Bioanalytical Community

Hi,
I was wondering if anyone has any thoughts on labeling of oligonucleotides to be used in assays for the detection of anti-drug antibodies? Here are some examples:

• For a labeled oligonucleotide, is there a preference of where the label should be such as 5' and/or internal modifications.
• What spacers could be used for labeling? 
  • How does the oligonucleotide format impact this decision? 
• Are there other types of labeling such as ruthenylation, DIG, etc. that can be problematic to incorporate during synthesis or post-synthesis.
• What level of purity of a reagent oligonucleotide is required?

I appreciate any thoughts or comments.
Best,
Michael Swanson

------------------------------
Michael Swanson
Spring House PA
[email protected]

Disclaimer: Opinions expressed are solely my own and do not express the views or opinions of my employer.
------------------------------

Hi Michael,

                AAPS has a discussion group on bioanalytical methods for immunogenicity of oligonucleotides (I can put you in touch with the right people if you want to join 😉) and it seems that nobody is using bridging format for ADA against oligos analogous to that used for protein drugs. Conjugating oligos with commercially available reagents (sulfo-Tag-NHS, biotin-LC-sulfo-NHS, etc.) is not straightforward due to limited number of reactive sites and relatively small size of oligonucleotides compared to the label. Attempts to label oligos were made, but the resulting assays were not very good and had to be scrapped. It doesn't mean that it can't be done. You would have to synthesize your oligonucleotide with the label attached to it or with a linker that could be labeled (e.g. a polylysine tail). You could let your imagination run wild and try all kinds of ideas, but the general consensus seems to be that the old-fashioned direct format works fine for detection of ADA to oligos: synthesize your oligo modified with NH₂ or SH at the 3' end, capture the oligo on an amino-reactive ELISA plate, add your sample and detect ADA with protein A/G or anti-human IgG conjugated with HRP. Main problem with these assays is their poor sensitivity, but this seems to be caused by difficulties in generating a decent positive control. Most oligos are not very immunogenic so trying to immunize animals often leads to rather low titers with low affinity. Bridging format typically has very good drug tolerance, but you don't really need that for oligo drugs which tend to have very low concentrations at trough when immunogenicity samples are collected.

In short, I'm very curious if anybody had any luck labeling oligos and how they did it, but if you want a cheap and easy ADA assay for oligos, you can't go wrong with the direct format.

------------------------------
Robert J. Kubiak, PhD
Director, Head of Bioanalytical Science
Third Arc Bio
[email protected]

Disclaimer: Opinions expressed are solely my own and do not express the views or opinions of my employer.
------------------------------

Original Message:
Sent: 02-04-2025 08:54
From: Michael Swanson
Subject: labeled oligonucleotides for immunogenicity assessment

Hi,
I was wondering if anyone has any thoughts on labeling of oligonucleotides to be used in assays for the detection of anti-drug antibodies? Here are some examples:

• For a labeled oligonucleotide, is there a preference of where the label should be such as 5' and/or internal modifications.
• What spacers could be used for labeling? 
  • How does the oligonucleotide format impact this decision? 
• Are there other types of labeling such as ruthenylation, DIG, etc. that can be problematic to incorporate during synthesis or post-synthesis.
• What level of purity of a reagent oligonucleotide is required?

I appreciate any thoughts or comments.
Best,
Michael Swanson

------------------------------
Michael Swanson
Spring House PA
[email protected]

Disclaimer: Opinions expressed are solely my own and do not express the views or opinions of my employer.
------------------------------

Hi Michael,

Echoing Robert's experience here.  In my experience supporting a large number of oligo programs over the years, we have ruled out feasibility of the bridging assay format (not due to labeling but due to relative size constraints and presumed steric hindrance) so have always employed a direct format. 

I can't help but add that we additionally have never observed clinically meaningful responses, although our sensitive assays could detect 'positives'.  So, a robust risk assessment is advised before investing heavily.  I refer you to the editorial by Chris Stebbins on this topic 

Stebbins, CC, Petrillo M, Stevenson LF, Immunogenicity for antisense oligonucleotides: a risk-based assessment, Bioanalysis, 2019 Nov; 11(21): 1913-1916.

Additionally, Chris will be presenting on this at NBC, so that will be a great place to continue the discussion.

------------------------------
Lauren Stevenson Ph.D.
Chief Scientific Officer
Immunologix Laboratories
Tampa FL
[email protected]

Disclaimer: Opinions expressed are solely my own and do not express the views or opinions of my employer.
------------------------------

Original Message:
Sent: 02-05-2025 13:46
From: Robert Kubiak
Subject: labeled oligonucleotides for immunogenicity assessment

Hi Michael,

                AAPS has a discussion group on bioanalytical methods for immunogenicity of oligonucleotides (I can put you in touch with the right people if you want to join 😉) and it seems that nobody is using bridging format for ADA against oligos analogous to that used for protein drugs. Conjugating oligos with commercially available reagents (sulfo-Tag-NHS, biotin-LC-sulfo-NHS, etc.) is not straightforward due to limited number of reactive sites and relatively small size of oligonucleotides compared to the label. Attempts to label oligos were made, but the resulting assays were not very good and had to be scrapped. It doesn't mean that it can't be done. You would have to synthesize your oligonucleotide with the label attached to it or with a linker that could be labeled (e.g. a polylysine tail). You could let your imagination run wild and try all kinds of ideas, but the general consensus seems to be that the old-fashioned direct format works fine for detection of ADA to oligos: synthesize your oligo modified with NH₂ or SH at the 3' end, capture the oligo on an amino-reactive ELISA plate, add your sample and detect ADA with protein A/G or anti-human IgG conjugated with HRP. Main problem with these assays is their poor sensitivity, but this seems to be caused by difficulties in generating a decent positive control. Most oligos are not very immunogenic so trying to immunize animals often leads to rather low titers with low affinity. Bridging format typically has very good drug tolerance, but you don't really need that for oligo drugs which tend to have very low concentrations at trough when immunogenicity samples are collected.

In short, I'm very curious if anybody had any luck labeling oligos and how they did it, but if you want a cheap and easy ADA assay for oligos, you can't go wrong with the direct format.

------------------------------
Robert J. Kubiak, PhD
Director, Head of Bioanalytical Science
Third Arc Bio
[email protected]

Disclaimer: Opinions expressed are solely my own and do not express the views or opinions of my employer.

Original Message:
Sent: 02-04-2025 08:54
From: Michael Swanson
Subject: labeled oligonucleotides for immunogenicity assessment

Hi,
I was wondering if anyone has any thoughts on labeling of oligonucleotides to be used in assays for the detection of anti-drug antibodies? Here are some examples:

• For a labeled oligonucleotide, is there a preference of where the label should be such as 5' and/or internal modifications.
• What spacers could be used for labeling? 
  • How does the oligonucleotide format impact this decision? 
• Are there other types of labeling such as ruthenylation, DIG, etc. that can be problematic to incorporate during synthesis or post-synthesis.
• What level of purity of a reagent oligonucleotide is required?

I appreciate any thoughts or comments.
Best,
Michael Swanson

------------------------------
Michael Swanson
Spring House PA
[email protected]

Disclaimer: Opinions expressed are solely my own and do not express the views or opinions of my employer.
------------------------------