Literature

Label-free HCP quantification using spiked-in proteins and sum all

June 25 2018, by Ejvind Mørtz, PhD

Ejvind Mørtz, co-founder and COO

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 a normal procedure to include an isotope-labeled peptide standard to obtain good HCP data [1].

Another solution is using chemical labeling (like iTRAQ® or SILAC) [2]. Some people even use isotope labeled recombinant proteins. You do this to consider the digestion efficiency of 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 the identification and quantification of HCPs [4].
  • Accurate absolute quantification of HCPs is now obtained by adding 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.

label-free quantification of Host Cell Proteins

The sum all method (now referenced in the USP <1132.1> General Chapter) was used to quantify all proteins in an E.Coli protein extract. This represents an HCP analysis without any drug substance; therefore, all proteins can be considered HCPs.

The E.coli proteins were quantified by spiking in seven (7) intact protein standards in known amounts, and the quantitative calculations were done using the Alphalyse SumAllTM algorithm that integrates the sum of all MS/MS peptide signals for each individual protein. So, if a specific protein has been identified by 10 peptides, the quantity of this protein is represented by the summed peptide signal from these 10 peptides.

SumAll used to quantify host cell proteins as described by USP 1132.1

The figure above shows the sum all, produced by the SumAll™ algorithm, peptide signals for a dilution series of 5 standard proteins. It illustrates the linear correlation between the protein amount in nanograms and the sum of all MS/MS peptide signals.

The response curve is individual for each protein, and the median response curve of the standards was used as a calibration curve. For HCP measurements in biopharmaceutical samples, the standards are typically spiked-in at 2000 nanograms (ng) per milligram (mg) or milliliter of drug substance. Therefore, the measurement unit becomes ng HCP/ mg DS, or ppm (w/w).

The SumAll™ method was developed by Alphalyse for HCP analysis and first presented by Ejvind Mørtz at The BEBPA 7th annual Host Cell Protein Conference May 15-17th, 2019 in Los Angeles, California, and published by Pilely et al in 2021 in Analytical and Bioanalytical Chemistry. https://doi.org/10.1007/s00216-021-03648-2

So to sum it up:

With the SWATH® technique, quantifying proteins reproducibly is now possible. And in addition, it is done without isotope labeling.

 

More information on the 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

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

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