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Implementation of UPLC as new technology for Biomolecules in Pharma

Mario HellingsAssociate Director Analytical Sciences & Centers of Excellence Janssen Pharmaceutical Companies of Johnson & Johnson, Beerse, Belgium

17th October 2012 – Waters BioLC Summit

Historical

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Before 2010:

• SM & LM separate

• Mixture of binary UPLC and quaternary H-Class system for SM applications

• No harmonization between SM applications (cleaning, assay/purity, CU, dissolution,...)

• No harmonization between R&D and commercial

• No use of UPLC for LM applications

After 2010:

• Merge of SM & LM

• Focus on cost/time reduction & efficiency improvement

• Platforming

• Standardization/Harmonisation of approaches

• Focus on easy transferability between sites

• New technology introduction team

• Explore UPLC for LM as new technology

J&J UPLC Implementation plan

Application for LM: Protein/MAb Characteristics

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• Is the biomolecule what I think it is?• Is it biologically active?• Is the quality ok?• Is product consistent for release?

Analytical Techniques used for biomoleculesTechnique Use

SDS-page / cSDS Identity, Purity (Proteins)

cIEF Identity, Purity, Isoform distribution

Size Exclusion (HPLC) Identity and purity (aggregation, size/shape variants)

Oligosaccharide mapping (HPLC) Characterization and quantification of N-, O-linked glycans

Peptide mapping (HPLC) Identity, characterization of post-translational modifications (e.g. oxidation, deamidation, etc.)

Cell-based bioactivity Bioactivity

ELISA Bioactivity and Impurity (Host cell protein, Residual Protein A, BovIgG, BSA)

qPCR Impurity (DNA)

Microbiology Bioburden, Mycoplasma, Endotoxin, etc.

Light Scattering, Circular Dichroism, LC-MS-MS, ultracentrifugation, Karl-Fisher,..

Characterization, purity, stability

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Analytical Techniques used for biomoleculesTechnique Use

SDS-page / cSDS Identity, Purity (Proteins)

cIEF Identity, Purity, Isoform distribution

Size Exclusion (HPLC) Identity and purity (aggregation, size/shape variants)

Oligosaccharide mapping (HPLC) Characterization and quantification of N-, O-linked glycans

Peptide mapping (HPLC) Identity, characterization of post-translational modifications (e.g. oxidation, deamidation, etc.)

Cell-based bioactivity Bioactivity

ELISA Bioactivity and Impurity (Host cell protein, Residual Protein A, BovIgG, BSA)

qPCR Impurity (DNA)

Microbiology Bioburden, Mycoplasma, Endotoxin, etc.

Light Scattering, Circular Dichroism, LC-MS-MS, ultracentrifugation, Karl-Fisher,..

Characterization, purity, stability

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Potential for UPLC

What is so special about Ultra Performance Liquid Chromatography (UPLC)?

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• Column has smaller particles:• Provide increased efficiency (lower pate

height H)• Maintain efficiency over a wider linear

range• Ability to increase resolution and speed

of separation• Need for a system with ultra-low

dispersion!

Well-known & implemented for SM applications

A cross-functional team created

Aim: Proof of concept of UPLC for LM, increase resolution (i.e better separation) & speed in robust manner in the LM applications

A well-known compound selected as test subject and DOE principles applied

HPLC UPLC

Size Exclusion Chromatography (SEC)

• Analysis of aggregates, monomer (IgG) and fragments

• Release and stability testing of biomolecules according platform method

• DOE:– Mobile phase

0.2M Sodium Phosphate pH 6.8 or pH 6.0;50mM Sodium Phosphate, 200mM Sodium Chloride pH 6.0

– Temperature30°C, 45°C, 60°C

– Flow rate0.2, 0.4 or 0.6 mL/min

– Sample concentration0.15, 0.5 or 0.75 mg/mL

– Sample volume3, 6 or 10 µL

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SEC – Mobile Phase

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Fragment A

A. 0.2M NaPi pH 6.8B. 0.2M NaPi pH 6.0C. 50mM NaPi, 0.2M NaCl pH 6.0

Fragment B

Histidine

Zoomed 4x

SEC DOE – Resolution Aggregate - Monomer(Mobile Phase A)

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604530

1.80

1.75

1.70

1.65

1.60

0.60.40.2

0.750.500.15

1.80

1.75

1.70

1.65

1.60

1063

Temperature

Mea

nFlow rate

Concentration Volume

Main Effects Plot for Raggr-monData Means

Optimized conditions

• Based on main effect plots:

– Mobile phase: A 0.2 M Sodiumphosphate pH =6.8

– Temperature: 30°, Higher temperature gives temperature induced fragments

– Flow: 0.4 mL a higher flow gives problems with the maximum pressure of the system.

