www.cordenpharma.com

Experts taking care.

Aggregation of Peptides in Preparative Chromatography

Anthony Zidell, A. Dalhitski, V. PaulsenAnalytical Development

CordenPharma Colorado

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

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

Crude 38mer1.0 cm i.d. columnReversed-phase sorbentMeCN/Low pH buffer

Crude 38mer45 cm i.d. columnReversed-phase sorbentMeCN/Low pH buffer

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

Nature of Aggregation

Noncovalent self-association1. Colloidal instability2. Conformational instability3. Interfacial instability

Nucleation event leads to aggregation InsolubilityPrecipitationGelation

Draper & Adams, 2016 Chem. Comm.

Factors to Consider

Sequence / Conformation

Concentration

Matrix

Temperature

Surface Effect

Materials of Construction

Degradation / Oxidation

Time

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

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

Aggregation Throughout Processing

Crude dissolution

Purification

Fractions

Pools

control

API

gelMALS

After one year!

MALDI-TOF

PreparativeChromatograms

AnalyticalChromatogram

Crude 32mer45 cm i.d. columnReversed-phase sorbentMeCN/Low pH buffer

Aggregation Within Fractions

Invisible aggregationcan act fast!

Assess stability

UndilutedDiluted 2×

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

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

$↓

Conclusion

Don’t underestimate aggregation

Spend the time/money up front to solve future problems

Be prepared to solve problems on the fly

www.cordenpharma.com

Experts taking care.

Anthony ZidellAnalytical ChemistCordenPharma ColoradoAnthony.Zidell@cordenpharma.comT: +1 303-938-6424

Inquirieswww.cordenpharma.com/contact-us/

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