[This blog post is centered around the paper “Biophysical properties of the clinical-stage antibody landscape” (http://www.pnas.org/content/114/5/944.abstract) by Tushar Jain and coworkers. It is designed as a very basic intro for computational scientists into the world of experimental biophysical assays.]

A major concern in the development of antibody therapies is being able to predict “developability issues” at the screening stage, to avoid costly developmental dead-ends. Examples of such issues include an antibody being difficult to manufacture, possessing unsuitable pharmacodynamic or pharmokinetic profiles, having a propensity to aggregate (both in storage and in vivo) and being highly immunogenic.

This post is designed to give a clear and concise summary of the principles behind some of the most common biophysical experimental assays used to assess antibody candidates for future developability issues.

1. Ease of manufacture

HEK Titre (HEKt): This assay tests the expression level of the antibody (the higher the better). The heavy and light chain sequences are subcloned into vectors (such as pcDNA 3.4+, ThermoFisher) and these vectors are subsequently transfected into a suspension of Human embryonic kidney (HEK293) cells. After a set number of days the supernatant is harvested to assess the degree of expression.

2. Stability of 3D structure

Melting temperature using Differential Scanning Fluorimetry (Tm with DSF) Assay: This assay tests the thermal stability of the antibody. The higher the thermal stability, the less likely the protein will spontaneously unfold and become immunogenic. The antibody is mixed with a dye that fluoresces when in contact with hydrophobic regions, such as SPYRO orange. The mixture is then taken through a range of temperatures (eg. 40°C -> 95°C at a rate of 0.5°C/2min). As the protein begins to unfold, buried hydrophobic residues will become exposed and the level of fluorescence will suddenly increase. The value of T when the increase in fluorescence intensity is greatest gives us a Tm value.

(Further reading: http://www.beta-sheet.org/resources/T22-Niesen-fingerprinting_Oxford.pdf)

3. Stickiness assays (Aggregation propensity/Low solubility/High viscosity)

Affinity-capture Self-interaction Nanoparticle Spectroscopy (AC-SINS) Assay: This assay tests how likely an antibody is to interact with itself. It uses gold nanoparticles that are coated with anti-Fc antibodies. When a dilute solution of antibodies is added, they rapidly become immobilised on the gold beads. If these antibodies subsequently attract one another, it leads to shorter interatomic distances and longer absorption wavelengths that can be detected by spectroscopy.

(Further reading: https://www.ncbi.nlm.nih.gov/pubmed/24492294)

Clone Self-interaction by Bio-layer Interferometry (CSI-BLI) Assay: A more high-throughput method that uses a label-free technology to measure self-interaction. Antibodies are loaded onto the biosensor tip and white light is shone down the instrument to yield an internal reflection interference pattern. Then the tip is inserted into a solution of the same antibody, and if self-interaction occurs, then the interference pattern shifts by an amount proportional to the change in thickness of the biological layer. Images from: http://www.fortebio.com/bli-technology.html

(Further Reading: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3896597/)

Hydrophobic Interaction Chromatography (HIC) Assay: Antibodies are mixed into a polar mobile phase and then washed over a hydrophobic column. UV-absorbance or other techniques can then be used to determine the degree of adhesion.

(Further Reading: https://www.ncbi.nlm.nih.gov/pubmed/4094424)

Standup Monolayer Chromatography (SMAC) Assay: Antibodies are injected onto a pre-packed Zenix HPLC column and their retention times are calculated. The longer the retention time, the lower their colloidal stability and the more prone they are to aggregate.

(Further Reading: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4622974/)

Size-exclusion Chromatography (SEC) Assay: Antibodies are flowed through a column consisting of spherical beads with miniscule pores. Non-aggregated antibodies are small enough to get trapped in the pores, whereas aggregated antibodies will flow through the column more rapidly. Percentage aggregation can be worked out from the concentrations of the different fractions.

4. Degree of specificity

Cross-Interaction Chromatography (CIC) Assay: This assay measures an antibody’s retention time as it flows across a column conjugated with polyclonal human serum antibodies. If an antibody takes longer to exit the column, it indicates that its surface is likely to interact with several different in vivo targets.

(Further Reading: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3896597/)

Enzyme-linked Immunosorbent Assay (ELISA) – with common antigens or Baculovirus Particles (BVPs): Common antigens or BVPs are fixed onto a solid surface and then a solution containing the antibody of interest linked to an enzyme (such as horseradish peroxidase, HRP) is washed over them. Incubation lasts for about an hour before any unreacted antibodies are washed off. When the appropriate enzyme substrate is then added, it triggers emission of a visible, fluorescent or luminescent nature, which can be detected. The intensity is proportional to the amount of antibody stuck to the surface.

(Further Reading: https://www.thermofisher.com/uk/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-elisa.html)

Poly-Specificity Reagent (PSR) Binding Assay: A more high-throughput method that uses fluorescence-activated cell sorting (FACS), a type of flow cytometry. A PSR is generated by biotinylating soluble membrane proteins (from Chinese hamster ovary (CHO) cells, for example) and then is incubated with IgG-presenting yeast. After washing a secondary labeling mix is added, and flow cytometry is used to determine a median fluorescence intensity – the higher the median intensity, the greater the chance of non-specific binding.

(Further Reading: https://www.ncbi.nlm.nih.gov/pubmed/24046438)