– Injection volume: 3 µL gives the best results in resolution both between aggregates and Monomer and between Monomer and fragments.

• Method parameters tested: LOQ, Linearity, Reproducibility

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SEC UPLC - Reproducibility

Reproducibility UPLC vs HPLC

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UPLC HPLC differencesample aggregate monomer fragment aggregate monomer fragment aggr mono fragBULK 0.15 99.83 0.02 0.09 99.91 0.00 0.06 -0.08 0.02BULK 0.13 99.85 0.02 0.07 99.93 0.00 0.06 -0.08 0.0212M/5°C 0.32 99.63 0.04 0.23 99.76 0.02 0.09 -0.13 0.0224M/5°C 0.29 99.62 0.09 0.20 99.75 0.04 0.09 -0.13 0.054M/5°C 0.38 99.54 0.08 0.34 99.62 0.04 0.04 -0.08 0.048M/5°C 0.20 99.72 0.08 0.11 99.85 0.04 0.09 -0.13 0.04BULK 0.11 99.88 0.01 0.06 99.94 0.00 0.05 -0.06 0.0117M/35°C 4.84 89.70 5.46* 3.89 93.35 2.76 0.95 -3.65 0.4431M/5°C 0.53 99.33 0.14 0.41 99.51 0.08 0.12 -0.18 0.0624M/25°C 1.12 97.68 1.20 0.89 98.14 0.97 0.23 -0.46 0.23

* = sum of 2 fragmentsfragment A = 2.24%fragment B = 3.20%

Change in trend : UPLC shows more aggregate and fragmentExplanation: less monomer tailing and better resolutionNo impact on specs

SEC UPLC – ReproducibilityReproducibility UPLC vs HPLC

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UPLC HPLC

UPLC HPLCsample aggregate monomer fragment aggregate monomer fragment17M/35°C 4.84 89.70 5.46* 3.89 93.35 2.76

* = sum of 2 fragmentsfragment A = 2.24%fragment B = 3.20%

Overlay GF Standard UPLC and HPLC

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SEC UPLC – Column Lifetime

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After ± 560 injections After ± 700 injections

UPLC SEC POC: Conclusions

• UPLC method gives better resolution and is more sensitive than HPLC

• Less difficult to integrate

• Duration of 1 run = 6 min instead of 20 min HPLC

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Peptide mapping

• The Identity Peptide Mapping assay is Final Bulk release assay requested by the Japanese Agency for some products

• The current method focuses on the Peak Area Ratio (PAR) and Relative Retention Time (RRT) for two product specific peptides for identity. One Reference peptide peak is used for the calculation.

• The peptides used are:

– HC60-65 (Identity Peptide)

– HC66-76 (Identity Peptide)

– HC284-297 (Reference Peptide)

• The goal for the POC for the Identity Peptide Map is to decrease the runtime of the assay with comparable resolution and same number of identified peaks.

16PDMS-AD-LMMD Leiden

Peptide Mapping HPLC with Vydac C18 vsUPLC with BEH300 column

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m in2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0

m A U

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

D A D 1 A , S i g = 2 1 4 ,4 R e f = 5 5 0 , 1 0 0 ( M A R T I J N T E N . .. T O 1 2 7 5 _ 1 4 8 L Y S C _ 0 7 D E C 1 0 2 0 1 0 -1 2 -0 8 1 7 -3 2 -0 9 \ M A 0 7 D E C 1 0 0 0 0 0 0 9 . D )

HC 60-65 (25min)

HC 66-76(38min)

HC 284-297Reference Peak (65 min)

HC 60-65

(13 min)

HC 66-76 (20min)

HC 284-297Reference Peak(31min)

HPLC

UPLC

Gradient optimization using LC simulation software

• To determine the gradient for the optimal separation of some of the peptides LC simulation software (LC simulator) was used.

• The software determined the optimal gradient to separate all the peptides based on different parameters (Retention time, Peak Area and Peak Height) from the first run using a linear gradient.

• The proposed optimal gradient by the LC simulation software was 3-8 %B (15 min); 8-18 %B (15min); 18-22%B (10min) and 22-50%B (50min).

• The proposed gradient by the simulation software was executed on a UPLC H-class using a flow at 0.2 mL/min and column temperature of 45°C.

