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The Third Modeling Workshop focusing on bioprocess modeling was held in Kenilworth, NJ in May 2019. A summary of these Workshop proceedings is captured in this manuscript. Modeling is an active area of research within the biotechnology community, and there is a critical need to assess the current state and opportunities for continued investment to realize the full potential of models, including resource and time savings. Beyond individual presentations and topics of novel interest, a substantial portion of the Workshop was devoted toward group discussions of current states and future directions in modeling fields. All scales of modeling, from biophysical models at the molecular level and up through large scale facility and plant modeling, were considered in these discussions and are summarized in the manuscript. Model life cycle management from model development to implementation and sustainment are also considered for different stages of clinical development and commercial production. The manuscript provides a comprehensive overview of bioprocess modeling while suggesting an ideal future state with standardized approaches aligned across the industry.

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 (0.01–0.1 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.

Protein aggregation is a common problem during the purification and formulation of therapeutic proteins. Here we report that polyphenolic disaccharides are unusually effective at preventing protein aggregation. We find that two polyphenolic glycosides-naringin and rutin-endow diverse proteins with the ability to unfold without aggregating when heated, as well as the ability to refold without aggregating when cooled at low glycoside concentrations (

The presence of even minute amounts of protein aggregates in solution can significantly alter the kinetics of amyloid formation. Removal of such pre-existing aggregates is critical for reproducible analysis of amyloid formation. Here we examine the effects of membrane filtration on insulin fibrillization. We find that filtration of insulin with large pore membranes (≥100 nm) generally slows fibril formation relative to unfiltered solutions by removing pre-aggregated protein. Unexpectedly, filtration with small pore membranes (

Substantial evidence suggests that soluble prefibrillar oligomers of the Aβ42 peptide associated with Alzheimer's disease are the most cytotoxic aggregated Aβ isoform. Limited previous work has revealed that aromatic compounds capable of remodeling Aβ oligomers into nontoxic conformers typically do so by converting them into off-pathway aggregates instead of dissociating them into monomers. Towards identifying small-molecule antagonists capable of selectively dissociating toxic Aβ oligomers into soluble peptide at substoichiometric concentrations, we have investigated the pathways used by polyphenol aglycones and their glycosides to remodel Aβ soluble oligomers. We find that eleven polyphenol aglycones of variable size and structure utilize the same remodeling pathway whereby Aβ oligomers are rapidly converted into large, off-pathway aggregates. Strikingly, we find that glycosides of these polyphenols all utilize a distinct remodeling pathway in which Aβ oligomers are rapidly dissociated into soluble, disaggregated peptide. This disaggregation activity is a synergistic combination of the aglycone and glycone moieties because combinations of polyphenols and sugars fail to disaggregate Aβ oligomers. We also find that polyphenolic glycosides and aglycones use the same opposing pathways to remodel Aβ fibrils. Importantly, both classes of polyphenols fail to remodel nontoxic Aβ oligomers (which are indistinguishable in size and morphology to Aβ soluble oligomers) or promote aggregation of freshly disaggregated Aβ peptide; thus revealing that they are specific for remodeling toxic Aβ conformers. We expect that these and related small molecules will be powerful chemical probes for investigating the conformational and cellular underpinnings of Aβ-mediated toxicity.