higher order mab aggregate analysis using new innovative ... · wcbp 2016 washington, dc. top 200...
TRANSCRIPT
Higher Order mAb Aggregate Analysis using New
Innovative SEC Technology
1
Ronald E. Majors, Ph.D.LCGC No. America
West Chester, PA USA
Linda Lloyd, Ph.D.Agilent TechnologiesChurch StrettonUnited Kingdom
WCBP 2016Washington, DC
Top 200 pharmaceutical products by U.S. retail sales (20062012)*
*Charts courtesy of the Njardarson Group, Univ. of Arizona, M. Brichacek, N. McGrath, E. Rogers, J.Morton, L.Batory, R. Bauer, J.A.Wurst, and J.T. Njardarson.
URL: http://cbc.arizona.edu/njardarson/group/top-pharmaceuticals-poster
2006 2012
World Preview Outlook to 2020, EvaluatePharma (2014), In 2020, the top 100 prescription drugs based on biologicals will account for 52% of the sales in the U.S.
(those in RED represent a biopharmaceutical product)
BioPharma trends: implications for liquid phase separations
• Require unique new technologies• Workflow driven
Traditional Pharma shifting to biopharmaceuticals
• Move to UHPLC and smaller particles• Superficially porous particle technologyNeed to speed up the analysis
• Require selectivity, resolution and reproducibility• Minimize degradation caused by analysis Accurate characterization
• Non-denaturing eluents – aqueous (Not RP)Analysis of native biomolecules in biocompatible environments
• LC techniques with MS friendly eluents• Low concentration of volatile salts
Connect LC to higher order LC/MS
• Require complete solution with documentation• Defined workflow solutions
Innovative columns/methods needed for biomolecules
• Need products, workflows suitable for biosimilars• Rapid analysis for reduced development time
Some biologics moving off patent
BioPharmaceutical Product Critical Quality Attribute (CQA)
SECCEX
HIC/CEXSEC
SECGlycan column
Glycan column
CEXGlycan column
HIC/RP
HIC/RP
CEX
Peptide mapping/RP
CEX
Reference: Amgen poster
Aggregation
• Proteins including mAbs are relatively unstable
• Do not always adopt native conformation
• Aggregates may form due to improper production, storage or handling
• Aggregates can be:-Tangled clusters of denatured mAbs- Order structures of native mAbs
• Can have important consequences for the safety and efficacy of biopharmaceuticals
- Will reduce the process yield- Will increase the cost of production- May cause patient immunogenicity
Factors that impact aggregation
Manufacturing ProcessesSteps and Products
• Fermentation• Purification• Formulation• Storage• Shipping• Administration
Stress Conditions
• Heat• Freeze-thaw• Cross-linking• Protein concentration• Formulation change –
pH, salt• Chemical modification• Mechanical stress/
surface
Size Exclusion Chromatography (SEC)
-Larger molecules spend less time in the pores and elute sooner.
-Smaller molecules spend longer in the pores and elute later.
Size in solution is related to retention time
Molecular weight determination
Apparent molecular weight is determined by running samples of known molecular weight and plotting a calibration curve
AdvanceBio SEC 300Å, 7.8 x 300 mm, 2.7µm
Unknown is β-LactoglobulinMW of 18.4 kD
β-Lactoglobulin dimer, apparentMW of 37 kD
Molecular weight determination
However - separation is based on hydrodynamic volume in solution and not molecular weight shape dependency
Light Scattering methods provide absolute molecular weights and increased sensitivity for aggregates
What does that mean for SEC columns?
• SEC columns must deliver accurate, precise quantitation for mAbsand mAb conjugates
• High resolution for more accurate quantitation
• Faster analysis speeds for delivery to deadlines
• Must be stable for long periods of time and high number of injections
• Should cause no change to sample integrity
• Sensitivity for quantitation at low levels - 1 to 5%
• Methods must be easily transferred to other locations, including QA/QC
“Pain points” for current SEC columns
Limited resolution over molecular size range of interest - incomplete resolution makes automated quantitation more difficult - will compromise reproducibility
SEC method impacts sample integrity - non-specific interactions drive mobile phase selection to achieve size separation and will contribute to poor peak shape, recovery and carry over - will compromise accuracy; also, shear forces may be caused by small porosity frits and particles
Long analysis times – large particle sizes and long columns run at low linear velocity - will compromise lifetime if run faster
Limited sensitivity - larger column IDs impact limits of detection and reduces the accuracy of the quantitation for low level aggregation
SEC columns are not very robust and have limited pressure stability
So, what is a good SEC column?
Need high pore volume and optimum pore size No interaction between
particle surface and samples
Mobile phases that do not impact the sample
composition
Introducing AdvanceBio SEC…a new generation of column technology
• A totally new way to make the base silica particle (fully porous) allowing better control of particle diameter, pore diameter and pore volume
• High degree of particle stability allowing for a robust column able to withstand up to 1500 injections, higher flow rates (speed) and no particulate bleed for LS detector
• Novel bonding chemistry minimizes non-specific interactions allowing improved peak shape, excellent recoveries, high degree of inertness and better quantitation (data accuracy), no or low salt in mobile phase and use of pure aqueous mobile phases (no organic).
