GEN Exclusives

More »

Feature Articles

More »
Oct 1, 2009 (Vol. 29, No. 17)

Advances in Quantitative Proteomics

Alternatives Designed to Overcome the Limitations of 2-D Electrophoresis Are Under Development

  • Host-Cell Protein Assays

    Biopharmaceuticals produced in recombinant cell lines can contain residual host-cell proteins (HCP) characteristic of the cell lines in which they were expressed. Present at extremely low concentrations, typically µg/g of recombinant protein, or ppm concentration, these contaminants can potentially elicit immune responses in individuals to whom they are administered.

    According to Waters scientists, current analytical methodologies may not completely identify and quantify individual host-cell proteins, as such assays typically only provide a total concentration of host-cell protein. Also, current methods may not respond equally to all host-cell proteins present in a sample. As each therapeutic protein utilizes a unique purification process, generic host-cell protein assays may miss key host-cell protein contaminants present in the sample.

    At the meeting, Catalin Doneau, Ph.D., senior scientist, described the development of a two-step approach to host-cell protein discovery and quantitation. In the discovery phase of a biopharmaceutical, 2D-nanoUPLC/MS/MS with a QTOF mass spectrometer, with database searching of tryptic peptides from the digested biopharmaceutical protein, is used to identify host-cell protein impurities. The identified proteins can be quantified using label-free quantitation against a spiked-in protein standard.

    Once identified, individual host-cell proteins can be quantitated on a tandem quadrupole system by multiple reaction monitoring (MRM) assays of signature peptides from each identified HCP. The methodology was successfully used to analyze five low-abundance proteins introduced into a stock solution of a humanized monoclonal antibody, according to Dr. Doneau.

    Jeff Mazzeo, Ph.D., biopharmaceutical business director, explained that the challenges inherent with quantitating HCP contaminants in recombinant protein production systems include never “really being sure whether you have detected all of the contaminating cell proteins in the product.”

    Typically, he said, companies may use polyclonal antibodies for ELISAs rather than a targeted monoclonal to detect a specific host-cell protein. Another approach is to use mixtures of monoclonal antibodies for specific contaminants, but the key step is discovering all the potential host-cell protein contaminants. The issue, he explained “is that you don’t know whether you are detecting all of the proteins present and you can miss things” Further, he noted, regulatory agencies are asking companies for very specific information, requiring not only determination of total HCPs but individual discrimination among them.

    Waters uses tools originally developed for proteomics and applies them to HCP detection and quantitation as well, Dr. Mazzeo said. For protein identification, “we perform two-dimensional LC separation, which allows us to load a lot of protein so we have a better chance of finding contaminants that are present in small amounts.”

    LC separation followed by tryptic digestion produces peptides that are sequenced, and then identified by searching against a database to find peptide matches with specific protein sequences. If a complete protein sequence database is unavailable, as he said is the case with CHO cells, “we use a combination of mouse and rat genomes to identify CHO proteins, relying on homology.”

    Once a process is established and an expected range of contaminants identified, HCPs can be quantitated directly by MRM. “Once you know what HCPs are present, they can be quantified with greater precision and a lot faster using a tandem quadrupole mass spectrometer. In an MRM experiment, one is monitoring the transition of the parent peptide ion to a specific fragment ion, which provides tremendous specificity and sensitivity. This approach has been used in proteomic studies to quantify biomarkers.” Once you know what the host-cell proteins are, you can use this MRM approach, he reported. “We suggest using the ID on a really dirty sample, then setting up a targeted MRM method for the next phase.”

Add a comment

  • You must be signed in to perform this action.
    Click here to Login or Register for free.
    You will be taken back to your selected item after Login/Registration.

Related content


GEN Jobs powered by connects you directly to employers in pharma, biotech, and the life sciences. View 40 to 50 fresh job postings daily or search for employment opportunities including those in R&D, clinical research, QA/QC, biomanufacturing, and regulatory affairs.
More »

Be sure to take the GEN Poll

The Triple Package and Success

One theory for explaining “success," put forward by Amy Chua Jed Rubenfeld, posits cultural traits such as a superiority complex, personal insecurity and impulse control. Union College professors Joshua Hart and Christopher Chabris counter that intelligence, conscientiousness, and economic advantage are the most likely elements of success, regardless of ethnicity. Do you think that Hart-Chabris make a better argument for achieving success than the Chua-Rubenfeld theory?

More »