This kit can be used for quantitative measurement of residual CHO cell-derived host proteins in biological products.
Contents of Kit
No.
Components
Size
Storage Conditions
1
CHO-K1 HCP Standard
100μL
2-8°C
2
Coated Plate
8 wells × 12 strips
2-8°C
3
Dilution Buffer 1
1 × 50 mL
2-8°C
4
Dilution Buffer 2
1 × 30 mL
2-8°C
5
Wash Buffer (20×)
1 × 50 mL
2-8°C
6
Detection Antibody (50×)
1 × 300 μL
2-8°C
7
Streptavidin-HRP (100×)
1 × 300 μL
2-8°C
8
TMB Substrate
1 × 12 mL
2-8°C
9
Stop Solution
1 × 10 mL
2-8°C
10
Sealing Film
5 pieces
2-8°C
11
Instructions for Use
1 copy
Storage
Sealed kits are valid for 12 months at 2-8°C.
Standard Curve
1. OD processing of the standard curve (See the following example, which is only for example purpose. Please refer to the actual measurement for details): 2. The standard curve is obtained by 4-parameter fitting with the theoretical standard concentrations and the corresponding OD values (as shown in the figure below).
Citations
Publication ()
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Background
Host cell proteins (HCPs) are among the process-related impurities monitored during recombinant protein pharmaceutical process development. Host cell proteins (HCPs) expressed by the hosts used for biologics production are a complex mixture with diverse physicochemical and immunological properties. The composition and abundance of HCPs present in various steps of a manufacturing process and in the final drug substance depend on multiple factors, with the expression system itself being the most significant. Commonly used host cells for production of therapeutic proteins include the Chinese Hamster Ovary (CHO) derived cells (e.g., CHO-K1) which have more than 24,000 predicted genes based on the latest genome sequencing efforts. To characterize HCP content during the production of therapeutic recombinant proteins, efforts have been made to investigate the HCP profile.
Figure 1. CHOP clearance at various purification steps. (Source: Biotechnology and Bioengineering. 2014.)
A plethora of cell lines are used for generating recombinant proteins for therapeutic purposes, including mammalian, insect, and plant cells, as well as prokaryotic cells such as E. coli. Chinese hamster ovary cells (CHO) are widely used in the biotechnology industry. Either the total lysate or cell culture supernatant (e.g., from CHO cells) may be used as an immunogen to challenge animals, typically goats or sheep, to generate antibodies. The resulting polyclonal antibodies (possible after further affinity fractionation for IgG) become an assortment of IgGs against multiple CHO protein (CHOP) species and are thus regarded as multi-analyte anti-CHOP antibodies.
HCP is a critical product quality attribute, and its clearance from biologic products is employed as a benchmark to demonstrate robust bioprocess. The challenges of HCP detection include (1) low levels of residual HCPs present in large excess of product protein, (2) the assay must measure a large number of different protein analytes, and (3) the population of HCP species may change during process development. Suitable methods for measuring process-related impurities are needed to support process development, process validation, and control system testing. HCP monitoring can facilitate process development, where large numbers of samples from various process pools are analyzed for HCP level.
References
1. Zhu-Shimoni, et al.. Host Cell Protein Testing by ELISAs and the Use of Orthogonal Methods. Biotechnology and Bioengineering. 2014.
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References
Identification and characterization of CHO host‐cell proteins in monoclonal antibody bioprocessing
Biotechnology and Bioengineering
Authors: Young Hoon Oh, Kerri M. Mendola, Leila H. Choe, Lie Min, Ashton R. Lavoie, Sobhana A. Sripada, Taufika Islam Williams, Kelvin H. Lee, Yinges Yigzaw, Alexander Seay, Jerome Bill, Xuanwen Li, David J. Roush, Steven M. Cramer, Stefano Menegatti, Abraham M. Lenhoff
Host-cell proteins (HCPs) are the foremost class of process-related impurities to be controlled and removed in downstream processing steps in monoclonal antibody (mAb) manufacturing. However, some HCPs may evade clearance in multiple purification steps and reach the final drug product, potentially threatening drug stability and patient safety. This study extends prior work on HCP characterization and persistence in mAb process streams by using mass spectrometry (MS)-based methods to track HCPs through downstream processing steps for seven mAbs that were generated by five different cell lines. The results show considerable variability in HCP identities in the processing steps but extensive commonality in the identities and quantities of the most abundant HCPs in the harvests for different processes. Analysis of HCP abundance in the harvests shows a likely relationship between abundance and the reproducibility of quantification measurements and suggests that some groups of HCPs may hinder the characterization. Quantitative monitoring of HCPs persisting through purification steps coupled with the findings from the harvest analysis suggest that multiple factors, including HCP abundance and mAb-HCP interactions, can contribute to the persistence of individual HCPs and the identification of groups of common, persistent HCPs in mAb manufacturing.
COA
Publication 
Figure 1. CHOP clearance at various purification steps.
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