Quality Control Overview
Shenandoah is proud to provide a step-by-step approach of ensuring high standards of quality for all of our products. In addition to these standards, Shenandoah offers electronic notebook documentation and traceability of each raw material and piece of equipment used. What set's Shenandoah apart from our competitors is our COA library that allows the customer see results for each specification BEFORE they buy!
Shenandoah’s Step-by-Step Quality Overview
Step 1: Purity and Quantitation
Shenandoah uses a combination of methods to determine protein purity and quantity. These methods are protein dependent and can include SDS-PAGE, analytical HPLC analysis, Western blotting, A280 spectrophotometry, and ELISA analysis. Due to the subjective nature of some methods and the inability to objectively distinguish between 96% and 97% purity, Shenandoah documentation denotes purity acceptance criteria and results as ≥90% or ≥95%.
Our primary method of determining product purity is by Coomassie Blue stained 4-20% Tris-Glycine SDS-PAGE gel under reducing and non-reducing conditions. SDS-PAGE analysis is an excellent tool for determining proper protein folding and sample purity.
Example of Coomassie-stained SDS-Page gel:
Analytical high-performance liquid chromotography (HPLC) is used to identify the quality and quantity of the protein sample.
Western blotting is a great tool that Shenandoah uses to validate product purity and protein identity. The protein samples used in western blotting are mainly mammalian-produced proteins that undergo multiple post-translational modifications, such as glycosylation. These types of proteins tend to run as a smear or multiple bands on a Coomassie-stained SDS-PAGE gel and require further validation using the western blot assay.
Example of Western Blotting Data:
Shenandoah’s primary method of protein quantitation is A280 spectrophotometry because it is fast, convenient, and does not require a protein standard. This technique is ideal for highly pure protein preparations like ours!
When available, protein quantity is determined by comparing Shenandoah samples to known protein standards in ELISA analysis. This method is often used to measure mammalian-produced proteins.
Step 2: Endotoxin Detection
Endotoxins are lipopolysaccharides (LPS) located on the cell wall of Gram-negative bacteria, such as E.coli. Endotoxins can provoke strong immune responses by binding LPS receptors on immune cells, and therefore are an important concern for immune-based assays. To ensure the reliability and quality of our endotoxin results, Shenandoah uses an FDA-licensed kinetic LAL endotoxin testing system to measure endotoxin levels in our protein samples. This endotoxin detection system has a detection range of 5.0 - 0.05 EUs/mL. Acceptance criteria for endotoxin levels in Shenandoah proteins are ≤1 EU/ug of protein, with the majority of our endotoxin results being <0.05 EUs/ug of protein.
Example of Endotoxin Results Report:
Step 3: Mass Spectrometry
After every new product is produced, Shenandoah validates that the purified protein is the target protein using mass spectrometry (mass spec) technology as part of our standard quality control. We generally utilize MALDI-TOF mass spec for small proteins that are approximately <35 kDa, and electrospray mass spec for larger proteins. Mass spec analysis confirms the predicted molecular weight of the protein and provides additional data regarding protein identity. We also perform mass spec on any samples that require additional processing (ex. tag or leader cleavage).
Step 4: Bioassay
Shenandoah’s In-House Bioassay Testing offers superior services to competitors:
- Cell-based assays – to measure proliferation, growth inhibition, cytotoxicity, cytokine production (ELISA), and chemotaxis.
- Extensive library of cell lines – over 25 cell lines are available for pre-qualified in-house bioactivity analysis and new assay development.
- Quality and speed of data analysis – The ability to assay our cytokines in-house provides consistency and control over the data analysis.
- Side-by-side comparison of products – to previous lots, competitor samples, and international standards.
- Lot-specific results are available on Certificates of Analysis – prior to order placement, when applicable. *
* Examples of bioassays that would not be found on Certificates of Analysis are assays performed in primary cells (ex. neutrophils, T cells) or assays that are qualitative in nature (ex. STAT phosphorylation). See the product pages or contact [email protected] to request example data of these assays.
Examples of cell-based proliferation and inhibition assays:
Examples of cell-based cytokine production assay: