sec/cex - separation of biomolecules · sec/cex - separation of biomolecules: high-throughput...
TRANSCRIPT
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.
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Overview
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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
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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
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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)
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• 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)
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• 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
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SEC platform method - method details
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• 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
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SEC platform method – suitability
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• 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
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Max out the data from one analytical method
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• 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
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CEX platform method - details
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• 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
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CEX platform method - suitability
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• 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
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• 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
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Summary
Questions? Thank you for your attention
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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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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
Turbidity Fill height SE-UHPLC
Turbidity Fill height SE-UHPLC
ISA-UHPLC CEX-UHPLC
pH DSF
Concentration
Conductivity* Turbidity Fill height SE-UHPLC
CEX-UHPLC
Viscosity* Particles*
* in development
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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
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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
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