Main Workshop, Day 2 (March 12th) Session 3:

Hybrid (LCMS & LBA) - Bioanalytical Challenges
Dr. Fabio Garofolo, VP Bioanalytical Services, Algorithme Pharma, will be presenting:

PEGylated Proteins Quantification by LCMS

Background:

  • PEG is a water-soluble, biocompatible, nontoxic, non-antigenic polymer
  • PEG is approved by FDA for human intravenous, oral, and dermal applications
  • PEGylation of proteins improves their pharmacokinetic, pharmacodynamic, and immunological profiles
  • 10 PEGylated biotherapeutics have already been marketed
  • All trends indicate that the use of PEG is able to increase biotherapeutics properties

LCMS challenges due to:

  • Size and charge distributions of PEG and proteins
  • Overestimate of PEGylated protein concentrations due to metabolite species

Sample preparation strategies to reduce sample complexity by depleting serum proteins and concentrate PEGylated proteins:

  • Protein precipitation in an organic solvent
  • Solid phase extraction
  • Immunoaffinity purification by anti-PEG antibody

Novel strategy for PEGylated proteins quantification in plasma using LCMS

Issues with PEG heterogeneity, site of PEG addition, and number of attached PEG moieties

Extraction of the PEGylated therapeutic protein from plasma

Case studies: Use of Surrogate peptide generated from the trypsin digestion for PEGylated protein quantification
and Direct Quantitation Using In-Source CID