Question:
How can I improve label-free quantification of HCP?
I find it difficult to get an accurate label-free quantification of Host Cell Proteins with traditional multiple reaction monitoring using LC-MS. It also seems that my data is difficult to reproduce. – Even if I perform multiple LC-MS runs for each sample.
Is there anything I can do to improve on the quantification and the reproducibility of the data?
Answer:
For quantitative proteomics experiments it has been normal procedure to include an isotope labeled peptide standard to obtain good HCP data [1].
Another solution is the use of chemical labelling (like iTRAQ® or SILAC) [2]. Some people even use isotope labeled recombinant proteins. You do this to consider the digest efficiency on a full length protein [3].
All of the above methods are time-consuming – and they are not always possible to use [2].
So what can you do instead?
With the new SWATH® LC-MS technology, label-free quantification of HCPs is no longer a problem
The reasons for this are:
- SWATH® analysis is a data-independent acquisition method. This results in very reproducible MS data – both for identification and quantification of HCPs [4].
- Accurate absolute quantification of HCPs is now obtained by the addition of standard proteins. They are added in known amounts and are digested within the sample.
- The SWATH® LC-MS is running at robust microflow rates. Therefore, there is no longer a problem with time-consuming nanoflow HPLC or carry-over.
Label-free quantification of thousands of proteins
In order to test the new LC-MS method, we applied SWATH® label free quantification to an E. coli cell lysate. With this method we could identify and quantify more than 1.000 proteins, up to a total sum of 969.158 ppm. The expected sum is 1.000.000 ppm – see graph below.
The analysis is so robust and reproducible that the standard deviation for individual proteins is below 20% in repeat experiments.
So to sum it up:
With the SWATH® technique it is now much easier to quantify proteins reproducibly. And in addition, it is done without isotope labeling.
More information on analysis of HCP:
The SWATH® technology is a form of data-independent analysis (DIA). You can learn more about the technology here: “What is SWATH”
Find out more on the identification and quantification by Host Cell Protein analysis on the Alphalyse website: www.alphalyse.com/hcp
References:
[1] Zhu-Shimoni et al: “Host Cell Protein Testing by ELISAs and the Use of Orthogonal Methods“, Biotechnology and Bioengineering, 2014
[2] Tscheliessnig et al: “Host cell protein analysis in therapeutic protein bioprocessing – methods and applications.“, Biotechnology Journal, 2013
[3] Bracewell et al: “The Future of Host Cell Protein (HCP) Identification During Process Development and Manufacturing Linked to a Risk-Based Management for Their Control“, Biotechnology and Bioengineering, 2015
[4] Heissel et al: “Evaluation of spectral libraries and sample preparation for DIA-LC-MS analysis of host cell proteins: A case study of a bacterially expressed recombinant biopharmaceutical protein.“, Protein Expression and Purification, 2018
