Sydnone-based prosthetic groups for radioiodination.

The potential of Strained-Promoted Sydnone-Alkyne Cycloaddition (SPSAC) for radioiodination was evaluated with model cyclooctyne-conjugated peptides. Starting with a series of sydnones with varying N3 and C4 substitution, a preliminary kinetic study with non-radioactive iodinated compounds highlighted the superiority of an arylsydnone substituted by a chlorine atom in C4 position. Interestingly, reaction rate up to 11 times higher than using an azide was achieved with the best system. Access to 125I-labelled sydnones was granted with high efficiency from arylboronic acid precursors by copper catalyzed nucleophilic substitution. Application of SPSAC on the model peptide in radiotracer conditions showed the same trend than in non-radioactive kinetic study and complete reactions could be achieved within less than an hour for the best systems. These results are favorable for use in the production of radiopharmaceuticals with heavy halogens and increase the diversity of available bioorthogonal reaction for nuclear imaging and therapy.

Preliminary Research of Radiolabeled Atezolizumab for the Noninvasive Evaluation of TNBC PD-L1 Expression In Vivo.

Programmed death-ligand 1 (PD-L1) expression is related to the efficacy and prognosis in triple-negative breast cancer. This study employed an indirect labeling method to synthesize [125I]PI-Atezolizumab. The in vitro stability of [125I]PI-Atezolizumab was assessed through incubation in phosphate buffered saline and fetal bovine serum, revealing sustained stability. Specific binding of [125I]PI-Atezolizumab to MDA-MB-231 cells expressing humanized PD-L1 was assessed through in vitro incubation, yielding a Kd value comparable to that of Atezolizumab. This suggests that the labeling process did not compromise the affinity of the Atezolizumab to PD-L1. Subsequently, pharmacokinetic studies were conducted in normal mice and biodistribution experiments in tumor-bearing mice. A comparison of the biodistribution results between [125I]PI-Atezolizumab and 125I-labeled Atezolizumab indicated better in vivo stability for the former. Single photon emission computed tomography (SPECT)/CT imaging further confirmed the targeted specificity of [125I]PI-Atezolizumab for PD-L1 in MDA-MB-231 xenografts, which were validated by immunohistochemistry staining. This research underscores the utility of [125I]PI-Atezolizumab, prepared via indirect labeling, for monitoring PD-L1 in triple-negative breast cancer models.

A practical protocol for large-scale copper-mediated radioiodination of organoboronic precursors: Radiosynthesis of [123 I]KX-1 for Auger radiotherapy.

Nucleophilic copper-mediated radioiodination (CMRI) of organoboronic precursors with radioiodides is a promising method of radioiodination. The previously reported CMRI has demonstrated its great potential and scope of labeling for the radiosynthesis of radioiodine-labeled compounds. However, the reported protocols (using a small amount/volume of radioactivity) are practically not reproducible in large-scale CMRI, in which the radioactivity was usually provided in a bulk alkaline solution. A large amount of water and a strong base are incompatible with CMRI. To overcome these issues in large-scale CMRI, we have developed a simple protocol for large-scale CMRI. The bulk water was removed under a flow of inert gas at 110°C, and the strong base (i.e., NaOH) was neutralized with an acid, pyridinium p-toluenesulfonate or p-toluenesulfonic acid. In the model reactions of [123 I]KX-1, a PARP-1 radioligand for Auger radiotherapy, radiochemical conversions were significantly improved after neutralization of the base, and the addition of additional acids was tolerated and favorable for the reactions. Using this protocol, [123 I]KX-1 was radiosynthesized from 20 mCi (0.74 GBq) of [123 I]iodide in high radiochemical yields, high radiochemical purity, and high molar activity. This protocol should be applicable to the radiosynthesis of other compounds with radioiodine via CMRI.

Radioiodination, purification, and evaluation of antihuman tumor-derived immunoglobulin G light chain monoclonal antibody in tumor-bearing nude mice.

