Identification of Surfactant Impact on a Monoclonal Antibody Characterization via HPLC-Separation Based and Biophysical Methods.

PURPOSE OR OBJECTIVE: Surfactants, including polysorbates and poloxamers, play a crucial role in the formulation of therapeutic proteins by acting as solubilizing and stabilizing agents. They help prevent protein aggregation and adsorption, thereby enhancing the stability of drug substance and products., However, it is important to note that utilizing high concentrations of surfactants in protein formulations can present significant analytical challenges, which can ultimately affect the product characterization. METHODS: In our study, we specifically investigated the impact of elevated surfactant concentrations on the characterization of monoclonal antibodies. We employed various analytical techniques including size-exclusion chromatography (SEC), capillary electrophoresis (CE-SDS), a cell based functional assay, and biophysical characterization. RESULTS: The findings of our study indicate that higher levels of Polysorbate 80 (PS-80) have adverse effects on the measured purity, biological activity, and biophysical characterization of biologic samples. Specifically, the elevated levels of PS-80 cause analytical interferences, which can significantly impact the accuracy and reliability of analytical studies. CONCLUSIONS: Our study results highlight a significant risk in analytical investigations, especially in studies involving the isolation and characterization of impurities. It is important to be cautious of surfactant concentrations, as they can become more concentrated during common sample manipulations like buffer exchange. Indeed, the research presented in this work emphasizes the necessity to evaluate the impact on analytical assays when there are substantial alternations in the matrix composition. By doing so, valuable insights can be gained regarding potential challenges associated with assay development and characterization of biologics with complex formulations.

Imaged capillary isoelectric focusing method development for charge variants of high DAR ADCs.

BACKGROUND: Charge heterogeneity is a critical quality attribute for therapeutic biologics including antibody-drug conjugates (ADCs). Developing an ion exchange chromatography (IEX) or an imaged capillary isoelectric focusing (icIEF) method for ADCs with high drug-to-antibody ratio (DAR) is challenging because of the increased hydrophobicity from the payload-linker, DAR heterogeneity, and payload-linker instability. A sub-optimal method can be poorly stability-indicating due to the inability to discern contributions from charge and size variants conjugated with different number of drugs/payloads. Systematic strategy and guidance on charge variant method development is highly desired for high DAR ADCs with various complex structures. RESULTS: This work encompasses the development and optimization of icIEF methods for high DAR ADCs of various DAR values (4-8) and payload linker chemistry. Method optimization focuses on improving resolution and stability indicating capabilities and differentiating contributions from the protein and payload-linker. Types, proportion, and combination of solubilizers and carrier ampholytes, as well as focusing parameters were interrogated. Our findings show that the structural units of the linker, the DAR, and the payload chemistry prescribe the selection of buffer, solubilizer, and ampholyte. We demonstrate that a stronger denaturant or solubilizer is needed for high DAR ADCs with polyethylene glycol (PEG)-containing linker structure compared to peptide linker. For unstable payload-linker, buffer system enhances sample stability which is vital to method robustness. In addition, a longer isoelectric focusing time is necessary for an ADC than its corresponding antibody to reach optimal focusing. SIGNIFICANCE: To the best of our knowledge, this is the first comprehensive study on icIEF method development for charge variant determination of high DAR ADCs with unique physicochemical properties.

Immunoliposomes that target endothelium in vitro are dependent on lipid raft formation.

