

> 11 minutes

• Obtain predictive scale-up

results 10x faster using 6x

less material than FPLC

Unique Hardware


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• Versatip dual purpose tools, provide

2x greater throughput

• One tool for both fixed cannula and

disposable tips

• Supports Atoll MediaScout ®,

Phynexus PhyTips ®, and Filter

Plates

Unique Hardware


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Small Scale Protein Purification Technologies N

umbe

r of

Sam

ples

per

Run

Filter Plates

Batch mode

PhyTip® Column

Separation in a disposable tip

Atoll MediaScout® RoboColumn®

Parallel chromatography

GraviTrap® Columns

1 ml column

Column Capacity

Pre-programmed methods automating

commercially available ion exchange and

affinity chromatography


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Vacuum-based Separations

Integrated vacuum manifold (Pro-Plus)

Parallel 96 channel Pipetting (MDT)

GE PredictorPlate & Pall Acroprep

Resin Screening-Prefilled

chromatography media

Control over vacuum pressure, cycles,

residence time


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Options for Sampling from ambr™ Bioreactiors

Use JANUS with 1 ml disposable tips to sample directly from ambr™ device


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Column Reproducibility

Capacity Screening / DBC Analysis

Resin Characterization

Case Study: Atoll Robocolumns on the Janus BioTx


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Column Reproducibility

Goal: Determine JANUS BioTx platform reproducibility

A step elution was done in parallel on 4 Atoll MediaScout® RoboColumns® (same resin). UV

monitoring of each collected fraction was used to evaluate system reproducibility. A similar

experiment was performed on GE AKTA platform (3.5mL scale) for comparison.

A high level reproducibility was obtained

on 4 different robocolumns and show

good similarity with Akta elution profile.


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Capacity Determination

Goal: Compare resins capacities vs. residence time obtained on JANUS BioTx and GE

AKTA platforms

5 CEX resins were tested (4 strong, 1 weak) on both platforms. Varied flow rates were used to

determine dynamic binding capacity on the JANUS BioTx (0.6 ml robocolumns). Similar experiment

was performed on AKTA GE system (3.5mL scale) for comparison.

Varied Flow Rated to Determine Dynamic Binding Capacity

A high correlation was found for all tested resins, irrespective of resin type or residence time for

both platforms

120100806040200

120

100

80

60

40

20

Capacity determined by Akta

Ca

pa

cit

y d

ete

rmin

ed

by

Ro

bo

tic

Resin 1

Resin 2

Resin 3

Resin 4

Resin 5

Resin type

Capacity correlation : impact of resin type

R2 = 0.9484

JANUS BioTx Pro Plus: 1.5 days

GE AKTA system: 5 days For research use only. Not intended for diagnostic procedures.

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Resin Characterization

High correlation between the results

obtained on both systems is observed: Yield

HMW purity from eluate

LMW purity from eluate

8 chromatographic runs performed: 1.5 hours using JANUS BioTx

1.5 day using GE AKTA

Protein consumption: About 120 mg for Robocolumns

About 0.7 g for GE AKTA

Results Comparison: Yield and Purity


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JANUS BioTx Workstation Offers:

Choice of multiple purification formats

Flexibility to process wide range of sample volumes and

elution conditions

Minimal programming

Simple user interface

Automated procedures

Applicable to multiple downstream characterization methods including

LabChip GXII, MS, HPLC, Immunoassays, Cellular Assays


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PerkinElmer Solutions for Accelerated Biotherapeutic Process Development

Automation

Automated Small Scale

Purification of Biotherapeutic

Proteins

LabChip GXII Touch

Automated electrophoretic separations of RNA, DNA,

Proteins and Glycans

LabChip DS

High Throughput

Microfluidic UV-VIS


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PerkinElmer, Inc. All rights reserved . Some of the information contained in this presentation was obtained from third party sources, as cited. PerkinElmer, LabChip, and the LabChip logo are registered trademarks of PerkinElmer, Inc. and/or its parent, affiliates, and/or subsidiary companies (collectively “PerkinElmer”). The PerkinElmer logo is a registered trademark of PerkinElmer, Inc. All references to other company names, products, trademarks and/or registered trademarks are the property of their respective holders

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Automated Small Scale Protein Purification and ... · Purification and Characterization for Accelerated Development of Protein ... Batch mode ... good similarity with Akta elution