We report the synthesis and biological evaluation of 131 I-labeled antihuman tumor-derived immunoglobulin G (IgG) light chain monoclonal antibody (4E9) ([131 I]I-4E9) as a promising probe for tumor imaging. [131 I]I-4E9 was synthesized in radiochemical yield of 89.9 ± 4.7% with radiochemical purity of more than 99%. [131 I]I-4E9 showed high stability in normal saline and human serum. In cell uptake studies, [131 I]I-4E9 exhibited favorable binding affinity and high specificity in HeLa MR cells. In biodistribution studies, [131 I]I-4E9 showed high tumor uptake, high tumor/non-tumor ratios, and specific binding in BALB/c nu/nu mice bearing human HeLa MR xenografts. Single-photon emission computerized tomography (SPECT) imaging of [131 I]I-4E9 in the HeLa MR xenograft model demonstrated clear visualization of tumor after 48 h and confirmed specific binding in tumor. These findings suggest that [131 I]I-4E9 possesses favorable biological characteristics and warrants further investigation as a prospective probe for imaging and treatment of cancers.

Copper-mediated radioiodination and radiobromination via aryl boronic precursor and its application to 125I/77Br-labeled prostate-specific membrane antigen imaging probes.

Prostate-specific membrane antigen (PSMA), expressed in prostate cancer cells, is being investigated extensively worldwide as a target for imaging and therapy of prostate cancer. Various radioiodinated PSMA imaging probes have been developed, and their structure has a peptidomimetic urea-based skeleton as a pharmacophore. For direct radioiodination of molecules containing these peptidomimetic structures, prior studies performed radioiododestannylation or electrophilic radioiodination of tyrosine residues. However, although these radiolabeling methods are frequently used, there are some issues with precursor toxicity and by-product production. Therefore, it is required to investigate a radiolabeling method that can be used for the radiosynthesis of radioiodinated PSMA imaging probes with urea-based peptidomimetic structures. We recently reported that copper-mediated radioiodination via a boronic precursor is an effective method for directly labeling a peptide. This radiohalogenation method was expected to be an effective method for radiosynthesis of PSMA imaging probes with a peptidomimetic structure. In this study, to confirm that this labeling method applies to the synthesis of the PSMA imaging probe, we synthesized PSMA imaging probes labeled with 125I and 77Br ([125I]mIB-PS and [77Br]mBrB-PS) using a copper-mediated radiohalogenation via common boronic precursors and investigated optimal boronic precursor and labeling conditions. As a result, the radiochemical yields of [125I]mIB-PS and [77Br]mBrB-PS were improved to > 93% at room temperature by optimizing the structure of the boronic precursor. We demonstrate that copper-mediated nucleophilic radiochemistry using a boronic precursor is a promising radiosynthetic method of PSMA imaging probes. Although we focused on the synthesis of PSMA imaging probes, the results in this study will also be useful for the synthesis of various radioiodine or radiobromine-labeled bioactive molecules.

Highly effective liquid and solid phase extraction methods to concentrate radioiodine isotopes for radioiodination chemistry.

Radioactive iodine isotopes play a pivotal role in radiopharmaceuticals. Large-scale production of multi-patient dose of radioiodinated nuclear medicines requires high concentration of radioiodine. We demonstrate that tetrabutylammonium chloride and methyltrioctylamonium chloride are effective phase transfer reagents to concentrate iodide-124, iodide-125 and iodide-131 from the corresponding commercial water solutions. The resulting concentrated radioiodide, in the presence of either phase transfer reagent, does not hamper the chemical reactivity of aqueous radioiodide in the copper (II)-mediated one-pot three-component click chemistry to produce radioiodinated iodotriazoles.

Radioiodination of balsalazide, bioevaluation, and characterization as a highly selective radiotracer for imaging of ulcerative colitis in mice.

This work focuses on tracking ulcerative colitis in mice. High labeling yield and radiochemical purity were achieved for the formation of a [125/131 I]balsalazide radiotracer at optimum conditions of oxidizing agent content (chloramines-T [Ch-T], 75 µg), substrate amount (100 µg), pH of reaction mixture (6), reaction time (30 min), and temperature (37°C), using radioactive iodine-125 (200-450 MBq). The radiolabeled compound, [125/131 I]balsalazide, was stable in serum and saline solution during 24 h. Balsalazide is acting as a peroxisome proliferator-activated receptor (PPARγ). Biodistribution studies were carried in normal and ulcerated colon mice. High uptake of 75 ± 1.90% injected dose/g organ (ID/g) observed in ulcerated mice confirmed the suitability of [131 I]balsalazide as a novel radiotracer for ulcerative colitis imaging in mice.

