sec/cex - separation of biomolecules · sec/cex - separation of biomolecules: high-throughput...

Kerstin Appenzeller Kathrin Schäker-Theobald, PhD 20. Juni 2018

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SEC/CEX - separation of biomolecules: High-throughput screening

(HTS) and data analysis

via platform methods

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Disclosure

Kerstin Appenzeller and Kathrin Schaeker-Theobald are employees of AbbVie Deutschland GmbH & Co KG.

The presentation reflects the view of the authors and not necessarily by any means the view of AbbVie Deutschland.

The design, study conduct, and financial support for this research was provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

Waters Pharma & Biopharmaceutical LC-MS Forum

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Overview


1. Complexity of biologics

formulation

2. Automation and Miniaturization

3. Automation requirements for

chromatographic analysis

4. SEC platform method

5. CEX platform method

6. Summary

mAb > 20.000 atoms

Paclitaxel 113 atoms

Protein function (efficacy) is coupled to 3D structure

Preserving stability = preserving structure

Complexity of Biologics Formulation

Class of Excipients Function Example

Buffer pH Adjustment & Maintenance

Histidine, Succinate, Citrate, Acetate, Phosphate

Surfactant Stabilization at interfaces

Polysorbate 20 Polysorbate 80

Sugars, Sugar alcohols

Tonicity Agent, Stabilizer Bulking Agent

Sucrose, Trehalose, Mannitol , Sorbitol

Salts Tonicity Adjustment, Stabilizer

Sodium chloride

Amino Acids Tonicity Adjustment, Stabilizer

Glycine, Arginine, Proline, Leucine

Antioxidant Oxidation Protection Methionine

Waters Pharma & Biopharmaceutical LC-MS Forum 4

Automation and Miniaturization – A Fully Automated Screening Line for Liquid Formulation Development

Liquid handler 96-channel

2 Heat Sealer

F/T device

De-Sealer

Centrifuge Shaker 3 Incubators

Liquid handler 8-channel

2 Plate hotels

Plate reader

Robot Spinnaker

Lightcycler

6 UHPLC

Customized solution with devices from >12 suppliers


Data Generation in High Throughput Formulation Screening

In Formulation development high throughput is referring to the number of tested environmental conditions, not the number of molecules.

molecule

conditions

formulation compositions

stress conditions

analytical techniques

unique data points per technique

resulting in 30.000 data points per screening module

1.000 10

3 10 100

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Automation requirements for chromatography in HT screening modus (1)


• Quick and robust – „one fits all“ platform methods (generic) • Miniaturized

• Plate based (96 well to 384 well format) • Sample volume reduction (none or less µL)

General pre-requesites

• Data processing steps ensure data consistency and comparison • Correction of data points by reference normalization and ‟delta“

values (difference to T0) => e.g. column changes, run to run variability are compensated

Screening data consistency

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Automation requirements for chromatography in HT screening modus (2)


• High-throughput vs high resolution: acceptance of lower resolution because of difference calulcation to T0; reduced preparation time for screenings

• Accelerated run time: UHPLC - SEC 10x faster than conventional HPLC • SEC run time 1 sample: 6 min vs 60 min • 100 samples: 10h vs 4 days

Platform methods - ‟universal“ analysis protocols

• Automated methods for column readiness integrated in sequence run

Rinsing and cleaning protocols

• Electronical readable report (export function) for subsequent automated data handling

• Automated chromatogram integration with lean results: • Grouping of pre/post main und main peak (3 data values)

CDS System

SEC platform method


SEC platform method - method details


• Isocratic gradient with phosphate buffered mobile phase at pH 7.0

• Column Waters ACQUITY UPLC BEH200 SEC 4.6 x 150 mm; 1.7 µm, 200 Å (Part

No. 186005225)

(Regeneration buffer as recommended in manual [high salt, low pH])

• Run time 6 min (vs run time HPLC 60 min)

• Column quality check (resolution criteria) with Gel Standard Mix

Method parameter

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SEC platform method – column requirements

• ACQUITY UPLC Protein BEH SEC Column,

200Å, 1.7 µm, 4.6 mm X 150 mm, 10K - 500K; [186005225]

Column type

• 200 to 1000 injections • Maximum injection number depends strongly on the lot of

column material; resolution criteria are monitored via the column check samples and other SSTs

• Pre-testing of column lots is substantial for smooth and qualitative screening

Column life span

• Mandatory regeneration (3h) after each sequence to maintain quality and column life time

• Exclusion of very turbid samples for SEC measurement to prevent column clogging

Column care and precautions


SEC platform method – suitability


• Thorough validation using in-house mAb projects has proven to be a very robust ‟one-fits-all“ method (due to relative constant product size of ~150 kDa)

Robustness

• Verification experiments take 2-3 days when platform method is suitable => quick implementation

• Lean verification testing for each new molecule: • Linearity, column load and LOQ-level are determined

