Bioanalytical strategy for the characterization and bioanalysis of biologics: a global, nonregulated bioanalytical lab perspective.

Over the past two decades, we have seen an increase in the complexity and diversity of biotherapeutic modalities pursued by biopharmaceutical companies. These biologics are multifaceted and susceptible to post-translational modifications and in vivo biotransformation that could impose challenges for bioanalysis. It is vital to characterize the functionality, stability and biotransformation products of these molecules to enable screening, identify potential liabilities at an early stage and devise a bioanalytical strategy. This article highlights our perspective on characterization and bioanalysis of biologics using hybrid LC-MS in our global nonregulated bioanalytical laboratories. AbbVie's suite of versatile, stage-appropriate characterization assays and quantitative bioanalytical approaches are discussed, along with guidance on their utility in answering project-specific questions to aid in decision-making.

Ablation of VLA4 in multiple myeloma cells redirects tumor spread and prolongs survival.

Multiple myeloma (MM) is a cancer of bone marrow (BM) plasma cells, which is increasingly treatable but still incurable. In 90% of MM patients, severe osteolysis results from pathological interactions between MM cells and the bone microenvironment. Delineating specific molecules and pathways for their role in cancer supportive interactions in the BM is vital for developing new therapies. Very Late Antigen 4 (VLA4, integrin α4ß1) is a key player in cell-cell adhesion and signaling between MM and BM cells. We evaluated a VLA4 selective near infrared fluorescent probe, LLP2A-Cy5, for in vitro and in vivo optical imaging of VLA4. Furthermore, two VLA4-null murine 5TGM1 MM cell (KO) clones were generated by CRISPR/Cas9 knockout of the Itga4 (α4) subunit, which induced significant alterations in the transcriptome. In contrast to the VLA4+ 5TGM1 parental cells, C57Bl/KaLwRij immunocompetent syngeneic mice inoculated with the VLA4-null clones showed prolonged survival, reduced medullary disease, and increased extramedullary disease burden. The KO tumor foci showed significantly reduced uptake of LLP2A-Cy5, confirming in vivo specificity of this imaging agent. This work provides new insights into the pathogenic role of VLA4 in MM, and evaluates an optical tool to measure its expression in preclinical models.

Copper-Catalyzed Covalent Dimerization of Near-Infrared Fluorescent Cyanine Dyes: Synergistic Enhancement of Photoacoustic Signals for Molecular Imaging of Tumors.

Photoacoustic (PA) imaging relies on the absorption of light by chromophores to generate acoustic waves used to delineate tissue structures and physiology. Here, we demonstrate that Cu(II) efficiently catalyzes the dimerization of diverse near-infrared (NIR) cyanine molecules, including a peptide conjugate. NMR spectroscopy revealed a C-C covalent bond along the heptamethine chains, creating stable molecules under conditions such as a wide range of solvents and pH mediums. Dimerization achieved >90% fluorescence quenching, enhanced photostability, and increased PA signals by a factor of about 4 at equimolar concentrations compared to the monomers. In vivo study with a mouse cancer model revealed that dimerization enhanced tumor retention and PA signal, allowing cancer detection at doses where the monomers are less effective. While the dye dimers highlighted peritumoral blood vessels, the PA signal for dimeric tumor-targeting dye-peptide conjugate, LS301, was diffuse throughout the entire tumor mass. A combination of the ease of synthesis, diversity of molecules that are amenable to Cu(II)-catalyzed dimerization, and the high acoustic wave amplification by these stable dimeric small molecules ushers a new strategy to develop clinically translatable PA molecular amplifiers for the emerging field of molecular photoacoustic imaging.

Selective imaging of solid tumours via the calcium-dependent high-affinity binding of a cyclic octapeptide to phosphorylated Annexin A2.

