Application of H-Class Bio in Method Development on ... Biopharma_Anna Izzo... · HPLC method for isoform analysis of a non-glycosylated protein. 14 Application of H-Class Bio in

Application of H-Class Bio in Method Development on Biotech Molecules in comparison with traditional chromatographic approaches.

Waters User Meeting UPLC16-17 October 2012

Anna Izzo, Cristoforo PellegrinoMerck Serono, Italy

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Who are we…..?

Application of H-Class Bio in Method Development on Biotech Molecules in comparison with traditional chromatographic approaches

Tiburtina site is part of Merck Serono Technical Operations

Analytical Development Biotech Products (Rome Tiburtina site):

• Development of Analytical Methods for QC, Formulation & Process Development

• Characterization & Comparability Testing

• Method Validation and Transfer• Quality Control of CTM

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Merck Serono is the Pharmaceutical Division of Merk KGaA

Over 17,000 employees worldwide

Merck Serono around the world


Application of H-Class Bio in Method Development on Biotech Molecules in comparison with traditional chromatographic approaches4

Outline

– Merck Serono: AD Biotech Products – Who are we– What do we do

– Case Study 1: Aggregation detection of a lipopeptide– Case study 2: IEX-UPLC for Isoform analysis – Case Study 3: IEX UPLC as Orthogonal Method to cIEF

Introduction

Analytical Method Development from HPLC to UPLC H-Class

Case study 1 Aggregation detection of a lipopeptide Aim of the study: Provide a high-resolution chromatographic method

HOW: Exploit the capabilities of the H-class system to rapidly screen different pH and ionic strength conditions in combination with novel SEC UPLC columns.

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membranemembraneplasmaplasma

Lipopeptide

aggregatesaggregates


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HPLC method development attempt

Case Study 1SEC method development on a Lipopeptide


Lipopeptide co-elutes with buffer salts

SE-HPLC conditions:Mobile Phase: 100 mM Sodium Phosphate pH 3.0

Analytical Column : TOSOH TSKgel

Column Temperature: 25°C

Column flow rate: 0.5 ml/min

7 Application of H-Class Bio in Method Development on Biotech Molecules in comparison with traditional chromatographic approaches

Experimental design

Method applicability for aggregation detection

Scouting of mobile phase conditions: pH and ionic strength

Comparison Standard TUV cell vs Titanium cell


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Mobile Phase: 25 mM Sodium Phosphate

Analytical Column:Acquity BEH 125 SEC 1.7 µm 4.6*150 mm

Stock Buffers:A: 0.125 M Monobasic Sodium Phosphate (Acidic Buffer) B: 0.125 M Diasic Sodium Phosphate (Basic Buffer) C: 1 M NaClD: Water

Column flow rate: 0.3 ml/min

Titanium TUV cell reduces peak tailing

SE-UPLC H-Class UV profile

Step I - Comparison of Standard vs Titanium TUV cell


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Standard TUV cell

Titanium TUV cell


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Analytical Column:Acquity BEH 125 SEC 1.7 µm 4.6*150 mmStock Buffers:A: 0.125 M Monobasic Sodium Phosphate (Acidic Buffer) B: 0.125 M Diasic Sodium Phosphate (Basic Buffer) C: 1 M NaClD: WaterColumn flow rate: 0.3 ml/min

Lower is pH, better is the profile in terms of peak shape


Step II - Scouting of mobile phase conditions - pH


Mobile Phase: 25 mM Sodium Phosphate (pH range 5.9-7.5)

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zoom


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Analytical Column:Acquity BEH 125 SEC 1.7 µm 4.6*150 mmStock Buffers:A: 0.1 M Acetic Acid (Acidic Buffer) B: 0.1 M Sodium Acetate(Basic Buffer) C: 1 M NaClD: WaterColumn flow rate: 0.3 ml/min

pH 3.9 minimizes undesired interactions of the lipopeptide with the column matrix

zoom


Mobile Phase: 10 mM Sodium Acetate (pH range 3.9-5.1)

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Step II - Scouting of mobile phase conditions - pH

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Peak tailing and broadening enhanced by Sodium Chloride


Analytical Column:Acquity BEH 125 SEC 1.7 µm 4.6*150 mmStock Buffers:A: 0.1 M Acetic Acid (Acidic Buffer) B: 0.1 M Sodium Acetate(Basic Buffer) C: 1 M NaClD: WaterColumn flow rate: 0.3 ml/min

Mobile Phase: 10 mM Sodium Acetate + NaCl (0 M to 0.4 M)



Step II - Scouting of mobile phase conditions – ionic strength


Mobile Phase: 10 mM Sodium Acetate pH 3.9Analytical Column:Acquity BEH 125 SEC 1.7 µm 4.6*150 mmStock Buffers:A: 0.1 M Acetic Acid (Acidic Buffer) B: 0.1 M Sodium Acetate(Basic Buffer) D: WaterColumn flow rate: 0.3 ml/min

Sample treatment:


• Separation of aggregates from lipopeptide achieved using H-Class

• The longer the UV treatment, the higher the peak 1 level


Step III – Method applicability for aggregation detection

Conclusions – Case study 1 (lipopeptide)

The use of the UPLC H-class system enabled us to achieve a separation of the aggregated species of the lipopetpide


Automated screening of buffer and salt concentration supported by Auto Blend Plus software significantly reduces time for method development

Titanium TUV cell yields superior peak shape

– A higher peak tailing was observed in the standard TUV detector presumably due to the interaction of the sample with the Teflon in the fluidic path

Case study 2IEX-HPLC for isoform analysis Aim of the study: Develop an improved chromatographic method for isoform

analysis

HOW: Explore the capabilities of the H-class system to improve an existing HPLC method for isoform analysis of a non-glycosylated protein.