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Gradient optimization using LC simulation software

Acquity BEH 300 C18 1.7um 2.1*150mm17:54:42

30-May-2011

ESI+ 50-2000 - MSE (high energy CE 20-60eV)Synapt HDMS G2Lys C digested CNTO 148 (Simponi)

Time10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00

AU

0.0

2.0

4.0

6.0

8.0

1.0e+1

CNTO 148_110530_MS011_03 4: Diode Array Range: 1.393e+17.30

64.62

42.84

41.16

21.38

12.35 13.2714.14

30.56

22.8725.23 29.06

28.5038.6134.55

37.96

46.11

45.79

56.2955.6149.35

48.94

50.77

63.56 68.4565.60 69.02

UV chromatogram @ 214nm

HC 66-76(25.2min)

HC 284-297Reference Peak(41.2min)

7472706866646260585654525048464442403836343230282624222018161412108642

Time, min

0.5

1.0

1.5

2.0

2.5

HC344-347LC185-189 HC223-227

HC60-65

LC209-215HC59-65 LC105-108

HC419-423LC147-150

LC151-170

HC66-76HC449-456

HC449-455

HC336-343LC192-208

HC143-156

HC350-369

HC131-142

HC370-379

LC171-184

HC424-448HC380-401

HC402-418

LC109-127

HC258-283

LC128-146

HC232-257

HC232-255

LC1-39HC298-326 Glycans

HC298-326 Glycans

LC40-104

HC157-219HC157-214 LC1-104

HC1-59 (N43 IsoD)HC1-58 (N43 IsoD)HC1-59

HC1-58HC1-59 (N43 D)

HC1-58 (N43 D)

MS TIC50 - 2000

HC 60-65(12.4min)

HC 66-76 (24.9min)

HC 60-65(12.3min)

HC 284-297Reference Peak(40.5min)

The simulated peptide Retention times and profile were very comparable to the determined Retention times and profile of the peptides from the experiment.

Peptide Mapping UPLC with BEH300 column and increased flow of 0.3mL/min

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Acquity BEH 300 C18 1.7um 2.1*150mm19:34:17

30-May-2011

ESI+ 50-2000 - MSE (high energy CE 20-60eV)Synapt HDMS G2Lys C digested CNTO 148 (Simponi)

Time10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00

AU

0.0

2.0e-1

4.0e-1

6.0e-1

8.0e-1

10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00

AU

0.0

2.0e-1

4.0e-1

6.0e-1

8.0e-1

CNTO 148_110530_MS011_04 4: Diode Array 214

Range: 2.3291.42

2.45

4.9143.56

29.0928.0120.5814.35

8.965.22 15.40 26.2323.29

31.3433.44 36.6442.87 46.12

CNTO 148_110530_MS011_03 4: Diode Array 214

Range: 2.3292.85

3.59

5.17

7.30 64.62

42.84

41.1630.5621.3813.277.66

22.8725.23 38.6134.55

46.1156.2949.35 54.37

50.76

63.56 68.45

m in2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0

m A U

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

D A D 1 A , S i g = 2 1 4 , 4 R e f = 5 5 0 , 1 0 0 ( M A R T I J N T E N . . . T O 1 2 7 5 _ 1 4 8 L Y S C _ 0 7 D E C 1 0 2 0 1 0 - 1 2 -0 8 1 7 - 3 2 - 0 9 \ M A 0 7 D E C 1 0 0 0 0 0 0 9 . D )

UPLC at 0.3mL/min runtime 60 min

UPLC at 0.2mL/min runtime 90 min

HPLC at 0.2mL/min runtime 180 min

Peptide map POC: Conclusions

• Runtime can be shortened from 180 min to 60 min.

• All peaks have been successfully identified by MS

• All peaks used for the release identity peptide map test calculation, are baseline separated and elute in the same order as in the HPLC peptide map platform method.

• The UPLC method is a suitable platform to replace the current HPLC Identity Peptide Map method

• Waters tests peptide mapping columns (PST) with peptide map mixture for reliable batch-to-batch reproducibility

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Oligo Saccharide map

• Analysis of neutral glycans, and charged glycans.

– Release of Glycans with PNGase F

– Label Anthranilic Acid (2-AA)

• Detection at Fluorimetric

– Emission : 425 nm

– Excitation: 360 nm

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Result Glycan standard

UPLC MethodBaseline separation between M5 and G2FG1F split into two peaksTotal run time 45 min.