Site-Specific Radioiodination of Oligonucleotides with a Phenolic Element in a Programmable Approach.

Radioiodination of oligonucleotides provides an extra modality for nucleic acid-based theranostics with potential applications. Herein, we report the design and synthesis of a phosphoramidite embedded with a phenolic moiety and demonstrate that oligonucleotides can be readily functionalized with phenol as a precursor by general DNA synthesis. It was identified that the introduction of the precursor does not block the specificity of an aptamer, and the radioiodination is applicable to both DNA and RNA oligonucleotides in a site-specific approach with a commercial kit.

Novel Radioiodinated and Radiofluorinated Analogues of FT-2102 for SPECT or PET Imaging of mIDH1 Mutant Tumours.

Isocitrate dehydrogenases (IDHs) are metabolic enzymes commonly mutated in human cancers (glioma, acute myeloid leukaemia, chondrosarcoma, and intrahepatic cholangiocarcinoma). These mutated variants of IDH (mIDH) acquire a neomorphic activity, namely, conversion of α-ketoglutarate to the oncometabolite D-2-hydroxyglutarate involved in tumourigenesis. Thus, mIDHs have emerged as highly promising therapeutic targets, and several mIDH specific inhibitors have been developed. However, the evaluation of mIDH status, currently performed by biopsy, is essential for patient stratification and thus treatment and follow-up. We report herein the development of new radioiodinated and radiofluorinated analogues of olutasidenib (FT-2102) as tools for noninvasive single photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging of mIDH1 up- and dysregulation in tumours. Nonradiolabelled derivatives 2 and 3 halogenated at position 6 of the quinolinone scaffold were synthesised and tested in vitro for their inhibitory potencies and selectivities in comparison with the lead compound FT-2102. Using a common organotin precursor, (S)-[125I]2 and (S)-[18F]3 were efficiently synthesised by radio-iododemetallation and copper-mediated radiofluorination, respectively. Both radiotracers were stable at room temperature in saline or DPBS solution and at 37 °C in mouse serum, allowing future planning of their in vitro and in vivo evaluations in glioma and chondrosarcoma models.

Preparation and biological evaluation of radioiodine-labeled triphenylphosphine derivatives as mitochondrial targeting probes.

The positive-charged lipophilic triphenylphosphonium cations (TPPs+ ) have been served as mitochondrial targeting vehicles for the delivery of various probes. In this study, we developed a new method for the preparation of radioiodine-labeled TPPs+ . Four 125 I-labeled TPPs+ , [125 I] 9-[125 I] 12, were prepared from the corresponding triphenylphosphine phenylborate precursors of B 5-B 8 via an optimized copper-catalyzed one-step procedure in high radiochemical yield (>95%). After radio-HPLC purification, the final products could be obtained with high specific activity. Their physicochemical properties, in vitro cellular uptake, and ex vivo mice biodistribution were investigated. The results suggested the 125 I-labeled TPPs+ were lipophilic and could specifically accumulate in the mitochondrial-rich myocardial cells through the mitochondrial membrane potential.

Copper-mediated radioiodination reaction through aryl boronic acid or ester precursor and its application to direct radiolabeling of a cyclic peptide.

A copper-mediated radioiodination using aryl boronic precursors is attracting attention as a solution to oxidative iododestannylation and nickel-mediated radioiodination drawbacks. The copper-mediated radiolabeling method allows radioiodination at room temperature with stable aryl boronic precursors without preparing complex starting materials or reagents and can be performed in a reaction vessel exposed to air. This method has good potential in radiochemistry; however, studies on the scope of copper-mediated radioiodination through boronic precursors are insufficient. In particular, few reports have demonstrated the effect of protecting groups on radiolabeling efficiency. Therefore, the effect of the protecting group of aryl boronic acids on the copper-mediated radioiodination was investigated. In addition, this method, which does not require heating, is expected to be useful for direct radiolabeling of peptides. Thus, we attempted direct radioiodination of c(RGDyk) as an example. The resulting radioiodination method was well tolerated in various substrates and was unaffected by the pinacol ester-type protecting group. Also, c(RGDyk) was labeled with 125 I via copper-mediated radioiodination using an aryl boronic acid precursor. The reaction time and yield were improved, compared with the indirect method. Furthermore, the large difference in polarity between the boronic acid precursor and the radiolabeled compound facilitated purification.

