Hi All,
I am trying to use DLS to verify that I am successfully reducing disulfide bonds responsible for the accumulation of mAbs in solution, yielding single uncoupled antibodies.
Has anybody done this successfully? I feel like the small amounts of mAbs that I have been using have been causing the strange, unreliable peaks I am seeing. Has anyone gotten away with using a small number of mAbs? Also, is the "Protein" setting on the Malvern DLS helpful for this?
Thank you!
EF
I think you would be better off using size-exclusion chromatography. DLS can be overly sensitive to minor amounts of aggregated protein.
Why would that be, I figured their algorithms accounted for the much larger (diameter to the 6th power) signals that larger particles yielded? Why would the percent composition results they give be inaccurate?
Thanks,
EF
I don't think these algorithms can accurately deal with the enormous difference (as you say, 6th power) between the light scattering signals of aggregates and monomers or small oligomers. I wouldn't trust it for a quantitative analysis of the sort you are trying. On the other hand, combining size exclusion chromatography with in-line multiangle light scattering (SEC-MALS) is a very powerful way to analyze the size distribution of a sample.
The Malvern Zetasizer performs two different analysis methods on DLS correlation data, Cumulants which and NNLS size distribution.
DLS is sensitive to aggregates as you say, and the values of ZAve and PdI from Cumulants will be highly skewed for an aggregated sample, the size distribution analysis should still report an appropriate size for the monomer, along with sizes of the aggregates.
The protein settings within the Zetasizer software perform a measurement with a longer correlation time in order to produce optimum results from low scattering samples, and also uses a higher resolution analysis method in determining the size distribution.
DLS may be able to resolve the mAb peak if there is enough scattering signal from that contribution. If there is more than 99% of the scattered light originating from larger aggregates, then the regularization algorithm will not resolve the contribution from the smaller size peak. However if there is significant signal from the small size then there are three different regularization schemes, and the Protein Analysis you mentioned could potentially be of use. The General Purpose should be the default mode to choose, a comparison is available from the Malvern site, links below.
As a first step centrifugation of filtration may be useful to improve the DLS result. Chromatography would be able to provide the highest resolution, either SEC-DLS or SEC-LS (for antibodies, while multi-angle light scattering MALS would work, a single angle should be sufficient to determine molecular weight).
Finally, the protein concentration should be appropriate to obtain enough signal from the sample, for antibodies this should be at least 0.01 mg/mL, and higher concentration will be easier due to scattering intensity.
http://www.malvern.com/en/support/resource-center/technical-notes/TN101124ZetasizerNanoAnalysisMethodsExplained.aspx
http://www.materials-talks.com/blog/2016/01/21/zetasizer-sensitivity-for-protein-dls/
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