Lipid rafts are plasma membrane microdomains rich in cholesterol, sphingolipids, and cell surface receptors. Recent studies demonstrated the upregulation and localization of two receptors, intercellular cell adhesion molecule-1 (ICAM, CD54) and endothelial leukocyte adhesion molecule-1 (E-selectin, CD64E), within lipid raft microdomains of inflamed or injured endothelial cells (ECs). We hypothesized that the localization of ICAM and E-selectin within lipid rafts may be essential for drug delivery vehicles labeled with antibodies against ICAM (aICAM) and E-selectin (aE-selectin). To eliminate localization of cell surface receptors, ECs were treated with a cholesterol depleting drug, methyl-ß-cyclodextrin. We also tested if antibody mobility and the ratio of aICAM to aE-selectin on immunoliposomes influenced binding to lipid-raft-depleted cells. Liposomes were prepared from either 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC, C(18:1), T(m) = -20 °C) or 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC, C(16:0), T(m) = 42 °C) which are in the liquid crystalline and gel phase at 37 °C, respectively. Mobility and the aICAM:aE-selectin ratio influenced cellular binding only when lipid rafts form. In the absence of lipid rafts, cellular binding of both DOPC and DPPC immunoliposomes was reduced to the nonspecific binding level. These results, which were obtained under static conditions, suggest that the presence of lipid rafts in ECs is critical for targeted drug delivery.

Development of homogeneous plasmonic potency assay using gold nanoparticle immunocomplexes.

We evaluated the use of gold nanoparticles (AuNPs) platform in a homogenous assay for a potency measurement of a therapeutic monoclonal antibody (mAb). The recombinant human ligand protein to the therapeutic mAb was immobilized on AuNPs via functionalized self-assembled monolayers. Binding of the mAb to ligand lead to plasmonic signals that were detected faster in a homogeneous assay than the conventional enzyme-linked immunosorbent assay (ELISA). In this study, we demonstrated that the AuNP-based homogeneous plasmonic immunoassay (HPI) generated comparable potency values of a therapeutic mAb to a conventional binding ELISA in relatively shorter assay time and steps. Binding HPI can be potentially implemented as a potency assay for therapeutic mAbs in quality control laboratories.

Complementary targeting of liposomes to IL-1α and TNF-α activated endothelial cells via the transient expression of VCAM1 and E-selectin.

Inflammation is in part defined by the transient upregulation of cell adhesion molecules on the surface of endothelial cells (ECs) in response to cytokines. We hypothesized that liposomes with a complementary surface presentation of antibodies to the pattern of molecules on the EC surface may enhance targeting. We quantified the expression of vascular cell adhesion molecule-1 (VCAM1) and endothelial leukocyte cell adhesion molecule-1 (E-selectin) on ECs upon exposure to either tumor necrosis factor-α (TNF-α) or interleukin-1α (IL-1α) as a function of time. Liposomes, composed of 95 mol% dioleoyl phosphatidylcholine (DOPC) and 5 mol% dodecanyl phosphatidylethanolamine (N-dod-PE), were prepared by conjugating different molar ratios of antibodies against VCAM1 (aVCAM1) and E-selectin (aE-selectin). Increased binding was observed when immunoliposomes complemented the presentation of VCAM1:E-selectin expressed on TNF-α activated ECs. The 1:1 aVCAM1:aE-selectin liposomes had maximal binding at both 6 and 24 h on IL-1α activated ECs due to differences in molecular organization. The results demonstrate that liposomes targeting to inflamed endothelium may be optimized by exploiting the dynamic expression of VCAM1 and E-selectin on the EC surface.

The role of antibody synergy and membrane fluidity in the vascular targeting of immunoliposomes.

Targeted drug delivery to inflamed or injured vascular endothelial cells (ECs) and smooth muscle cells (SMCs) may provide a precise and effective therapeutic treatment for cardiovascular diseases. Upregulation of cytokine-regulated cell surface receptors, intercellular cell adhesion molecule-1 (ICAM) and endothelial-leukocyte adhesion molecule-1 (ELAM), on ECs and SMCs are used to target drug delivery vehicles. Recent studies demonstrate clustering of these molecules in lipid rafts may affect binding due to a nonhomogenous presentation of antibodies. We hypothesized that altering the antibody ratio for ICAM and ELAM (aICAM:aELAM) and mobility would influence cellular targeting. To alter antibody mobility, liposomes were prepared from either 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC, C(18:1), T(m)=-20 degrees C) or 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC, C(16:0), T(m)=42 degrees C) which are in the liquid crystalline (L(alpha)) and gel phase (L(beta)) at 37 degrees C, respectively. We report that cellular binding of DOPC immunoliposomes by ECs is maximal at an equimolar ratio of aICAM:aELAM whereas DPPC immunoliposomes showed no ratio dependence and binding was reduced by more than 2-fold. SMCs, which do not express ELAM, show a dependence on aICAM surface density. These results suggest that antibody mobility and molar ratio play a key role in increasing receptor-mediated cell targeting.

