Conexiant
Login
  • The Analytical Scientist
  • The Cannabis Scientist
  • The Medicine Maker
  • The Ophthalmologist
  • The Pathologist
  • The Traditional Scientist
The Analytical Scientist
  • Explore

    Explore

    • Latest
    • News & Research
    • Trends & Challenges
    • Keynote Interviews
    • Opinion & Personal Narratives
    • Product Profiles
    • App Notes

    Featured Topics

    • Mass Spectrometry
    • Chromatography
    • Spectroscopy

    Issues

    • Latest Issue
    • Archive
  • Topics

    Techniques & Tools

    • Mass Spectrometry
    • Chromatography
    • Spectroscopy
    • Microscopy
    • Sensors
    • Data & AI

    • View All Topics

    Applications & Fields

    • Clinical
    • Environmental
    • Food, Beverage & Agriculture
    • Pharma & Biopharma
    • Omics
    • Forensics
  • People & Profiles

    People & Profiles

    • Power List
    • Voices in the Community
    • Sitting Down With
    • Authors & Contributors
  • Business & Education

    Business & Education

    • Innovation
    • Business & Entrepreneurship
    • Career Pathways
  • Events
    • Live Events
    • Webinars
  • Multimedia
    • Video
Subscribe
Subscribe

False

The Analytical Scientist / App Notes / 2014 / Characterisation Studies of PEGylated Lysozyme

Characterisation Studies of PEGylated Lysozyme

09/29/2014

Share

Featured Image
Abstract

PEGylation, the process by which polyethylene glycol (PEG) chains are attached to protein and peptide drugs is a common practice in the development of biopharmaceuticals to prolong serum half-life and improve pharmacokinetics of a drug. There is increasing demand for chromatographic methods to separate the modified isoforms from the native protein. This application note describes the use of size exclusion and ion exchange chromatography for the characterization of PEGylated lysozyme.

TOSOH LOGO
Introduction
Chemical modification of therapeutic proteins in order to enhance their biological activity is of increasing interest. One of the most frequently used protein modification method is the covalent attachment of poly (ethylene glycol) which is called PEGylation. This polymeric modification changes the biochemical and physicochemical properties of the protein, which decreases the in vivo clearance rate and reduces toxicity and immunogenicity of therapeutic proteins.After PEGylation the reaction mixture has to be purified in order to remove non-reacted protein and undesired reaction products. Chromatography as the most common purification method is influenced by PEGylation because of masking and shield effects of the covalently linked PEG molecule. Lysozyme is a well known standard protein, which is often used to determine the dynamic binding capacity of Ion Exchange Chromatography (IEC) resins; therefore we decided to use PEG-lysozyme as a model protein in our study. PEGylated lysozyme was produced out of methoxy- PEG-aldehyde (with a MW of 5 kDa, 10 kDa and 30 kDa) and chicken egg white lysozyme in phosphate buffer in presence of sodium-cyano-borohydrid (NaCNBH3) as reducing agent. The PEGylation reaction takes place between the aldehyde group of methoxy-PEGaldehyde and free amino acid group (NH2-group) of lysine residues within the lysozyme (see Fig. 1).
app-note-108-14-fig.1.pngFigure 1. Lysozyme has six lysine residues as possible PEGylation reaction sides.
The product mixture was analysed by a TSKgel G3000SWxl SEC HPLC-column, SDS-PAGE (not shown), IEC (TSKgel SP-5PW (20) and TSKgel SPNPR strong cation exchange (SCX)) and subsequent MALDI-TOF MS analysis (not shown).
>> Download the full Application Note as PDF

Newsletters

Receive the latest pathology news, personalities, education, and career development – weekly to your inbox.

Newsletter Signup Image

Explore More in Pathology

Dive deeper into the world of pathology. Explore the latest articles, case studies, expert insights, and groundbreaking research.

False

Advertisement

Recommended

False

Related Content

Comprehensive Analysis of Drug Residues from a Confiscated Pipe
Comprehensive Analysis of Drug Residues from a Confiscated Pipe

May 1, 2014

This application note shows the utility of high resolution mass spectrometry with soft ionization to facilitate identification of unknown compounds which were present in extracted residues from a confiscated pipe...

Extraction and Analysis of Neonicotinoid Pesticides from Flower Blossoms
Extraction and Analysis of Neonicotinoid Pesticides from Flower Blossoms

May 6, 2014

In this application, the Quick, Easy, Cheap, Effective, Rugged, Safe (QuEChERS) approach was used to develop an extraction and cleanup method for the analysis of seven neonicotinoid pesticides in flower blossoms...

Ultrapure water: LC-MS suitability tests
Ultrapure water: LC-MS suitability tests

May 7, 2014

Fresh ultrapure water used in all the experiments was produced from a Milli-Q® water purification system fed by an Elix system...

GC/MS analysis of a complex sample in DCM using hydrogen carrier gas
GC/MS analysis of a complex sample in DCM using hydrogen carrier gas

May 8, 2014

This application note aims to demonstrate the injection of a complex, 76-component sample diluted in Dichloromethane (DCM) using hydrogen carrier gas...

False

The Analytical Scientist
Subscribe

About

  • About Us
  • Work at Conexiant Europe
  • Terms and Conditions
  • Privacy Policy
  • Advertise With Us
  • Contact Us

Copyright © 2025 Texere Publishing Limited (trading as Conexiant), with registered number 08113419 whose registered office is at Booths No. 1, Booths Park, Chelford Road, Knutsford, England, WA16 8GS.