Discovery of New Coumarin-Based Lead with Potential Anticancer, CDK4 Inhibition and Selective Radiotheranostic Effect: Synthesis, 2D & 3D QSAR, Molecular Dynamics, In Vitro Cytotoxicity, Radioiodination, and Biodistribution Studies.

Novel 6-bromo-coumarin-ethylidene-hydrazonyl-thiazolyl and 6-bromo-coumarin-thiazolyl-based derivatives were synthesized. A quantitative structure activity relationship (QSAR) model with high predictive power r2 = 0.92, and RMSE = 0.44 predicted five compounds; 2b, 3b, 5a, 9a and 9i to have potential anticancer activities. Compound 2b achieved the best ΔG of -15.34 kcal/mol with an affinity of 40.05 pki. In a molecular dynamic study 2b showed an equilibrium at 0.8 Å after 3.5 ns, while flavopiridol did so at 0.5 Å after the same time (3.5 ns). 2b showed an IC50 of 0.0136 µM, 0.015 µM, and 0.054 µM against MCF-7, A-549, and CHO-K1 cell lines, respectively. The CDK4 enzyme assay revealed the significant CDK4 inhibitory activity of compound 2b with IC50 of 0.036 µM. The selectivity of the newly discovered lead compound 2b toward localization in tumor cells was confirmed by a radioiodination biological assay that was done via electrophilic substitution reaction utilizing the oxidative effect of chloramine-t. 131I-2b showed good in vitro stability up to 4 h. In solid tumor bearing mice, the values of tumor uptake reached a height of 5.97 ± 0.82%ID/g at 60 min p.i. 131I-2b can be considered as a selective radiotheranostic agent for solid tumors with promising anticancer activity.

A Novel Reagent for Radioiodine Labeling of New Chemical Entities (NCEs) and Biomolecules.

Radioiodine labeling of peptides and proteins is routinely performed by using various oxidizing agents such as Chloramine T, Iodobeads, and Iodogen reagent and radioactive iodide (I-), although some other oxidizing agents were also investigated. The main objective of the present study was to develop and test a novel reagent, inorganic monochloramine (NH2Cl), for radioiodine labeling of new chemical entities and biomolecules which is cost-effective, easy to make and handle, and is selective to label amino acids, peptides, and proteins. The data presented in this report demonstrate that the yields of the non-radioactive iodine labeling reactions using monochloramine are >70% for an amino acid (tyrosine) and a cyclic peptide (cyclo Arg-Gly-Asp-d-Tyr-Lys, cRGDyK). No evidence of the formation of N-chloro derivatives in cRGDyK was observed, suggesting that the reagent is selective in iodinating the tyrosine residue in the biomolecules. The method was successfully translated into radioiodine labeling of amino acid, a peptide, and a protein, Bovine Serum Albumin (BSA).

Synthesis and bioevaluation of radioiodinated nitroimidazole hypoxia imaging agents by one-pot click reaction.

Eight radioiodinated 2-nitroimidazole derivatives for use as hypoxia imaging agents were synthesized by one-pot click reaction using four azides, two alkynes, and [131I]iodide ions and evaluated by hypoxic cellular uptake and biodistribution experiments. The results suggested that radiotracers with suitable partition coefficients (log P: -0.2-1.2) were more likely to have higher hypoxic cellular uptake. Among these eight molecules, [131I]15 ([131I]-(5-iodo-1-(2-(2-(2-nitro-1H-imidazol-1-yl)ethoxy)ethyl)-4-((2-nitro-1H-imidazol-1-yl)methyl)-1H-1,2,3-triazole)) had a suitable log P (0.05 ± 0.03) and contained two 2-nitroimidazole groups. The hypoxic/aerobic cellular uptake ratio of [131I]15 was 4.4 ± 0.5, and the tumor/blood (T/B) and tumor/muscle (T/M) ratios were 2.03 ± 0.45 and 6.82 ± 1.70, respectively. These results suggested that [131I]15 was a potential hypoxia imaging agent.

Design of iodinated radioligands for SPECT imaging of central human 5-HT4R using a ligand lipophilicity efficiency approach.

