Corporate Banner
Satellite Banner
Chromatography
Scientific Community
 
Become a Member | Sign in
Home>News>This Article
  News
Return

Analysis of Host-Cell Proteins in Biotherapeutic Proteins by Comprehensive Online Two-Dimensional Liquid Chromatography/Mass Spectrometry

Published: Friday, March 02, 2012
Last Updated: Friday, March 02, 2012
Bookmark and Share
This paper presents assays for identification and quantification of host-cell proteins in biotherapeutic proteins over 5 orders of magnitude in concentration.

Abstract
The host-cell protein (HCP) assays consist of two types: HCP identification using comprehensive online two-dimensional liquid chromatography coupled with high resolution mass spectrometry (2D-LC/MS), followed by high-throughput HCP quantification by liquid chromatography, multiple reaction monitoring (LC-MRM). The former is described as a "discovery" assay, the latter as a "monitoring" assay. Purified biotherapeutic proteins (e.g., monoclonal antibodies) were digested with trypsin after reduction and alkylation, and the digests were fractionated using reversed-phase (RP) chromatography at high pH (pH 10) by a step gradient in the first dimension, followed by a high-resolution separation at low pH (pH 2.5) in the second dimension. As peptides eluted from the second dimension, a quadrupole time-of-flight mass spectrometer was used to detect the peptides and their fragments simultaneously by alternating the collision cell energy between a low and an elevated energy (MSE methodology). The MSE data was used to identify and quantify the proteins in the mixture using a proven label-free quantification technique ("Hi3" method). The same data set was mined to subsequently develop target peptides and transitions for monitoring the concentration of selected HCPs on a triple quadrupole mass spectrometer in a high-throughput manner (20 min LC-MRM analysis). This analytical methodology was applied to the identification and quantification of low-abundance HCPs in six samples of PTG1, a recombinant chimeric anti-phosphotyrosine monoclonal antibody (mAb). Thirty three HCPs were identified in total from the PTG1 samples among which 21 HCP isoforms were selected for MRM monitoring. The absolute quantification of three selected HCPs was undertaken on two different LC-MRM platforms after spiking isotopically labeled peptides in the samples. Finally, the MRM quantitation results were compared with TOF-based quantification based on the Hi3 peptides, and the TOF and MRM data sets correlated reasonably well. The results show that the assays provide detailed valuable information to understand the relative contributions of purification schemes to the nature and concentrations of HCP impurities in biopharmaceutical samples, and the assays can be used as generic methods for HCP analysis in the biopharmaceutical industry.

The article is published online in Landes Bioscience and is free to access.


Further Information

Join For Free

Access to this exclusive content is for Technology Networks Premium members only.

Join Technology Networks Premium for free access to:

  • Exclusive articles
  • Presentations from international conferences
  • Over 3,400+ scientific posters on ePosters
  • More than 4,900+ scientific videos on LabTube
  • 35 community eNewsletters


Sign In



Forgotten your details? Click Here
If you are not a member you can join here

*Please note: By logging into TechnologyNetworks.com you agree to accept the use of cookies. To find out more about the cookies we use and how to delete them, see our privacy policy.