• A 2.7µm particle to allow good resolution, moderate pressure (can use 400 bar systems) and use of 2µm frits (ruggedness)
Introducing AdvanceBio SEC…a new generation in column technology
• Initial pore size offering: 130 Å and 300Å
• Column dimensions: 4.6mm and 7.8mm i.d’s.150mm and 300mm lengths
• Guard columns: same i.d’s but 50mm length
• Recommended for proteins and peptides with emphasis on monoclonal antibodies analysis and quantification including higher order mAb aggregates and degradation fragments*
• Suitability for different sample types - modified proteins including ADCs and pegylated proteins*
*with same mobile phase
Typical operating conditions
Parameter Conditions
Mobile phases Aqueous buffers150 mM phosphate buffer, pH 7.0Aqueous organic mixes
pH 2 - 8.5
Operating temp 20 - 30oC (recommended)80oC (maximum)
Operating pressure
<200 bar per column (recommended)400 bar (maximum)
Flow rate 0.1 - 2.0 ml/min for 7.8 mm ID0.1- 0.7 ml/min for 4.6 mm ID
Protein resolving ranges
0.1 - 100 kD for 130Å5 - 1,250 kD for 300Å
Recommended starting conditions
No compromise in operating conditions
Features of AdvanceBio SEC and resulting benefits
Feature Advantages BenefitsNew high pore volume silica Increased resolution Accuracy of quantitation
300Å Pore size Higher mAb aggregates are not excluded
Quantitation of dimer and higher aggregates in single run
130Å Pore size Resolution of mAb fragments, small proteins, peptides
More information about a wider range of samples
Unique–hydrophilic polymeric bonded phase
Narrow symmetrical peaks Gain accuracy and reproducibility of results
2.7µm particle High resolution with lower back pressure
Performance benefits with HPLC and UHPLC systems
300 mm column length High resolution Increased accuracy of quantitation
150 mm column length Shorter analysis times Increased sample throughput
7.8 mm ID Robust separations Reproducibility
4.6 mm ID Increased sensitivity Able to quantify low level aggregation
To increase speed of analysis Sample: IgG I9640 Reduce the column length from 250 to 150 mmIncrease Flow: 0.5mL/min, 1.0mL/min, 1.5mL/min Normalized and aligned
Run time reduced from 12 to 4 minutes
3X sample throughput
Resolution maintained
0.5 mL/min
1.0 mL/min1.5 mL/min
Reproducibility protein standards:four production batches
Batch RS Oval/Myo6273369 2.12
6273380 2.17
6279525 2.12
6277528 2.12
Oval Myo
No batch or column qualification is
required -Replace and go
Protein StandardsBioRad Gel Filtration test mix
Reproducibility mAb aggregate samples:four production batches
Batch RS Dim/Mono6273369 1.92
6273380 1.99
6279525 1.90
6277528 1.96
MonoDim
Multiple batches available for
customer validation
Target application
Sample integrity: recovery
Injection Monomer Peak Area
1 50.5
2 49.6
3 50.6
4 50.0
5 50.2
Five replicate injections of BSA 0.1mg/mL, 1µL equivalent to 0.1 µg of BSA on column
BSA peak area is consistent even with 0.1 µg on-column loads
• No protein recovery issues observed• Sample integrity maintained• Accurate data
Sample integrity: run-to-run
Injection 1
Injection 1500
2
1
2
1
1. Dimer2. Monomer Injection
1Injection
1500
Rsmonomer/dimer
2.19 2.11
Monomer 73.9% 74.3%
Dimer 12.2% 11.5%
Target application: mAb monomer and aggregate(s)
Reproducible quantitation from injection 1 to injection 1500- reduces rework as column ages
Separations of different sample types using same columns and conditions
Retention Time Peak Area
Samples Mean(min)
RSD Mean(mAU/min)
RSD
RituximabInnovator 8.28 0.04 99.33 1.21
RituximabBiosimilar 8.29 0 100 0
CetuximabInnovator 7.86 0 99.60 0.96
HerceptinInnovator 8.034 0 100 0
ADC (T-DM1) 8.106 0.005 98.91 0.33
Biotherapuetic mAbs, innovators, biosimilars and ADC
The same column [AdvanceBio SEC-3, 300Å, 7.8 x 300 mm, 2.7 μm] and mobile phase: [(PBS) 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.4]for mAbs and ADCs - Eliminates stocking multiple column types - Eliminates multiple methods
min2 4 6 8 10 12 14
mAU
0
200
400
600
800
1000
1200
1400
1600
220nm, Heat/pH stressed ADC220nm, Intact ADC
Aggregated ADC
Intact ADC
Degraded ADC
min2 4 6 8 10 12 14
mAU
02004006008001000120014001600
RituximabInnovator
min2 4 6 8 10 12 14
mAU
02004006008001000120014001600 Rituximab
Biosimilar
min2 4 6 8 10 12 14
mAU
0100200300400500600700 , Cetuximab
Innovator
min2 4 6 8 10 12 14
mAU
02505007501000125015001750 Herceptin
Innovator
A B
C D
Different types of samples: complex conjugated proteins
PEGylated Proteins - increase bioavailability, increase serum half-life, decrease immunogenicity
Pegfilgrastin (PEG GCSF)
Summary: AdvanceBio SEC delivers
Accuracy of quantitation:• Unique chemistry: improved peak shapes with high efficiency for increased resolution and
provides better sensitivity for low level aggregate/fragment analysis.• Unique chemistry: reduced non-specific interactions over a wide range of pH and salt
concentrations to maintain sample integrity and provide high data accuracy• Narrow particle size distribution and high pore volume: increased column efficiency and
resolution• Increase resolution: run two columns of the same pore size in series increases resolution • Extended resolving range: run two columns of different pore size in series and
enable analysis of multiple attributes in one run• Decreased analysis time: run columns at higher flow rates; increases sample throughput
Method robustness:• Particle integrity: reduces soluble and insoluble column bleed for mass spec and light
scattering detectors • Control of manufacturing process: ensures batch-to-batch and column-to-column
reproducibility• Particle size and 2µm frit: reduces the risk of column clogging and early failure; operates
at lower pressure.• Increased pressure stability: reduces risk of column collapse with high viscosity eluents
or during solvent transfers• QA testing: application specific protein test mix