WAX methodNo good separation between M5 and G2FTotal run time 120 min.

SA2F

SA1F

G2F

M5

G1F

G0F

Test substance reference standard

UPLC methodG1F split into two peaksSA2F 30.6 min.

WAX methodSA2F 84.4 min.

SA2F

SA1F

G2F

G1F

G0F

POC UPLC Oligo: Conclusions

• Faster and gives a better (baseline) resolution than the WAX method (30 minutes compared to 120 minutes).

• Results are comparable.

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Overall summary UPLC for biomolecules

• UPLC Proof Of Concept was successful for:

– SEC

– Identity Peptide Map

– Oligo Saccharide Map

• Compared to classical HPLC UPLC provides:

– equal or better resolution

– faster analysis for all three methods

• UPLC could be a suitable new platform for SEC, Identity Peptide Map and Oligo Sacharride Map for future NME’s

Furter implementation

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Succesfull proof of concept, how to push technology further?:

• Implementation on R&D portfolio • NME’s easy, late phase needs check for spec setting and comparison• Low impact and can be ran parallel with LC samples • Test more products to gain experience => 5 R&D compounds selected

• Implementation on legacy portfolio• Filter interesting products with customer, typically those were method issues

or improvement is desired => 2 extra compounds selected• Perform testing and check if spec changes• Check impact with regulatory

• Demonstrate System/Column/Vendor reliability• Extensive column/system testing => high volume characterization groups

ideally suited for this as they can perform parallel testing• Vendor needs to give insight in manufacturing process and associated quality

control => Waters provided this information

Outcome

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• POC SEC method could be applied for all 8 compounds (3 legacy, 5 R&D)

• For legacy although excellent results and no spec impact several pitfalls 1. Batches are not split and products were global

• Use of technology only applicable if all countries accepted (US, EU, ROW, Canada, Japan)

• Takes up to 3 years• Parallel testing is useless and expensive for commerial sites

2. Just no business benefit (e.g. product sales decrease, filing cost to high)• E.g. products sales decrease, filing cost to high to do effort

3. No spec changes but trend breaches• Related to better separation, better integration • Hesitance as this might trigger more questions, comparison etc.

• Technology implemented within the R&D functional groups responsible for the LM test package

Non-scientific part

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• Platform selection throughout entire J&J • Bio-H-Class for LM applications and mixed groups

• Absolute requirement for SEC

• H-class for SM applications • Quaternary system provided required flexibility• Can run traditional LC methods

• Available binary systems• Used for non-gmp characterization purposes only and if broken replaced

by I-Class

Non-scientific part

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• Define global site roll-out map • # units/site/year• Replacement program• SIPOC’s for ownership/maintenance/training etc.

• Price negotiation with vendor Platform prices Exchange of old systems / Trade-in’s (max 2 for 1) Training packages, consumables, support, ....

Lessons learned

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• Global Capital Request hard to achieve• Different organizations (R&D – Commercial) => Finance pushed to take up

into CAPEX• Not taken up in CAPEX

• System Lease => loss of all benefits because third party interaction, also not desired by internal finance

• Get now, pay later => very interesting, but book values need to be captured upon delivery so not a solution for finance

• Reprioritize and free up CAPEX

• Numbers fluctuate• Remain ball-park figures, Pharma is just not predictable (budget cuts, new

licensed projects, issues, etc.)• Incorporate in site master plans

• Strong alignment internally needed + alignment in vendor’s organization• SIPOC’s for J&J (Project lead + Procurement) and SIPOC’s for Waters• Strong internal communication needed• Follow-up of delivery and installation (problems)

General Conclusion

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Benefits by selecting platform

• Global Alignment• Easy and fast transfer of methods• Training more effective, less confusion with analysts• Standard for new compounds, switch for old methods with quality or

efficiency issues• Flexibility (SM/LM, quaternary/binary, LC/UPLC)

• Cost saving• Operational cost related to transfers or analyst mistakes• 2 for 1 trade • Maintenance• Energy [HPLC 56W vs (Bio)-H-Class 29W]• Increased throughput

• Space saving• HPLC 58.4cm vs (Bio)-H-Class 34.3cm (with 30cm oven 54cm)

Acknowledgements

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• Werner Claes• Tom Vennekens• Hein Van Nunen• Martijn Ten Asbroek• Herman Bentlage• Jan Plasse• Koen Demoen• Ivan Somers• Koen Vanhoutte• Els Poupeye• Davy Petit• Kim De Kock• Marleen van Wingerden• ....