Journal: International Journal of Pharmaceutics: X

Article Title: Roadmap for the accelerated development and clinical translation of fluorescent tracers: Adalimumab-680LT as a proof of concept

doi: 10.1016/j.ijpx.2026.100514

Figure Lengend Snippet: Results for the feasibility testing and lab development of adalimumab-680LT . SE-HPLC chromatograms of (A) unmodified adalimumab and (B) adalimumab-680LT at 280 nm, (C) chromatogram of adalimumab-680LT and free IRDye 680LT at 676 nm and (D) results of the indirect ELISA of unmodified adalimumab and adalimumab-680LT.

Article Snippet: Briefly, to remove excipients in the solution and to optimise the pH for labelling, adalimumab registered product (Humira®, AbbVie) was buffer exchanged to a 50 mM sodium phosphate buffer pH 8.5 (Apotheek A15, Gorinchem, the Netherlands) using pre-equilibrated PD-10 columns (Cytiva lifesciences, Chicago, IL, USA). cGMP grade IRDye 680LT NHS-ester (LI-COR Biosciences, Lincoln, NE, USA), dissolved in dimethyl sulfoxide (DMSO) (Sigma Aldrich, Darmstadt, Germany) 5 mg/mL, was added to the adalimumab solution in a molar dye-to-protein ratio of 2:1.

Techniques: Indirect ELISA

Journal: International Journal of Pharmaceutics: X

Article Title: Roadmap for the accelerated development and clinical translation of fluorescent tracers: Adalimumab-680LT as a proof of concept

doi: 10.1016/j.ijpx.2026.100514

Figure Lengend Snippet: Stability results of adalimumab-680LT. Release specifications are displayed with dotted lines, and end of shelf-life specifications are displayed with dashed lines. (A) Protein concentration, (B) percentage of free dye, (C) percentage of aggregates, and (D) target binding affinity of lab run 1 and 2 were tested during 3 months. For the technology transfer batch, (E) protein concentration, (F) percentage of free dye, (G) percentage of aggregates and (H) target binding affinity were tested at two different temperatures during 18 or 24 months. The stability study of the technology transfer batch at 2–8 °C is still ongoing. (A-C) are means of two different measurements, ( E -G) are means + standard deviations of three different measurements, and (D and H) are means of two measurements.

Article Snippet: Briefly, to remove excipients in the solution and to optimise the pH for labelling, adalimumab registered product (Humira®, AbbVie) was buffer exchanged to a 50 mM sodium phosphate buffer pH 8.5 (Apotheek A15, Gorinchem, the Netherlands) using pre-equilibrated PD-10 columns (Cytiva lifesciences, Chicago, IL, USA). cGMP grade IRDye 680LT NHS-ester (LI-COR Biosciences, Lincoln, NE, USA), dissolved in dimethyl sulfoxide (DMSO) (Sigma Aldrich, Darmstadt, Germany) 5 mg/mL, was added to the adalimumab solution in a molar dye-to-protein ratio of 2:1.

Techniques: Protein Concentration, Binding Assay

Journal: bioRxiv

Article Title: Thousandfold Expansion Microscopy

doi: 10.64898/2026.05.31.729018

Figure Lengend Snippet: (A) Schematic of four-round ionic-in-ionic casting sequence used to construct the interpenetrating charged hydrogel architecture. The recursive casting process yields ∼1000× linear (∼10⁹ volumetric) expansion and enables separation of biomolecular building blocks anchored within the gel matrix following macromolecule bond cleavage, including (but not limited to) individual amino acid residues within proteins. (i) Biomolecules are functionalized with polymer-incorporable groups to enable covalent anchoring to the hydrogel network. For proteins, primary amines can be modified with acryloyl-X, SE (AcX) to install polymerizable acrylate groups. (ii) Modified biomolecules are embedded in a swellable charged hydrogel network generated by reacting sodium acrylate (25.60% w/v), N,N-dimethylacrylamide (DMAA, 44.20% v/v), N,N,N′,N′-tetramethylethylenediamine (TEMED, 0.037% v/v), and potassium persulfate (KPS, 0.145% w/v). (iii) Anchored biomolecules are fragmented by cleavage between anchoring points. For proteins, proteinase K (8 U/mL) generates anchored peptide fragments. The gel is expanded in water to ∼18× linear expansion, separating covalently anchored biomolecular fragments (e.g., peptides, individual amino acid residues, or other subunits), depending on the anchoring and fragmentation strategy. (iv) The fully expanded ∼18× gel is infiltrated with a charged monomer solution and polymerized, yielding a composite gel that contracts to ∼10× linear expansion. (v) The composite gel is expanded in water to ∼100× linear expansion. (vi) The fully expanded ∼100× gel is infiltrated with a charged monomer solution and polymerized, yielding a composite gel that contracts to ∼50× linear expansion. (vii) The composite gel is expanded in water to ∼500× linear expansion. (viii) The fully expanded ∼500× gel is infiltrated with a charged monomer solution and polymerized, yielding a composite gel that contracts to ∼250× linear expansion. (ix) The composite gel is expanded in water to ∼1500× linear expansion. (x) Gel-anchored biomolecular fragments are labeled through reactive functional groups generated during fragmentation. For proteins, primary amines exposed by proteinase K digestion can be labeled with NHS-ester fluorophores. The gel is then equilibrated in 1× DPBS, contracting to ∼1000× linear expansion, which stabilizes the sample and enables stable imaging over extended durations. (B) Physical expansion factors measured after full expansion at each round of gelation, shown as boxplots: (i) ∼18× round 1 gelation (n = 11 separate gelations), (ii) ∼100× round 2 gelation (n = 9 separate gelations), (iii) ∼500× round 3 gelation (n = 7 separate gelations), (iv) ∼1000× round 4 gelation (n = 4 separate gelations). Boxplots in this figure: the center line denotes the median, the box spans the interquartile range (IQR), whiskers extend to the farthest data point within 1.5×IQR of each box edge, and individual measurements are shown as open circles. Expansion proceeded through four sequential rounds of gelation and expansion, with a subset of the gels that made it through round N, going through round N+1 (simply for convenience); for a set of gels that were all advanced in parallel, using a slightly modified protocol (two monomer incubations per round), see Supp Fig. 31.