The heterogeneity and continuous genetic adaptation of tumours complicate their detection and treatment via the targeting of genetic mutations. However, hallmarks of cancer such as aberrant protein phosphorylation and calcium-mediated cell signalling provide broadly conserved molecular targets. Here, we show that, for a range of solid tumours, a cyclic octapeptide labelled with a near-infrared dye selectively binds to phosphorylated Annexin A2 (pANXA2), with high affinity at high levels of calcium. Because of cancer-cell-induced pANXA2 expression in tumour-associated stromal cells, the octapeptide preferentially binds to the invasive edges of tumours and then traffics within macrophages to the tumour's necrotic core. As proof-of-concept applications, we used the octapeptide to detect tumour xenografts and metastatic lesions, and to perform fluorescence-guided surgical tumour resection, in mice. Our findings suggest that high levels of pANXA2 in association with elevated calcium are present in the microenvironment of most solid cancers. The octapeptide might be broadly useful for selective tumour imaging and for delivering drugs to the edges and to the core of solid tumours.

Cellular Trafficking of Sn-2 Phosphatidylcholine Prodrugs Studied with Fluorescence Lifetime Imaging and Super-resolution Microscopy.

While the in vivo efficacy of Sn-2 phosphatidylcholine prodrugs incorporated into targeted, non-pegylated lipid-encapsulated nanoparticles was demonstrated in prior preclinical studies, the microscopic details of cell prodrug internalization and trafficking events are unknown. Classic fluorescence microscopy, fluorescence lifetime imaging microscopy, and single-molecule super-resolution microscopy were used to investigate the cellular handling of doxorubicin-prodrug and AlexaFluor™-488-prodrug. Sn-2 phosphatidylcholine prodrugs delivered by hemifusion of nanoparticle and cell phospholipid membranes functioned as phosphatidylcholine mimics, circumventing the challenges of endosome sequestration and release. Phosphatidylcholine prodrugs in the outer cell membrane leaflet translocated to the inner membrane leaflet by ATP-dependent and ATP-independent mechanisms and distributed broadly within the cytosolic membranes over the next 12 h. A portion of the phosphatidylcholine prodrug populated vesicle membranes trafficked to the perinuclear Golgi/ER region, where the drug was enzymatically liberated and activated. Native doxorubicin entered the cells, passed rapidly to the nucleus, and bound to dsDNA, whereas DOX was first enzymatically liberated from DOX-prodrug within the cytosol, particularly in the perinuclear region, before binding nuclear dsDNA. Much of DOX-prodrug was initially retained within intracellular membranes. In vitro anti-proliferation effectiveness of the two drug delivery approaches was equivalent at 48 h, suggesting that residual intracellular DOX-prodrug may constitute a slow-release drug reservoir that enhances effectiveness. We have demonstrated that Sn-2 phosphatidylcholine prodrugs function as phosphatidylcholine mimics following reported pathways of phosphatidylcholine distribution and metabolism. Drug complexed to the Sn-2 fatty acid is enzymatically liberated and reactivated over many hours, which may enhance efficacy overtime.

Preclinical Development of CD38-Targeted [89Zr]Zr-DFO-Daratumumab for Imaging Multiple Myeloma.