A series of iodinated ligands for the SPECT imaging of 5-HT4 receptors was designed starting from the previously reported hit MR-26132. We focused on the modulation of the piperidine-containing lateral chain by introducing hydrophilic groups in order to decrease the liphophilicity of the new ligands. All the synthesized compounds were tested for their binding affinities on 5-HT4Rs and based on the Ligand Lipophilicity Efficiency approach, compound 13 was further selected for radioiodination with iodine-125 and imaging experiments. Compound 13 showed its ability to displace the specific signal of the reference compound [125I]SB-207710 but no significant detection of [125I]13 was observed in vivo in SPECT experiments.

"Tau Protein Targeting Via Radioiodinated Azure A For Brain Theranostics: Radiolabeling, Molecular Docking, in vitro And in vivo Biological Evaluation".

Azure-A is one of the phenothiazines (PTZs) derivatives which for decades have been used as antipsychotic drugs due to good lipophilic characteristics which enable them to pass through the blood brain barrier (BBB), besides the important property of enabeling investigation of the pathological forms of aggregated tau protein found in the neurons of the central nervous system. Radioiodination of Azure-A was carried out via an electrophilic substitution reaction using chloramine-T as oxidizing agent. The influence of various reaction parameters and conditions on radioiodination efficiency was investigated, and a high radiochemical yield of 92.07 ± 0.9 % was obtained. An in vitro cytotoxicity study of iodinated Azure-A on three cell lines (HCT-116, human colon carcinoma cell line; Hep-G2, liver carcinoma cell line and HFB-4, normal human melanocytes) was carried out, and the data revealed that ioiodinated Azure A has no to very low toxic effect. The in vivo biodistribution study of 131 I-Azure A showed a high brain uptake of 6.15 ± 0.09 % injected dose/g tissue organ at 30 minutes post-injection, and its retention in brain remained high up to 2 hours, whereas the clearance from the body appeared to proceed via the renal system. The experimental data were confirmed by the molecular docking studies to predict the effect of radioiodination on the binding affinity of the parent molecule (Azure A) to tau paired helical filaments (PHFs). Both ligands showed better binding to S2 and S3 pockets of (PHFs). Consequently, radioiodinated Azure A seems to be a good candidate as an imaging agent for taupathies such as Alzheimer's disease, chronic traumatic encephalopathy, and corticobasal degeneration. Furthermore, it could be a very potent theranostics agent for brain tumors.

Radioiodinated Portable Albumin Binder as a Versatile Agent for in Vivo Imaging with Single-Photon Emission Computed Tomography.

In this study, radioiodinated 4-( p-iodophenyl)butyric acid ([131I]IBA) was synthesized and evaluated as a portable albumin-binder for potential applications in single photon emission computed tomography imaging of blood pool, tumor, and lymph node with significantly improved pharmacokinetic properties. The [131I]IBA was prepared under the catalyst of Cu2O/1,10-phenanthroline. After that, the albumin-binding capability of [131I]IBA was tested in vitro, ex vivo, and in vivo, respectively. [131I]IBA was obtained with very high radiolabeling yield (>99%) and good radiochemical purity (>98%) within 10 min. It binds to albumin effectively with high affinity (IC50= 46.5 µM) and has good stability. The results of biodistribution indicated that the [131I]IBA was mainly accumulated in blood with good retention (10.51 ± 2.58%ID/g at 30 min p.i. and 4.63 ± 0.17%ID/g at 4 h p.i.). In the SPECT imaging of mice models with [131I]IBA, blood pool, lymph node, and tumors could be imaged clearly with high target-to-background ratio. Overall, the radioiodinated albumin binder of [131I]IBA with long blood half-life and excellent stability could be used to decorate diversified albumin-binding radioligands and developed as a versatile theranostic agent.

Synthesis and evaluation of radioactive/fluorescent peptide probes for imaging of legumain activity.

