Characterization of Characterization of PEGylatedPEGylated Products Products by LC/MS and LC/MS/MSby LC/MS and LC/MS/MS

Lihua Huang and Michael De FelippisEli Lilly and Companyy p y

Approved Approved PEGylatedPEGylated BioproductsBioproducts in USAin USA

Brand Name PEG Conjungate Company PEGylation Year

Adgene® PEG‐ bovine ademase Enzon Ran, Linear, >1×5KDa   1990

Oncaspar® PEGylated L‐asparaginase Enzon Ran, Linear, >1×5KDa 1994

Pegintron® PEG interferon alpha‐2b Schering‐Plough Ran, Linear, 1×12KDa 2001

Neulasta® PEGylated GCSF Amgen Sel, Linear, 1×20 KDa 2002

Pegasys® PEG interferon alpha‐2b Hoffman‐La Roche Ran, Branch, 1×40KDa 2002g y p , ,

Somavert® PEG visomant Pharmacia/Upjohn Ran, Linear, 4‐6×5KDa 2003

Macugen® PEG aptanib sodium OVI/Pfizer Sel, branch, 1×40 KDa 2004

Mircera® mPEG epoetin beta Roche Ran, Linear, 1×30KDa  2007

Cimzia® Certolizumab Pegol UCB Sel, branch, 1×40KDa 2008

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Expert Opinion Emerging Drugs (2009) 14(2)

Potential Importance of Potential Importance of PEGylationPEGylation

Improve circulation time (PK) of biopharmaceuticals

Increase solubility of biopharmaceuticals

Decrease immunogenicity and antigenicity of biopharmaceuticals biopharmaceuticals

Increase chemical and physical stability of p y ybiopharmaceuticals

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Characterization ChallengesCharacterization Challenges

♦ PEGylation heterogeneity• Accurate molecular weight• Polydispersity

♦ Number of attached PEG moieties

♦ Identification of Pegylation sites

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OutlineOutline♦ Methodology for Mass Measurement of PEGylated Products

1 LC/MS with post column addition of amines1. LC/MS with post-column addition of amines2. Applications

I. Impurity of PEGI. Impurity of PEGII. Peptide or protein degradationsIII. PEG linker degradation

♦ Methodology for PEGylation site identification1. LC/MS with in-source fragmentation and tandem MS2. Applications

I. Maleimide linker P i h l b (PNP) li k

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II. Para-nitrophenylcarbonate (PNP) linker

Methodology for Mass Measurement ofMethodology for Mass Measurement of PEG Products

-- LC/MS with Post-Column Addition of Amines

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Chemistry of Chemistry of PegylationPegylation

PEG para-nitrophenyl carbonate (PNP)

PEG-maleimide

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TIC of TIC of LC/MS for Degraded 40 LC/MS for Degraded 40 kDakDa PEGPEG

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Mass Spectra at the Different Positions

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Mass Spectrometry Analysis for Mass Spectrometry Analysis for PEGylationPEGylation

• MALDI -TOF – Average massg

• High End Mass Spectrometer – Fourier transform ion cyclotron resonance (FTICR) MS – Ion Mobility (IM) MS

• Charge ReductionAft PEG l ti i i i d– After PEGylation is ionized

– Strong base– Corona discharge

– Before PEGylation is ionized– Using charge reducing reagents

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Mass spectra of 40 Mass spectra of 40 kDakDa mPEGmPEG 40kDa40kDa--PNPPNP

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Huang, et al., Analytical Chemistry, 2009, 81, 567-577.

Effect of Amine Amount on Mass spectra of PEGEffect of Amine Amount on Mass spectra of PEG

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Huang, et al., Analytical Chemistry, 2009, 81, 567-577.

Deconvoluted Mass Spectra of 40kD PEGDeconvoluted Mass Spectra of 40kD PEG

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TIC of 20kDa PEGTIC of 20kDa PEG--PNP and Deconvoluted MSPNP and Deconvoluted MS

P2

P3

P1P2

P1 P2 P3

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LC/MS Analysis for the Stressed DiLC/MS Analysis for the Stressed Di--20kD20kD--PEG PeptidePEG Peptide

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Deconvoluted Mass Spectra of 20 Deconvoluted Mass Spectra of 20 kDakDa PEG GlucagonPEG Glucagon

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22××20kDa 20kDa PEGylatedPEGylated Peptide at Different ConditionsPeptide at Different Conditions

pH60 ºC-4w0 C-4w

pH340 ºC-4w

pH5 5+18Da

pH5.540 ºC-4w

pH840 ºC-4w

+36Da

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SummarySummary

♦ LC/MS with post-column addition of amines, such as DEMA and TEA effective for reducing the charges of PEGDEMA and TEA, effective for reducing the charges of PEG.

♦ Accurate mass measurement of PEGylated products were y psuccessfully obtained by the methodology.

Th th d l f ll li d f l ♦ The methodology was successfully applied for several PEGylated products to identify PEG impurity, peptide and PEG linker degradations of PEGylated productsPEG linker degradations of PEGylated products

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Methodology for PEGylation Site ElucidationElucidation

-- LC/MS with In-Source Fragmentation andLC/MS with In Source Fragmentation and Tandem MS

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Mass Spectrum of Mass Spectrum of PEGylatedPEGylated ProductProduct

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Mass Spectrum of 5 Mass Spectrum of 5 kDakDa PEGylatedPEGylated GlucagonGlucagon

O NH

OGlucagonO

H n

n=15

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Lu, et al. JASMS, 2010, 21, 810-818

MS/MS Spectrum of Ion at m/z 1396.7MS/MS Spectrum of Ion at m/z 1396.7

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Mass Spectra of 20 Mass Spectra of 20 kDakDa PEGylatedPEGylated GlucagonGlucagon

O NH

OGlucagonO

H n

n=13

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Tandem MS Spectra of Ion at m/z 1367.3Tandem MS Spectra of Ion at m/z 1367.3

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Mass Spectrum of 20 Mass Spectrum of 20 kDakDa PEGylatedPEGylated IgG4 LCIgG4 LC

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Mass Spectrometry Analysis for the LysMass Spectrometry Analysis for the Lys--C Digest of C Digest of PEGylatedPEGylated Partially Reduced IgG4Partially Reduced IgG4yy y gy g

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Deconvoluted MS for 20 Deconvoluted MS for 20 kDkD PEG PEG FabFab HC with ISFHC with ISF

NN

O

O

OH

O226 Da =

HC:1-218+226Da

n=2

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SummarySummary

♦ The small PEG fragment attachment generated by ISF was stable during the CID activationby ISF was stable during the CID activation.

♦ Methodology is suitable for PEGylation site gy ymapping of various PEGylated products.

Th li ti f th d l t i t♦ The application of our methodology to a mixture of different positional isomers successfully distinguishes the distinct PEGylation sitesdistinguishes the distinct PEGylation sites.

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AcknowledgementsAcknowledgements

♦ Xiaojun Lu♦ Clayton Gough♦ Shun Li♦ Jirong Lu♦ Rohn Millican♦ Jerry Kinzel

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