Multiple myeloma (MM) is a plasma B-cell hematologic cancer that causes significant skeletal morbidity. Despite improvements in survival, heterogeneity in response remains a major challenge in MM. Cluster of differentiation 38 (CD38) is a type II transmembrane glycoprotein overexpressed in myeloma cells and is implicated in MM cell signaling. Daratumumab is a U.S. Food and Drug Administration-approved high-affinity monoclonal antibody targeting CD38 that is clinically benefiting refractory MM patients. Here, we evaluated [89Zr]Zr-desferrioxamine (DFO)-daratumumab PET/CT imaging in MM tumor models. Methods: Daratumumab was conjugated to DFO-p-benzyl-isothiocyanate (DFO-Bz-NCS) for radiolabeling with 89Zr. Chelator conjugation was confirmed by electrospray ionization-mass spectrometry, and radiolabeling was monitored by instant thin-layer chromatography. Daratumumab was conjugated to Cyanine5 (Cy5) dye for cell microscopy. In vitro and in vivo evaluation of [89Zr]Zr-DFO-daratumumab was performed using CD38+ human myeloma MM1.S-luciferase (MM1.S) cells. Cellular studies determined the affinity, immunoreactivity, and specificity of [89Zr]Zr-DFO-daratumumab. A 5TGM1-luciferase (5TGM1)/KaLwRij MM mouse model served as control for imaging background noise. [89Zr]Zr-DFO-daratumumab PET/CT small-animal imaging was performed in severe combined immunodeficient mice bearing solid and disseminated MM tumors. Tissue biodistribution (7 d after tracer administration, 1.11 MBq/animal, n = 4-6/group) was performed in wild-type and MM1.S tumor-bearing mice. Results: A specific activity of 55.5 MBq/nmol (0.37 MBq/µg) was reproducibly obtained with [89Zr]Zr-daratumumab-DFO. Flow cytometry confirmed CD38 expression (>99%) on the surface of MM1.S cells. Confocal microscopy with daratumumab-Cy5 demonstrated specific cell binding. Dissociation constant, 3.3 nM (±0.58), and receptor density, 10.1 fmol/mg (±0.64), was obtained with a saturation binding assay. [89Zr]Zr-DFO-daratumumab/PET demonstrated specificity and sensitivity for detecting CD38+ myeloma tumors of variable sizes (8.5-128 mm3) with standardized uptake values ranging from 2.1 to 9.3. Discrete medullar lesions, confirmed by bioluminescence images, were efficiently imaged with [89Zr]Zr-DFO-daratumumab/PET. Biodistribution at 7 d after administration of [89Zr]Zr-DFO-daratumumab showed prominent tumor uptake (27.7 ± 7.6 percentage injected dose per gram). In vivo blocking was achieved with a 200-fold excess of unlabeled daratumumab. Conclusion: [89Zr]Zr-DFO- and Cy5-daratumumab demonstrated superb binding to CD38+ human MM cells and significantly low binding to CD38low cells. Daratumumab bioconjugates are being evaluated for image-guided delivery of therapeutic radionuclides.

Noninvasive depth estimation using tissue optical properties and a dual-wavelength fluorescent molecular probe in vivo.

Translation of fluorescence imaging using molecularly targeted imaging agents for real-time assessment of surgical margins in the operating room requires a fast and reliable method to predict tumor depth from planar optical imaging. Here, we developed a dual-wavelength fluorescent molecular probe with distinct visible and near-infrared excitation and emission spectra for depth estimation in mice and a method to predict the optical properties of the imaging medium such that the technique is applicable to a range of medium types. Imaging was conducted at two wavelengths in a simulated blood vessel and an in vivo tumor model. Although the depth estimation method was insensitive to changes in the molecular probe concentration, it was responsive to the optical parameters of the medium. Results of the intra-tumor fluorescent probe injection showed that the average measured tumor sub-surface depths were 1.31 ± 0.442 mm, 1.07 ± 0.187 mm, and 1.42 ± 0.182 mm, and the average estimated sub-surface depths were 0.97 ± 0.308 mm, 1.11 ± 0.428 mm, 1.21 ± 0.492 mm, respectively. Intravenous injection of the molecular probe allowed for selective tumor accumulation, with measured tumor sub-surface depths of 1.28 ± 0.168 mm, and 1.50 ± 0.394 mm, and the estimated depths were 1.46 ± 0.314 mm, and 1.60 ± 0.409 mm, respectively. Expansion of our technique by using material optical properties and mouse skin optical parameters to estimate the sub-surface depth of a tumor demonstrated an agreement between measured and estimated depth within 0.38 mm and 0.63 mm for intra-tumor and intravenous dye injections, respectively. Our results demonstrate the feasibility of dual-wavelength imaging for determining the depth of blood vessels and characterizing the sub-surface depth of tumors in vivo.

Molecular probes for fluorescence lifetime imaging.

