Maya Wright1, Luca Groß1, Sean Devenish1
With the monoclonal antibody (mAb) market growing, analytical methods must be ready to provide accurate but rapid quality control. Size presents a simple metric to assess quality as an indicator of aggregation, degradation, oligomerization or misfolding. In this work six commercially available antibodies are analysed by SEC and MDS. Both techniques are successful in identifying problem samples, but MDS took 80% less time and 90% less sample per test.
As the demand for antibodies as therapeutics and diagnostics rises (1), biomanufacturing techniques chase ever higher yields to ensure supply (2).
This growing market must be supported by quality control methods that are accurate, reliable but also convenient and allow for rapid decision making.
In addition to this, the variable nature of biologics means that more tests must be performed during manufacture; typically a chemical drug has 40 to 50 critical tests during manufacture, while a biologic has around 250 (3).
Early stage laboratory research underpins this growing area, with the same needs for quality, consistency and speed of analysis present at a smaller scale.
Here we evaluate two methods for measuring the size of antibodies; size exclusion chromatography (SEC) and microfluidic diffusional sizing (MDS). We find that both can identify clean, aggregated and degraded samples but that MDS took 80% less time and 90% less sample per analysis.
Six commercially available antibodies were purchased and analysed by Microfluidic Diffusional Sizing (MDS) using a Fluidity One instrument, and by SEC using an AKTA. The details of each antibody are given in Appendix 1.
For MDS analysis, the solutions were desalted to remove contaminants and then diluted to 50%. 5 µL of the resulting solution was pipetted onto a testing chip and run in the Fluidity One instrument. Results (size, hydrodynamic radius (Rh), and concentration) were obtained in 8 minutes. No instrument setup or calibration was required.
For SEC analysis, 40 µL of the sample was introduced to the column. Each chromatogram was obtained in around 50 minutes, although additional setup and calibration was required which took around 1 hour at the beginning of the day. Detection was by UV-280.
Table 1: The requirements for size analysis by SEC and MDS
MDS testing reports the average Rh of all primary amine containing species present. This means the presence of fragments reduce the average, while aggregates increase it. Figure 1 compares the measured and predicted Rh for each antibody.
Figure 1: The size of each antibody as measured by MDS on the Fluidity One vs the predicted size. We observe that Anti-Insulin and Rabbit-IgG are likely aggregated, while Anti-EGFR appears slightly degraded. The dotted line shows the expected Rh (4). Each point represents the average of 3 MDS tests.
The SEC analysis provides a chromatogram indicating the relative abundance of each species detected by UV-280 absorbance. Figure 2 shows the chromatogram obtained for each antibody.
Figure 2: Chromatograms from SEC analysis of each antibody.
For all species an elution volume of 12 ml is expected for the intact monomer; elution at lower volumes indicates oligomerization or aggregation, elution at higher volumes indicates fragmentation or degradation. It is clear that the SEC results agree with those obtained by MDS; Anti-BSA, IL2 and His all appear to be monomeric and free from aggregates or fragments, while the anti-insulin and Rabbit IgG show signs of aggregation and the anti-EGFR appears to have small fragments present in addition to the monomer.
MDS and SEC were used to assess six commercially available antibodies. Both techniques draw the same conclusions, identifying clean, aggregated and degraded products - but MDS testing was able to provide these insights much faster, in just 8 minutes per test vs the 50 minutes per test of SEC. In addition, the MDS test used significantly less sample (just 2.5 µg vs 40 µg for SEC per test in this instance – though MDS analysis can be performed with as little as 50 ng).
The complexity of setting up SEC analysis should also be noted – with column equilibration, buffer degassing, and experimental parameter setting required before the test begins. Further, SEC does not provide an absolute size – only elution time. Either a column calibration or running known samples on the same column is needed to obtain absolute size.
By contrast MDS tests simply require pipetting of the sample onto a chip, then selection of the run setting from 3 pre-set options via the integrated touchscreen. After this the hydrodynamic radius is reported directly on screen in 8 minutes.
Overall MDS presented results in 80% less time and using 90% less sample than the SEC analysis.
For rapid accept/reject testing of antibodies, MDS testing on Fluidity One provides a far simpler and faster method with significantly lower sample consumption than SEC.