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Pharmaceutical Research

, Volume 29, Issue 1, pp 209–224 | Cite as

Elucidation of Degradants in Acidic Peak of Cation Exchange Chromatography in an IgG1 Monoclonal Antibody Formed on Long-Term Storage in a Liquid Formulation

  • Sejal Gandhi
  • Da Ren
  • Gang Xiao
  • Pavel Bondarenko
  • Christopher Sloey
  • Margaret Speed Ricci
  • Sampathkumar Krishnan
Research Paper

ABSTRACT

Purpose

An IgG1 therapeutic monoclonal antibody showed an increase in acidic or pre-peak by cation exchange chromatography (CEX) at elevated temperatures, though stable at 2–8°C long-term storage in a liquid formulation. Characterization effort was undertaken to elucidate the degradants in CEX pre-peak and effect on biological activity.

Methods

Purified CEX fractions were collected and analyzed by peptide mapping, size exclusion, intact and reduced-alkylated reversed phase techniques. Biophysical characterization, isoelectric focusing and Isoquant analysis were also performed to determine nature of degradants. Bioassay and surface plasmon resonance experiments were performed to determine the impact on biological activity of the degradants.

Results

No major degradation due to oxidation, clipping or aggregation was detected; conformational differences between purified fractions observed were not significant. Sialic acid, N-terminal glutamine cyclization and glycation differences contributed to the CEX pre-peak in the mAb control sample; increase in CEX pre-peak at 25°C and higher was caused by additive degradation pathways of deamidation, related isomerization and clipping.

Conclusions

The observed CEX pre-peak increase was caused by multiple degradations, especially deamidation and clipping. This elucidation of degradants in CEX peaks may apply to other therapeutic IgG1 monoclonal antibodies.

KEY WORDS

acidic-peak cation exchange chromatography deamidation IgG1monoclonal antibody protein formulation 

ABBREVIATIONS

Asn

asparagine

Asp

aspartic acid

bis-ANS

4,4′-dianilino-1,1′-binapthyl-5,5′-disulfonic acid dipotassium salt

CD

circular dichroism

CDR

complementarity-determining region

CEX

cation exchange chromatography

cIEF

capillary isoelectric focusing

FcRn

neonatal Fc receptor

FTIR

Fourier transform infrared spectroscopy

HIC

hydrophobic interaction chromatography

HPLC

high performance liquid chromatography

IgG1

immunoglobulin gamma 1

mAb

monoclonal antibody

PIMT

protein L-isoaspartyl methyltransferase

RP

reversed-phase

SAH

S-adenosyl homocysteine

SEC

size exclusion chromatography

SPR

surface plasmon resonance

TFF

tangential flow filtration

Notes

ACKNOWLEDGMENTS & DISCLOSURES

Our sincere thanks to Scott Smallwood, Jeffrey Reichert, Himanshu Gadgil, Gary Pipes, Ramil Latypov, David Hambly, Jaymi Lee, Lynn Peabody, Renuka Thirumangalathu, Thomas Dillon and process team members all within Amgen for their technical support and discussion.

Supplementary material

11095_2011_536_MOESM1_ESM.doc (110 kb)
ESM 1 (DOC 110 kb)

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Sejal Gandhi
    • 1
  • Da Ren
    • 1
  • Gang Xiao
    • 1
  • Pavel Bondarenko
    • 1
  • Christopher Sloey
    • 1
  • Margaret Speed Ricci
    • 1
  • Sampathkumar Krishnan
    • 1
    • 2
  1. 1.Formulation and Analytical Resources Process and Product DevelopmentAmgen Inc.Thousand OaksUSA
  2. 2.Amgen Inc.Thousand OaksUSA

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