Surface presentation of bioactive ligands in a nonadhesive background using DOPA-tethered biotinylated poly(ethylene glycol).

We have developed surfaces for the selective presentation of biotinylated peptides and proteins in a background that resists nonspecific protein adsorption; controlled amounts of biotinylated poly(ethylene glycol) (MW 3400 Da; PEG3400) anchored to titanium-dioxide-coated surfaces via an adhesive tri-peptide sequence of L-3,4-dihydroxyphenylalanine (DOPA3-PEG3400-biotin; DPB) were incorporated within a DOPA3-PEG2000 background. Using optical waveguide lightmode spectroscopy, we found that the amounts of sequentially adsorbed NeutrAvidin and singly biotinylated molecules increased proportionally with the amount of DPB in the surface. Biotinylated peptides (MW approximately 2000 Da) were able to fill all three of the remaining avidin-binding sites, while only one molecule of biotinylated PEG5000 or stem cell factor bound to each avidin. The resulting biotin-avidin-biotin linkages were stable for prolonged periods under continuous perfusion, even in the presence of excess free biotin. Hematopoietic M07e cells bound to immobilized peptide ligands for alpha5beta1 (cyclic RGD) and alpha4beta1 (cylic LDV) integrins in a DPB-dose-dependent manner, with near-maximal binding to cylic LDV for surfaces containing 1% DPB. Multiple ligands were adsorbed in a controlled manner by incubating NeutrAvidin with the respective ligands in the desired molar ratio and then adding the resulting complexes to DPB-containing surfaces. Cell adhesion to surfaces containing both cylic LDV and cyclic RGD increased in an additive manner compared to that for the individual ligands. The bioactivity of adsorbed biotinylated stem cell factor was retained, as demonstrated by DPB-dose-dependent M07e cell adhesion and ERK1/2 activation.

Cell migration and polarity on microfabricated gradients of extracellular matrix proteins.

This paper explores the effects of the surface density and concentration profiles of extra cellular matrix proteins on the migration of rat intestinal IEC-6 cells. Microfluidic devices were used to create linear, immobilized gradients of laminin. This study investigated both the impact of the steepness and local concentrations on the directedness of cell migration. The bulk concentrations of proteins in the feed streams in the mixing device determined the gradient profile and the local concentration of laminin in the device. Two sets of gradients were used to explore cell migration directedness: (i) gradients with similar change in local concentration, i.e., the same gradient steepness, and (ii) different gradients with similar local concentrations. Cells migrated up the gradients, independent of the steepness of the gradients used in this study. At the same local laminin concentration, the migration rate was independent of the gradient steepness. However, cell directedness decreased significantly at high laminin densities.

Regiospecific control of protein expression in cells cultured on two-component counter gradients of extracellular matrix proteins.

This work describes the use of microfluidic tools to generate covalently immobilized counter gradients of extracellular matrix (ECM) proteins laminin and collagen I. Using these platforms, we demonstrate control of the expression levels of two proteins linked to cell cycle progression by virtue of the spatial location of cells on the gradients, and hence by the local ECM environments in these devices. In contrast to physisorbed gradients, covalently immobilized protein patterns preserved the gradient fidelity, making long term cell studies feasible. This method of precisely controlling local cell environments is simple and broadly portable to other cell types and to other ECM proteins or soluble factors. Our approach promises to enable new investigations in cell biology that will contribute to the establishment of biological design rules for controlling cell growth, differentiation, and function.