Sub-optimal separation of isoforms by HPLC

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Case Study 2IEX method improvement on a Non–glycosylated protein


IEX-HPLC conditions:Analytical Column : Tosoh TSK- gel SP-5PW (Strong CationExchange)Column Temperature: 25°C Mobile Phase: A: 50 mM TRIS, 500 mM NaCl; pH 6.5B: 50 mM TRIS, 1M mM NaCl; pH 6.5

Despite development work, resolution still poor...

Background information

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Analytical Columns: Protein pack Hi Res Q 4.6*100 mm

Protein pack Hi Res CM 4.6*100 mm

Protein pack Hi Res SP 4.6*100 mm

Scouting of different buffering systems

Screening of mobile phase concentration

Screening of pH

Screening of ion strenght gradient


Selection of Weak Cation Exchange column

Selection of final chromatographic conditions


Experimental approach

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Improved UPLC method:

Mobile Phase: 10 mM Sodium Phosphate pH 7.0

Analytical Column:Protein Pak Hi Res CM (Weak CationExchange 4.6 Ø x 100 mm)

Stock Buffers:A: 100 mM NaH2PO4 (Acidic Buffer) B: 100 mM Na2HPO4 (Basic Buffer) C: 1 M NaClD: Water


IEX-UPLC H-Class UV profile


Isoforms separation improved

Faster scouting of the chromatographic conditions



Comparison of HPLC vs UPLC H-Class:

Increased separation/ sensitivity of main Isoforms with UPLC H-Class

Conclusions – Case study 2 (IEX isoform profiling) The use of the UPLC H-class system enabled us to

achieve a better isoform resolution

Automated screening of buffer and salt concentration by Auto Blend Plus software reduces method development time

This separation permitted us to follow up and go ahead with fraction collection and isoform characterization, as described in the next case study


Case study 3IEX UPLC as orthogonal method to cIEF Aim of the study: Isoform identification of a cIEF (iCE280) isoform profile

emplying the UPLC H-Class system

HOW: Isoform collection from UPLC combined with peptide mapping / MS


iCE280 method available to separate the isoforms of a non-glycosiyated protein

Identification of isoforms detected by capillary IEF not possible (collection not possible)

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Case Study 3IEX UPLC as orthogonal method to cIEF


capillary IEF profile

Orthogonal separation by a IEX-UPLC H-Class method yields a comparable profile with the possibility to collect fractions

Background information

14.3

57 19.7

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29.1

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35.9

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37.7

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50.9

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IEX-UPLC H-Class profile


Optimized IEX-UPLC H-Class to collect fractions

Fraction desalting

Isoform identification workflow


Peptide mapping (LC-MS)Verification of migration time by injection in cIEF

cIEF peak identification

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Working Conditions:

Mobile Phase: 10 mM Sodium Phosphate pH 7.0

Analytical Column:Protein Pak Hi Res CM (Weak CationExchange 4.6 Ø x 100 mm)

Stock Buffers:A: 100 mM NaH2PO4 (Acidic Buffer) B: 100 mM Na2HPO4 (Basic Buffer) C: 1 M NaClD: Water


IEX-UPLC H-Class UV profile

10 fractions collected


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Primary structure and PTM verification of collected fractions by LC-MS peptide mapping

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Protein Truncated species



Reference SampleFraction 1

UPLC Peptide Mapping profile

Capillary IEF profile

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Verification of migration time by re-injection in cIEF in comparison with a control sample

Blank

pI marker

Control Sample

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pI marker

Control Sample

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pI marker

Control Sample

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pI marker

Control Sample

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cIEF profile isoform attribution


Capillary IEF profile

IEX-UPLC H-Class

An identity was attributed to all IEX-UPLC fractions Cross-identification of the iCE280 profile

Conclusions – Case study 3 (Isoform identification) UPLC H-class IEX separation yields comparable profile

compared to cIEF

The improved chromatographic separation represents an orthogonal separation of isoforms

Collection of fractions allows cross-identification versus the cIEF profile


Overall conclusions

The UPLC H-class system successfully fills the gap between HPLC and UPLC

The flexibility of the system addresses the needs for rapid and automated method development

Integration of the systems ensures effortless re-development / optimisation and Tech-transfer

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HPLCHPLC UPLC HUPLC H--classclass UPLCUPLC


Acknowledgments

Mara Rossi

29Application of H-Class Bio in Method Development on Biotech Molecules in comparison with traditional chromatographic approaches

Cristoforo Pellegrino Alessia Pellegrini

Horst Bierau

Gabriella Angiuoni


Application of H-Class Bio in Method Development on ... Biopharma_Anna Izzo... · HPLC method for isoform analysis of a non-glycosylated protein. 14 Application of H-Class Bio in

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