It sounds like you need to prepare a series of conjugates with different sizes of PEG and do a full molecular and kinetic evaluation (on rates, off-rate, affinity). So you will need equipment like a Biacore or Octet.

The prep of conjugates, however, is not necessarily straightforward, as you are trying to examine the impact of a single parameter (PEG size). You therefore also need to know the degree of labelling for each conjugate, and the DoL will be the SAME (or very similar) irrespective of PEG size. Unfortunately, the size of the PEG may influence DoL during conjugate production, potentially making data interpretation difficult. You will therefore probably need to aim for a relatively low DoL otherwise you will struggle to achieve the same DoL with both large and small PEGs, because of steric factors.

How easy/difficult it will be to maintain constant DoL will also depend to some extent on your method of conjugation. However, you could prepare a few conjugates with each size of PEG and select conjugates with the same/similar DoL for further detailed kinetic assessments.

This brings me onto how you assess DoL - not easy with PEG unless you have an inbuilt fluorescent reporter. However, SDS PAGE, though qualitative, can certainly rule out grossly overlabelled or underlabelled samples easily enough. If you have the equipment, mass spec is a more sophisticated way of assessing molecular weight of the conjugates/DoL.

Nick Gee, thanks for your answer. Is there any tool I can use to first do a molecular simulation before going ahead to prepare different PEG conjugates? That may give me an idea of what to expect.

Sorry, molecular simulation is not my area of knowledge but hopefully someone else can advise.

Nick Gee Thank you