Utility of High Throughput Screening Techniques to Predict Stability of Monoclonal Antibody Formulations During Early Stage Development.

Selecting optimal formulation conditions for monoclonal antibodies for first time in human clinical trials is challenging due to short timelines and reliance on predictive assays to ensure product quality and adequate long-term stability. Accelerated stability studies are considered to be the gold standard for excipient screening, but they are relatively low throughput and time consuming. High throughput screening (HTS) techniques allow for large amounts of data to be collected quickly and easily, and can be used to screen solution conditions for early formulation development. The utility of using accelerated stability compared to HTS techniques (differential scanning light scattering and differential scanning fluorescence) for early formulation screening was evaluated along with the impact of excipients of various types on aggregation of monoclonal antibodies from multiple IgG subtypes. The excipient rank order using quantitative HTS measures was found to correlate with accelerated stability aggregation rate ranking for only 33% (by differential scanning fluorescence) to 42% (by differential scanning light scattering) of the antibodies tested, due to the high intrinsic stability and minimal impact of excipients on aggregation rates and HTS data. Also explored was a case study of employing a platform formulation instead of broader formulation screening for early formulation development.

Plasmonic measurements of monoclonal antibody self-association using self-interaction nanoparticle spectroscopy.

One of the most significant challenges in developing therapeutic monoclonal antibodies (mAbs) is their unpredictable solubilities and viscosities at the high concentrations required for subcutaneous delivery. This challenge has motivated the development of screening assays that rapidly identify mAb variants with minimal self-association propensities and/or formulation conditions that suppress mAb self-association. Here we report an improved version of self-interaction nanoparticle spectroscopy (SINS)capable of characterizing both repulsive and attractive self-interactions between diverse mAbs. The basis of SINS is that self-interactions between mAbs immobilized on gold nanoparticles increase (repulsion) or decrease (attraction)interparticle distances, which shift the wavelength of maximum absorbance (plasmon wavelength) in opposite directions.We find that the robustness of SINS is improved by varying the amount of immobilized mAb by co-adsorbing a polyclonal antibody. The slopes of the plasmon wavelength shifts as a function of the amount of immobilized mAb (0.01­0.1 mg/mL) are correlated with diffusion interaction parameters measured at two to three orders of magnitude higher antibody concentrations. The ability of SINS to rapidly screen mAb self-association in a microplate format using dilute mAb solutions makes it well suited for use in diverse settings ranging from antibody discovery to formulation.

Formulation development of therapeutic monoclonal antibodies using high-throughput fluorescence and static light scattering techniques: role of conformational and colloidal stability.

In this work, we describe the application of two different high-throughput screening (HTS) techniques that can be used to determine protein stability during early formulation development. Differential scanning fluorescence (DSF) and differential static light scattering (DSLS) are used to determine the conformational and colloidal stability of therapeutic monoclonal antibodies (mAbs) during thermal denaturation in a high-throughput fashion. DSF utilizes SYPRO Orange, a polarity-sensitive extrinsic fluorescent probe, to monitor protein unfolding. We found that melting temperatures determined by DSF have a linear correlation with melting temperatures of the first domain unfolding determined by differential scanning calorimetry, establishing DSF as a reliable method for measuring thermal stability. The DSLS method employs static light scattering to evaluate protein stability during thermal denaturation in a 384-well format. Overall comparison between mAb aggregation under typical accelerated stress conditions (40°C) and the thermal stability obtained by DSF and DSLS is also presented. Both of these HTS methods are cost effective with high-throughput capability and can be implemented in any laboratory. Combined with other emerging HTS techniques, DSF and DSLS could be powerful tools for mAb formulation optimization.

G3.5 PAMAM dendrimers enhance transepithelial transport of SN38 while minimizing gastrointestinal toxicity.

Poly(amido amine) (PAMAM) dendrimers have shown promise in oral drug delivery. Conjugation of SN38 to PAMAM dendrimers has the potential to improve its oral absorption while minimizing gastrointestinal toxicity. In this work we evaluated G3.5 PAMAM dendrimer-SN38 conjugates with ester-linked glycine and ß-alanine spacers for their suitability in oral therapy of hepatic colorectal cancer metastases. G3.5-ßAlanine-SN38 was mostly stable while G3.5-Glycine-SN38 showed 10%, 20%, and 56% SN38 release in simulated gastric, intestinal and liver environments for up to 6, 24 and 48 hours, respectively. Short-term treatment of Caco-2 cells with G3.5-SN38 conjugates did not reduce cell viability, while comparable concentrations of SN38 caused significant cytotoxicity. G3.5-Glycine-SN38 and G3.5-ßAlanine-SN38 showed IC50 values of 0.60 and 3.59 µM, respectively, in HT-29 cells treated for 48 h, indicating the efficacy of the drug delivery system in colorectal cancer cells with longer incubation time. Both conjugates increased SN38 transepithelial transport compared to the free drug. Transport of G3.5-Glycine-SN38 was highly concentration-dependent whereas transport of G3.5-ßAlanine-SN38 was concentration-independent, highlighting the influence of drug loading and spacer chemistry on transport mechanism. Together these results show that PAMAM dendrimers have the potential to improve the oral bioavailability of potent anti-cancer drugs.

Cellular entry of G3.5 poly (amido amine) dendrimers by clathrin- and dynamin-dependent endocytosis promotes tight junctional opening in intestinal epithelia.

PURPOSE: This study investigates the mechanisms of G3.5 poly (amido amine) dendrimer cellular uptake, intracellular trafficking, transepithelial transport and tight junction modulation in Caco-2 cells in the context of oral drug delivery. METHODS: Chemical inhibitors blocking clathrin-, caveolin- and dynamin-dependent endocytosis pathways were used to investigate the mechanisms of dendrimer cellular uptake and transport across Caco-2 cells using flow cytometry and confocal microscopy. RESULTS: Dendrimer cellular uptake was found to be dynamin-dependent and was reduced by both clathrin and caveolin endocytosis inhibitors, while transepithelial transport was only dependent on dynamin- and clathrin-mediated endocytosis. Dendrimers were quickly trafficked to the lysosomes after 15 min of incubation and showed increased endosomal accumulation at later time points, suggesting saturation of this pathway. Dendrimers were unable to open tight junctions in cell monolayers treated with dynasore, a selective inhibitor of dynamin, confirming that dendrimer internalization promotes tight junction modulation. CONCLUSION: G3.5 PAMAM dendrimers take advantage of several receptor-mediated endocytosis pathways for cellular entry in Caco-2 cells. Dendrimer internalization by dynamin-dependent mechanisms promotes tight junction opening, suggesting that dendrimers act on intracellular cytoskeletal proteins to modulate tight junctions, thus catalyzing their own transport via the paracellular route.