Visualization of biological processes and pathologic conditions at the cellular and tissue levels largely relies on the use of fluorescence intensity signals from fluorophores or their bioconjugates. To overcome the concentration dependency of intensity measurements, evaluate subtle molecular interactions, and determine biochemical status of intracellular or extracellular microenvironments, fluorescence lifetime (FLT) imaging has emerged as a reliable imaging method complementary to intensity measurements. Driven by a wide variety of dyes exhibiting stable or environment-responsive FLTs, information multiplexing can be readily accomplished without the need for ratiometric spectral imaging. With knowledge of the fluorescent states of the molecules, it is entirely possible to predict the functional status of biomolecules or microevironment of cells. Whereas the use of FLT spectroscopy and microscopy in biological studies is now well-established, in vivo imaging of biological processes based on FLT imaging techniques is still evolving. This review summarizes recent advances in the application of the FLT of molecular probes for imaging cells and small animal models of human diseases. It also highlights some challenges that continue to limit the full realization of the potential of using FLT molecular probes to address diverse biological problems and outlines areas of potential high impact in the future.

Noninvasive imaging of focal atherosclerotic lesions using fluorescence molecular tomography.

Insights into the etiology of stroke and myocardial infarction suggest that rupture of unstable atherosclerotic plaque is the precipitating event. Clinicians lack tools to detect lesion instability early enough to intervene, and are often left to manage patients empirically, or worse, after plaque rupture. Noninvasive imaging of the molecular events signaling prerupture plaque progression has the potential to reduce the morbidity and mortality associated with myocardial infarction and stroke by allowing early intervention. Here, we demonstrate proof-of-principle in vivo molecular imaging of C-type natriuretic peptide receptor in focal atherosclerotic lesions in the femoral arteries of New Zealand white rabbits using a custom built fiber-based, fluorescence molecular tomography (FMT) system. Longitudinal imaging showed changes in the fluorescence signal intensity as the plaque progressed in the air-desiccated vessel compared to the uninjured vessel, which was validated by ex vivo tissue studies. In summary, we demonstrate the potential of FMT for noninvasive detection of molecular events leading to unstable lesions heralding plaque rupture.

Electron tomography of cryo-immobilized plant tissue: a novel approach to studying 3D macromolecular architecture of mature plant cell walls in situ.

Cost-effective production of lignocellulosic biofuel requires efficient breakdown of cell walls present in plant biomass to retrieve the wall polysaccharides for fermentation. In-depth knowledge of plant cell wall composition is therefore essential for improving the fuel production process. The precise spatial three-dimensional (3D) organization of cellulose, hemicellulose, pectin and lignin within plant cell walls remains unclear to date since the microscopy techniques used so far have been limited to two-dimensional, topographic or low-resolution imaging, or required isolation or chemical extraction of the cell walls. In this paper we demonstrate that by cryo-immobilizing fresh tissue, then either cryo-sectioning or freeze-substituting and resin embedding, followed by cryo- or room temperature (RT) electron tomography, respectively, we can visualize previously unseen details of plant cell wall architecture in 3D, at macromolecular resolution (∼ 2 nm), and in near-native state. Qualitative and quantitative analyses showed that wall organization of cryo-immobilized samples were preserved remarkably better than conventionally prepared samples that suffer substantial extraction. Lignin-less primary cell walls were well preserved in both self-pressurized rapidly frozen (SPRF), cryo-sectioned samples as well as high-pressure frozen, freeze-substituted and resin embedded (HPF-FS-resin) samples. Lignin-rich secondary cell walls appeared featureless in HPF-FS-resin sections presumably due to poor stain penetration, but their macromolecular features could be visualized in unprecedented details in our cryo-sections. While cryo-tomography of vitreous tissue sections is currently proving to be instrumental in developing 3D models of lignin-rich secondary cell walls, here we confirm that the technically easier method of RT-tomography of HPF-FS-resin sections could be used immediately for routine study of low-lignin cell walls. As a proof of principle, we characterized the primary cell walls of a mutant (cob-6) and wild type Arabidopsis hypocotyl parenchyma cells by RT-tomography of HPF-FS-resin sections, and detected a small but significant difference in spatial organization of cellulose microfibrils in the mutant walls.