Article Snippet: Gels were post-expansion labeled with NHS-ester fluorescein (46409, Thermo Fisher Scientific).

Techniques: Sequencing, Construct, Polymer, Modification, Generated, Labeling, Functional Assay, Imaging

Journal: bioRxiv

Article Title: Thousandfold Expansion Microscopy

doi: 10.64898/2026.05.31.729018

Figure Lengend Snippet: Gray boxes indicate reactions performed inside the expansion hydrogel; all other steps were performed in solution. A. Chemical structure of the mCLING peptide. B.i NHS-acrylate (Acryloyl-X) reacts with primary amines via NHS ester–amine coupling. B.ii All primary amines on the mCLING peptide, ideally, are functionalized with polymerizable acrylate groups. C. Hydrogel polymerization (gray box). Acrylate-functionalized amines (abbreviated A) are covalently attached to the gel network, anchoring the peptide at the labeled sites. D. Proteinase K digestion in which the amide bond between the anchoring site and Atto647N is cleaved. All peptide backbone amide bonds are digested. Fragments lacking an anchoring site are removed during expansion. Because Atto647N is not anchored, that dye is lost. E. Proteinase K digestion in which the amide bond between the anchoring site and Atto647N is not cleaved. Atto647N remains attached to an anchored fragment and is therefore retained and displaced during expansion according to the anchoring atom. Cleavage of this bond is unlikely due to steric constraints of the Atto647N substituent in the Proteinase K substrate recognition site . F. NHS-ester fluorescein labeling of remaining free primary amines. The blue sphere denotes a primary amine that has been conjugated with fluorescein. In this panel, the full chemical conjugation product is shown. G. I–III. Labeling outcomes for case D (Atto647N cleaved and removed). In G and H, the blue sphere is used as a symbolic shorthand indicating an NH₂ group conjugated with fluorescein. G.I Both the N-terminal amine and the lysine side-chain amine are anchored. No free primary amines remain; no fluorescein labeling occurs. G.II Only the lysine side-chain amine is anchored. The free N-terminal amine is fluorescein-labeled (blue sphere). Upon expansion, the dye is displaced to the position of the lysine anchoring atom. G.III Only the N-terminal amine is anchored. The lysine side-chain amine is fluorescein-labeled (blue sphere). The dye is displaced to the position of the N-terminal anchoring atom. H. I–III. Labeling outcomes for case E (Atto647N retained). Configurations are the same as G.I–III, except that Atto647N remains attached and is displaced according to its anchoring site.

Article Snippet: Gels were post-expansion labeled with NHS-ester fluorescein (46409, Thermo Fisher Scientific).

Techniques: Labeling, Conjugation Assay

Journal: bioRxiv

Article Title: Thousandfold Expansion Microscopy

doi: 10.64898/2026.05.31.729018

Figure Lengend Snippet: Primary amines were functionalized with NHS–ester acrylate to covalently anchor the nitrogen atom to the polymer network. In the schematic, the nitrogen of the first primary amine bearing the Atto647N dye is shown as an orange sphere; adjacent lysine ε-amine nitrogens are shown as blue spheres. These nitrogen atoms are attached to the hydrogel and displaced during expansion. Thus, the measured positions of Atto647N and fluorescein correspond to the displaced coordinates of their respective anchoring nitrogens. Inter-lysine distances were quantified by molecular dynamics. (A) Distribution of distances (nm). (B) Minimum distance (∼0.44 nm), parallel side chains. (C) Peak distance (∼0.88 nm), staggered orientation. (D) Larger distance (∼1.41 nm), antiparallel orientation.

Article Snippet: Gels were post-expansion labeled with NHS-ester fluorescein (46409, Thermo Fisher Scientific).

Techniques: Polymer