Purified mAb with difficult-to-remove HCP impurity (ubiquitin)

How a detailed analysis enabled risk assessment of Host Cell Proteins in a mAb DS


A clinical-stage biopharmaceutical company wanted a thorough analysis of their process’ ability to remove Host Cell Proteins (HCPs) from their CHO-expressed monoclonal antibody (mAb).

Even though their HCP-ELISA reported no detectable HCPs in the purified drug substance, LC-MS analysis identified one HCP impurity, as can be seen in the table below.

Problematic HCP impurity in antibody
HCP quantifications (ppm) of the top 10 HCPs, quantified by MS signal in a sample prepared with standards. Green indicates levels above LLOQ.

The specific HCP detected by LC-MS – but missed by ELISA – was ubiquitin.

Ubiquitin is a protein that sticks to protein substrates through ubiquitination, and it is a prevalent posttranslational modification. Ubiquitination marks other proteins for degradation via the proteasome pathway, and it can thus affect protein activity and cellular location.

In this blog post, you can learn more about why the purification process did not remove ubiquitin. It also includes reasons why the process-specific ELISA did not detect it >>>

How the client used the LC-MS data

The client wanted to do a risk assessment of discovered and documented ubiquitin in their drug substance with the LC-MS data. During their risk assessment, they found that you also find this common HCP in many other mAb products.

It is still uncertain if they can or should reduce the ubiquitin level further. However, now the client knows the absolute quantity of this specific HCP and can monitor the ubiquitin levels from batch to batch.

Oncology-focused biopharmaceutical company_logo
"We did not expect any host cell proteins in the purified drug substance since our ELISA reported a level below LLoQ. To be honest, we were very surprised to see the mass spec results. The detailed report enables us to optimize our CMC process and remove specific HCPs if we find it relevant."

Oncology-focused biopharmaceutical company- Director, Regulatory CMC

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