Legumain or asparaginyl endopeptidase is an enzyme overexpressed in some cancers and involved in cancer migration, invasion, and metastasis. We have developed radioiodine- ([125I]I-LCP) or fluorescein-labeled peptides (FL-LCP) with a cell-permeable d-Arg nonamer fused to an anionic d-Glu nonamer via a legumain-cleavable linker, to function as peptide probes that measure and monitor legumain activity. Non-cleavable probes of FL-NCP and [125I]I-NCP were similarly prepared and evaluated as negative control probes by altering their non-cleavable sequence. Model peptides with the legumain-cleavable or non-cleavable sequence (LCP and NCP, respectively) reacted with recombinant human legumain, and only LCP was digested by this enzyme. [125I]I-LCP uptake in legumain-positive HCT116 cells was significantly higher than that of [125I]I-NCP (11.2 ±â€¯0.44% vs 1.75 ±â€¯0.06% dose/mg). The accumulation of FL-LCP in the HCT116 cells was rather low (4.75 ±â€¯0.29% dose/mg protein), but not significantly different from the levels of FL-NCP. It is possible that low concentrations of [125I]I-LCP (40 pM) can be effectively internalized after legumain cleavage. On the other hand, the cellular uptake of much higher concentrations of the FL-LCP derivative (1 mM) may be restricted by high concentrations of polyanions. The in vivo biodistribution studies in tumor-bearing mice demonstrated that the tumor uptake of [125I]I-LCP was 1.34% injected dose per gram (% ID/g) at 30 min. The tumor/blood and tumor/muscle ratios at 30 min were 0.63 and 1.77, respectively, indicating that the [125I]I-LCP accumulation in tumors was inadequate for in vivo imaging. Although further structural modifications are necessary to improve pharmacokinetic properties, [125I]I-LCP has been demonstrated to be an effective scaffold for the development of nuclear medicine imaging probes to monitor legumain activity in living subjects.

Radioiodinated progesterone derivative for progesterone receptor targeting with enhanced nucleus uptake via phenylboronic acid conjugation.

A novel 131 I-radiolabeled probe with aromatic boronate motif (131 I-EIPBA) was designed to target progesterone receptor (PR)-positive breast cancer with enhanced nucleus uptake. Acetylene progesterone was conjugated with pegylated phenylboronic acid via click reaction and radiolabeled with 131 I to afford 131 I-EIPBA. Meanwhile, 131 I-EIPB without boronate was prepared as control agent. After determination of the lipophilicity and stability of these tracers, in vitro cell uptake studies and in vivo biodistribution in rats were performed to verify the enhanced nucleus uptake and PR targeting ability of 131 I-EIPBA. 131 I-EIPBA was obtained with moderate radiochemical yield (40.35 ± 3.52%) and high radiochemical purity (>98%). As expected, the high binding affinity (39.58 nM) of 131 I-EIPBA for PR was determined by cell binding assay. The internalization ratio of 131 I-EIPBA was remarkably higher than that of 131 I-EIPB in PR-positive MCF-7 cells. Furthermore, the enhanced nucleus uptake of 131 I-EIPBA (0.59 ± 0.02%) was found to be significantly higher than that of 131 I-EIPB (0.13 ± 0.01%) in MCF-7 cells. A novel 131 I-EIPBA compound was developed for PR targeting with improved cellular nucleus uptake. Furthermore, the introduction of aromatic boronate motif provides a worthwhile strategy for enhancing the nuclear receptor targeting of tracers.

Radiolabeled IgG antibodies: Impact of various labels on neonatal Fc receptor binding.

The number of therapeutic antibodies in research and development as well as their complexity increases from year to year. Novel therapeutic protein formats, such as Fc-fusions, bispecific, or multivalent antibodies, are currently in preclinical and clinical development. Therefore, the need for biodistribution and imaging studies, eg, with radiolabeled proteins are very high. However, the labeling process or the label itself can have an impact on binding to cellular receptors, eg, to neonatal Fc receptor (FcRn), which can lead to altered PK properties compared with the unlabeled antibody. FcRn affinity chromatography allows the assessment of immunoglobulin G (IgG) samples with respect to their pH-dependent FcRn interaction. We analyzed IgGs with different types of labels, namely, direct iodination with 125 I; chelating agents, such as DOTA and DOTAM; and [3 H]propionate. Direct radio-iodination leads to shifts in FcRn column retention time, which might indicate a potentially faster clearance. Furthermore, high conjugation ratios of chelator lower the affinity to FcRn successively and thus may influence the lysosomal degradation of the antibody in endothelial cells. In contrast, IgGs labeled with [3 H]propionate did not show any timeshifts in FcRn affinity chromatography. This article is based on the oral presentation at the IIS 2018 Prague and highlights the importance of an affinity chromatography for characterization of potential changes in affinity to FcRn itself or charge and hydrophobicity.