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Host Cell Protein (HCP) Assay: Product Purity
Host Cell Protein (HCP) Assay: Antibody Coverage

 

 

 

Click to view a complete list of Applied Biomics HCP assays

Monitoring product purity is another important application of HCP assay. Due to the different spot patterns from samples in the purification process, External Protein Markers (EPMs) can be added to the sample and used to adjust the protein quantitation. Applied Biomics has developed several EPMs at different pH and Mw for you to choose from.

The EPMs can serve as a standard to accurately assess product purity in the following areas:

Evaluating of the purity of therapeutic antibodies, recombinant proteins and vaccines
 Monitoring and optimizing of bioprocess
  Characterizing batch to batch cell lines
Evaluating storage conditions of biological samples


Example-1: Adjust Protein Ratios Using Applied Biomics EPMs
Example-2: Monitor Product Purity Using Applied Biomics EPMs

Applied Biomics External Protein Markers (EPMs)


Cy3-EPMs

Cy3 - EPMs


Cy5-EPMs

Cy5 - EPMs

Overlay image
Cy3-EPMs

Cy5-EPMs
Cy3 Cy5 EPMs image overly

* EPM_1: 76 kDa, pI=6.0, 6.2, 6.6
* EPM_2: 30 kDa, pI=5.4 – 6.0
* EPM_3: 14 kDa, pI=6.8 - 7.2

   

Locations of Applied Biomics EPMs Relative to HCPs


EPMS

EMPS


Cell Lysates

Cell Lysate

Green: EPMS
Red: Cell Lysates

EMPS Cell Lysates image overlay


Example-1: Adjust Protein Ratios Using Applied Biomics EPMs

NO EPM Adjustment

protein ratios without EPM adjustment

With EPM Adjustment

Protein ratios with EPM adjustment

Spots Ratios Without EPM Adjustment Ratios With EPM Adjustment
1 1.6 3.9
2 -6.3 -2.6
3 -18.6 -7.6
4 9.2 22.5
5 6.4 15.7
6 -11.8 -4.8
7 -2.2 1.1
EPM -2.4 1.0


Example-2: Monitor Product Purity Using Applied Biomics EPMs

DeCyder Image

Purification Process

monitor product purity using Applied Biomics EPMs

1) Use DIGE Western blot information to locate all product protein (and their isoforms) - blue spots

2) Calculate % purity of Product

3) Adjust the product ratios between different purification steps using EPMs - green spots