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www.cordenpharma.com
Experts taking care.
Aggregation of Peptides in Preparative Chromatography
Anthony Zidell, A. Dalhitski, V. PaulsenAnalytical Development
CordenPharma Colorado
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Integrated Resource for the Development & Manufacture of Advanced Intermediates and APIs for Global Pharmaceutical and Biotechnology Industries
Development & Manufacture of Complex Synthetic Peptides & Conjugates
Development & Manufacturing of Highly Potent, Cytotoxic & Non-Cytotoxic APIs
Excellent Track Record in Development from Early Stage to Launch
SafeBridge® IV Certified
Location: Boulder, Colorado USA Acquisition: September 2011 Employees: 205
Core Competencies• Leader in Development &
Manufacture of Synthetic Peptides• Process Analytical Technology &
Track Record in Regulatory Filing• Development & Scale-up of Highly
Potent & Oncology APIs
CordenPharma Colorado
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CordenPharma Colorado
Core Competencies• Leader in Development &
Manufacture of Synthetic Peptides• Process Analytical Technology &
Track Record in Regulatory Filing• Development & Scale-up of Highly
Potent & Oncology APIs
Small-scale development Synthesis Purification Precipitation
Location: Boulder, Colorado USA Acquisition: September 2011 Employees: 205
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Crude 38mer1.0 cm i.d. columnReversed-phase sorbentMeCN/Low pH buffer
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Crude 38mer45 cm i.d. columnReversed-phase sorbentMeCN/Low pH buffer
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Peptide Behavior
Peptide–peptide interaction influences physical properties of solutionViscosityClarity
Critical for performance of purification systemPressure spikesPlugged column frits
Aggregation can be irreversible
Can affect solubility of API in formulation
FilterabilitySolubility
Clogged sorbent pores
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Nature of Aggregation
Noncovalent self-association1. Colloidal instability2. Conformational instability3. Interfacial instability
Nucleation event leads to aggregation InsolubilityPrecipitationGelation
Draper & Adams, 2016 Chem. Comm.
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Factors to Consider
Sequence / Conformation
Concentration
Matrix
Temperature
Surface Effect
Materials of Construction
Degradation / Oxidation
Time
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Signs of AggregationVisual inspection
Turbidity Increased viscosity
Pressure rise during loading
Excessive back-pressure spiking during elution
UV trace is irregularChange in retentionSplit peak
Mass balance is off
Gelation beadsObscuration of light
Excessive tailing
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Prevention is Key
Assess aggregation potential in early development Is there a tendency? Is it reversible?
Use the proper toolsSEC, MALS, DLS, MS-MSCD, IR and UV spectroscopy
Keep scale-up in mindColumn cleaningDissolutionProcess flow (filters, vessels, agitation, storage, QC time)
Under what conditions?Can it be seeded?
StabilityChromatographic capability
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Aggregation Throughout Processing
Crude dissolution
Purification
Fractions
Pools
control
API
gelMALS
After one year!
MALDI-TOF
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PreparativeChromatograms
AnalyticalChromatogram
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Crude 32mer45 cm i.d. columnReversed-phase sorbentMeCN/Low pH buffer
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Aggregation Within Fractions
Invisible aggregationcan act fast!
Assess stability
UndilutedDiluted 2×
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Troubleshooting
Dilution is the solution!Aqueous MP or DI water
pH adjustment
Additional filtration
Reduced agitation speed
Additional column cleaning/flushing
Column repacking and frit cleaning
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Consequences of Aggregation
Reduced performanceChanges in retentionSplitting of main peak
Low throughput↑ Filtration↑ CIP
Yield loss↓ Recovery↓ Processability of recovered material
↓ Flow rate↑ Recycling
↓ Sorbent life
$↓
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Conclusion
Don’t underestimate aggregation
Spend the time/money up front to solve future problems
Be prepared to solve problems on the fly
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www.cordenpharma.com
Experts taking care.
Anthony ZidellAnalytical ChemistCordenPharma [email protected]: +1 303-938-6424
Inquirieswww.cordenpharma.com/contact-us/
THANK YOU!