Verification process

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SEC platform method – exemplary chromatograms overview

mAb1

mAb2


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mAb1

SEC platform method - exemplary chromatograms zoomed


Max out the data from one analytical method


• All extractable informations are pulled together to characterize the molecule and its stability in the different formulations

• Calculations performed by CDS (optimized report template for automated data export and further processing)

o Size distribution = > %area of monomer, aggregates and

fragments o Protein concentration => calibration with total area samples

vs total area standard o DAR-estimation (for ADCs) => wavelength ratio indicates

stability of bounded war head; optional

1x run => 3 analytical data sets

CEX platform method


CEX platform method - details


• 500 mM NaCl salt gradient with MES buffered mobile phase at pH 6.5

• Column Waters Protein-Pak Hi Res CM, 7 µm, 4.6 x 100 mm (Part No. 186004929)

• Run time 15 min (vs run time HPLC 30 min), 2x faster

• Column quality check (resolution criteria) using internal protein standard

CEX method parameter

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CEX platform method – column requirements

• Protein-Pak Hi Res CM, 7µm, 4.6x100 mm [186004929]

Column type

• 200 to 2000 injections • Maximum injection number depends strongly on the lot of

column material; resolution criteria are monitored via the column check samples and other SSTs

• Pre-testing of column lots is substantial for smooth and qualitative screening

Column life span

• Storage in NaN3 • Exclusion of very turbid samples for CEX measurement to

prevent column clogging

Column care and precautions


CEX platform method - suitability


• Thorough validation using in-house mAb projects has proven to be a very robust method although some method adaption may be necessary depending on pI

• Higher effort for screening preparation when compared to SEC platform method

Robustness

• Prediction of retention time dependent on calculated surface charge and pre-experiment to check for suitability of platform method

• Adaption of pH and/or gradient if necessary • Verification experiments of adapted method take 2-3 days • Lean verification testing for each new molecule:

• Linearity, column load and LOQ-level are determined

Verification process

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CEX platform method – exemplary chromatograms overview

mAb1

mAb2


• Robust and fast • Generic, less adaption necessary • Low volume • High analytical data gain • Column quality as a critical factor for HTS

Platform methods - ‟universal“ analysis protocols

• Automated data handling is enabled by: • Automated chromatogram integration • Electronical readable reports

• Further chromatography data processing is performed in an automated fashion using in-house developed algorithms

CDS System and automated data handling


Summary

Questions? Thank you for your attention


29,000 Employees worldwide

8 research &

development sites

13 manufacturing

sites

Approx. 17 % of sales reinvested in R&D (2017)

~50 Programs in

clinical development

28.2 billion $ sales in 2017

ABBVIE – AN OVERVIEW > ABBVIE TODAY – FACTS AND FIGURES

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ABBVIE – AN OVERVIEW > INNOVATIVE SPECIALTY DRUGS

AbbVie works in many indications, such as:

Parkinson‘s disease

HIV

HCV Ulcerative colitis

Rheumatic diseases

Respiratory Syncytial Virus

Psoriasis

Crohn‘s disease

Secondary Hyperparathyreoidism

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Hidradenitis suppurativa

Uveitis chronic lymphocytic leukemia (CLL)

Generic HTS workflow

analytics (block 2)

analytics (block 3)

analytics (block 4)

Plate number and type

96 well 384 well

analytics (block 1b, t0)

analytics (block 1a, t0)

plate preparation (compounding)

F/T stress (4 cycles)

mechanical stress (shaking)

plate preparation #2 (subaliquotation)

temperature stress (2 levels)

25°C 5°C

MASTER

2 3

40°C

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26 Waters Pharma & Biopharmaceutical LC-MS Forum

Generic HTS workflow

analytics (block 2)

analytics (block 3)

analytics (block 4)

Plate number and type

96 well 384 well

analytics (block 1b, t0)

analytics (block 1a, t0)

plate preparation (compounding)

F/T stress (4 cycles)

mechanical stress (shaking)

plate preparation #2 (subaliquotation)

temperature stress (2 levels)

25°C 5°C

MASTER

2 3

40°C

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Turbidity Fill height SE-UHPLC



ISA-UHPLC CEX-UHPLC

pH DSF

Concentration

Conductivity* Turbidity Fill height SE-UHPLC

CEX-UHPLC

Viscosity* Particles*

* in development

27 Waters Pharma & Biopharmaceutical LC-MS Forum

Column resolution quality is monitored by the column check samples and further SSTs.

Specification compliance is checked automatically during sequence run and is listed in the report.

SST criteria for reference sample, unstressed:

• RSD of Area and RT of references (n=6)

• Drift reference (Area and Retention time) max. deviation 5%

Column Check citeria

• SEC: Peak asymmetry of Column check sample (Vit B12 –Peak)

• CEX: Resolution (USP) between Main Isoform Peak and following Peak

Data consistency during column change is maintained by our data processing (T0 normalized) with reference data


Maintaining data quality using platform methods

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Max out the data – report design

1x run => 3 analytical data sets

Reference raw data

Sample raw data table


sec/cex - separation of biomolecules · sec/cex - separation of biomolecules: high-throughput...

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