The Pichia pastoris expression system is a highly efficient eukaryotic expression system with post-translational processing modifications, which is commonly used for the stable expression of functional biological products such as recombinant proteins. Products using the Pichia pastoris expression system will inevitably introduce host cell protein (HCP) impurities during the production and purification process, which can reduce the efficacy of the product and lead to adverse toxicity or immune reactions, so it is necessary to ensure that the residual level of HCP in the final product of the biologic product is as low as possible.

ELISA is a simple to use, sensitive and objective test for HCPs. This kit uses the principle of double-antibody sandwich enzyme-linked immunosorbent assay (sandwich ELISA) to detect the residual Pichia pastoris HCP in samples. First, add the Pichia pastoris HCP standard (36720-B) and test sample to the Anti-Pichia pastoris HCP coated microtiter strips (36720-A), then add the diluted biotin labeled Anti-Pichia pastoris HCP antibody (36720-C), and finally add Streptavidin-HRP (36720-D) to form an antibody + antigen + antibody-Biotin + SA-HRP complex. Subsequently, add TMB substrate (36720-H) into the complex to observe color reaction after washing the complex. TMB was converted from colorless to blue and finally to yellow in the presence of termination solution (36720-I) catalyzed by HRPase. Shades of yellow were positively correlated with the amount of Pichia pastoris HCP detected in the sample. This kit can be used for the optimization of the purification process of biologics, the control of impurities in intermediate processes, and the release testing of final products.

The detection range of this kit is 3.125~200 ng/mL; the lower detection limit is 0.5 ng/mL.

Feature

• Highly sensitive: Detecting as little as 0.5 ng/mL of Pichia pastoris HCP residuals in in-process and final biological samples.
• Ensures accuracy: The Pichia pastoris HCP residuals can be accurately detected with a recovery of 75%-125%.
• Specificity: Specific detection of Pichia pastoris HCP without interference from other exogenous proteins.
• Stable: The difference between lot-to-lot is low, the kit property is not impacted under 37℃ for 7 days.
• Easy signal collection: Using Biotin-Streptavidin system, the signal can be stably enlarged.

Application

Residual Pichia pastoris HCP test in biological products

Specification

 Product Performance

Components

Storage

This product should be stored at 2°C ~8°C. Unopened product is valid for one year.

^*Upon receipt of the kit, please check whether all components are complete and immediately store them in corresponding condition.

Figures

Precision Display

A) Repeatability

B) Intermediate precision

Table 1. The precision of Pichia pastoris HCP ELISA was assayed as the above scheme

10 duplicated were tested at 3 concentrations on the standard curve, and all the CV% were lower than 10% (Table 1A). 3 lot of Pichia pastoris HCP ELISA kits were tested and the CV% were lower than 15% (Table 1B).

Accuracy Validation

A) Dilution linearity

The intermediate purified samples using recombinant proteins expressed by Pichia pastoris (sample protein concentration of 20 mg/mL, HCP concentration of 1024 μg/mL) were subjected to dilution multiplicity mapping to make their HCP residue values within the range of the standard curve. From the following data, it can be concluded that the dilution linearity of the samples determined by this kit was good, and the differences between the measured values of HCP and the theoretical concentration after different multiplicities of dilution were small, the linear recoveries were in the range of 80%~120%, and the detection values of HCP were linearly correlated with the multiplicities of dilution.

Table2 Sample dilution linearity

B) Spiking Recovery Rate

After 32,000-fold dilution of the intermediate purified sample using recombinant protein expressed by Pichia pastoris (sample protein concentration 20 mg/mL, HCP concentration 1024 μg/mL), the content of Pichia pastoris HCP in the sample was 12.48 ng/mL.This 32,000-fold diluted sample (referred to as S) was mixed with the standards of each concentration in equal volume, and the results showed that all of the sample spiked recoveries ranged from 80% to 120%. sample spiked recovery experiment, the results showed that the spiked recoveries were all between 80% and 120%.

Spiked recovery % = (measured value of spiked samples - measured value of unspiked samples) / theoretical value (amount of spiked standard) x 100%.

Table3 Sample spiking recovery rate

Documents:

Safety Data Sheet

36720_MSDS_HB250310.pdf

Manuals 

36720_Manual_Ver.EN20250310.pdf