Good image analysis will maximize the output of your HCP coverage experiment. In this chapter we provide advice and tips to assist researchers to make more reproducible decisions when analysing their 2D gels and Western blots to achieve percentage coverage which can help to develop a more meaningful and useful analysis result.
Living host organisms are used in biological drug manufacturing to produce biologics and biosimilars. However, for safety of the final drug product, host cell proteins are impurities and can affect drug efficiency and can lead to undesirable effects related to immunogenicity in patients. This makes the HCP risk an industry-wide challenge and it is of particular interest to regulatory bodies. They state that adequate evaluation, monitoring and removal of these host cell proteins during bioprocess development (HCP) must be performed. Regarding the level of acceptable limits of host cell proteins: No common limit for HCP contamination has been set by regulatory bodies. However, the regulatory bodies state that process-related impurities such as residual host cell proteins (HCPs) must be removed to minimum level during the drug manufacturing process. Current best practice for determining the quality of the anti-host cell protein antibodies is 2D gel electrophoresis followed by Western blotting.
For the article we will assume you have already ran and captured your samples and you are at the image analysis stage.
Before our reproducible tips, we want to outline what software practices you might be using that can actually cause a lack of reproducibility for HCP coverage determination.
Spot matching techniques are inherently unreliable and require hours of tedious subjective decisions. Migration of spots, low abundance spots and the general distortion of spot patterns can make it near impossible to accurately define which spots on the gel correspond to which spots on the blot.
Spot warping techniques are only accurate to the spot level whereas an alignment technique which is used in our HCP coverage analysis software is accurate to the near pixel level.
As expected, whenever a user decision is required it reduces the objectivity of an analysis. However, decisions are often inevitable in HCP coverage analysis with 2D gel western blotting, due to the absence of ground-truth for real-gel spots. Decisions such as “is that spot a real protein spot” are often required.
In order to make these decisions you will need as much factual information and flexibility as possible to make an informed decision. The more visual representations of your data alongside software aided tools the more confident you can be when spotting the real proteins in your images (get in touch if you’d like to find out which tools you can use to recognise a real spot).
We recommend creating a standard operating procedure (SOP) for the analysis of 2D gel and Western blots as a way to further reduce the subjectivity of the analysis and variation between users and labs.
When completing an analysis there are a few things you should consider to generate a reproducible analysis.
As previously discussed reproducible software should account for and demonstrate reliability of final results, but to ensure you are using your software to the fullest extent there are a few considerations.
Don’t worry, we have highlighted practices you can use to improve your reproducibility today:
The reproducibility problems suffered by researchers can be reduced using the right analysis software and putting reproducible procedures into practice. The guidelines presented here are based on problems encountered by our customers and are by no means exhaustive. However simple adjustments, data management and knowing when subjectivity is introduced in image analysis can greatly improve your experiments.
An example report to illustrate how quickly you can analyse challenging 2D gels and Western blots to calculate host cell protein (HCP) percentage coverage.View Poster