Related Content

Determination of 13 Organic Toxicants in Human Blood
Researchers have utilised liquid-liquid extraction coupling HPLC-MS/MS to identify and quantify organic toxicants in human blood.
Friday, July 22, 2016
Plasma Biomarkers for Breast Cancer Diagnosis
Plasma lipidomics profiling identified lipid biomarkers in distinguishing early-stage breast cancer from benign lesions.
Wednesday, May 11, 2016
A Mechanistic Tool for Studying Fungal Pathogens
This study presents an overview of lipid extraction, chromatographic separation and mass spectrometry employed in qualitative and quantitative sphingolipidomics in fungi.
Wednesday, May 11, 2016
Acetaldehyde and Formaldehyde Content in Foods
Korean researchers have determined the content of the toxic and carcinogenic aldehydes, acetaldehyde and formaldehyde, in a variety of food groups.
Monday, November 09, 2015
Metabolomics Data Normalization with EigenMS
In this study, a singular value decomposition-based method, called EigenMS, for normalization of metabolomics data was tested.
Wednesday, January 07, 2015
Bridging Quantitative Analysis and Untargeted Metabolomics
Untargeted metabolomics has become a popular tool in metabolite biomarker discovery and in the evaluation of metabolic pathway changes associated with disease.
Thursday, December 04, 2014
Proteomics for Systems Toxicology
MS-based proteomics is maturing into a robust technology for the measurement of proteome-wide exposure effects.
Monday, November 24, 2014
Single-Step Isolation of Extracellular Vesicles by SEC
Researchers from the University of Amsterdam have developed a single-step protocol to isolate vesicles from human body fluids.
Thursday, October 16, 2014
Comparison of Caffeinated and Decaffeinated Coffee - Implications for Alzheimer's Disease
Researchers from the National University of Singapore successfully demonstrated the viability of GC-TOF-MS as an analytical platform for metabolomics analysis of coffee samples.
Monday, September 08, 2014
RP HPLC Retention Behaviours in Carotenoids Analysis
The efficiency of the separation process in reversed-phase HPLC is highly dependent on the binary hydro-organic mobile phase composition and surface properties of the packing material.
Thursday, August 14, 2014
Identification of Psoriatic Arthritis Mediators in Synovial Fluid
This study represents the most comprehensive proteomic analysis of psoriatic arthritis synovial fluid, to date.
Friday, August 08, 2014
Simultaneous Determination of Six Flame Retardants in Water
The work aimed to develop an analytical procedure for the extraction and simultaneous determination of selected flame retardants.
Thursday, July 24, 2014
Assessing Anticancer Drugs Efficacy by Amino Acid Metabolomics
Researchers aimed to develop a method that determines the therapeutic efficacy of anticancer drugs based on the changes in amino acid metabolism at the cellular level.
Thursday, July 17, 2014
UHPLC: The Greening Face of Liquid Chromatography
Greening pathways for liquid chromatography is especially significant for drug analysis in the pharmaceutical matrix.
Thursday, June 26, 2014
Evaluating Acute Hydrogen Sulfide Poisoning in Rats through Serum Metabolomics
Through the use of GC/MS-based metabolomics, researchers have evaluated the effect of acute hydrogen sulfide poisoning on the metabolic profiles of rats.
Thursday, June 12, 2014
Scientific News
Shark Fins & Meat Contain High Levels of Neurotoxins Linked to Alzheimer’s Disease
UM research team says restricting shark consumption protects human health and shark populations.
Diverse Fungi Secrete Similar Suite of Decomposition Enzymes
A recent study reveals different fungal species secrete a rich set of enzymes that share similar functions, despite species-specific differences in the amino acid sequences of these enzymes.
Lower Mortality with Polyunsaturated Fat
In a study from Uppsala University the fatty acid linoleic acid (Omega 6) in subcutaneous adipose tissue was linked to lower mortality among older men followed over a 15-year period.
How Cloud Connectivity Can Combat the Reproducibility Crisis
This infographic explains the reproducibility crisis, and how cloud connectivity can help overcome this problem.
Simplifying Drug Development
Scientists have for the first time revealed the nanostructure of the mesoporous magnesium carbonate Upsalite® and controlled pore size without organic molecules as templates or swelling agent.
Two New Carcinogens Found in E-cig Vapor
Berkeley Lab study identifies two additional carcinogens not previously reported in e-cigarette vapor.
Quantifying Water Content in Solid Pharma Drugs
UTA chemists devise cheaper, faster, more accurate, automated method to quantify water content in solid pharmaceutical drugs.
Core-Shell Columns in HPLC: Food Analysis Applications
Explore the most recent applications of core-shell columns in food analysis.
Review of the Analysis of Haemoglobin A1c for Diabetes Diagnostics
This paper aims to clarify methods, units, quality requirements, reference and cutoff limits for hemoglobin A1c (HbA1c) and ratio of blood glucose/HbA1c on the basis of the results from Finnish quality control surveys by comparing them to the literature.
Determination of 13 Organic Toxicants in Human Blood
Researchers have utilised liquid-liquid extraction coupling HPLC-MS/MS to identify and quantify organic toxicants in human blood.
Skyscraper Banner

SELECTBIO Market Reports
Go to LabTube
Go to eposters
 
Access to the latest scientific news
Exclusive articles
Upload and share your posters on ePosters
Latest presentations and webinars
View a library of 1,800+ scientific and medical posters
3,400+ scientific and medical posters
A library of 2,500+ scientific videos on LabTube
4,900+ scientific videos
Close
Premium CrownJOIN TECHNOLOGY NETWORKS PREMIUM FOR FREE!