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Controlling viral contamination is an important issue in the process development of monoclonal antibodies (MAbs) produced from mammalian cell lines. Virus filtration (VF) has been demonstrated to be a robust and effective clearance step which can provide ≥4 logs of reduction via size exclusion. The minimization of VF area by increasing flux and filter loading is critical to achieving cost targets as VFs are single use and often represent up to 10% of total purification costs. The research presented in this publication describes a development strategy focused on biophysical attributes of product streams that are directly applicable to VF process performance. This article summarizes a case study where biophysical tools (high-pressure size exclusion chromatography, dynamic light scattering, and absolute size exclusion chromatography) were applied to a specific MAb program to illustrate how changes in feed composition (pH, sodium chloride concentration, and buffer salt type) can change biophysical properties which correlate with VF performance. The approach was subsequently refined and expanded over the course of development of three MAbs where performance metrics (i.e., loading and flux) were evaluated for two specific virus filters (Viresolve Pro and Planova 20N) during both unspiked control runs and virus clearance experiments. The analyses of feed attributes can be applied to a decision tree to guide the recommendation of a VF filter and operating conditions for use in future MAb program development. The understanding of the biophysical properties of the feed can be correlated to virus filter performance to significantly reduce the mass of product, time, and costs associated with virus filter step development.

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Enhanced bioactive anti-oxidant formulations are critical for treatment of inflammatory diseases, such as atherosclerosis. A hallmark of early atherosclerosis is the uptake of oxidized low density lipoprotein (oxLDL) by macrophages, which results in foam cell and plaque formation in the arterial wall. The hypolipidemic, anti-inflammatory, and antioxidative properties of polyphenol compounds make them attractive targets for treatment of atherosclerosis. However, high concentrations of antioxidants can reverse their anti-atheroprotective properties and cause oxidative stress within the artery. Here, we designed a new class of nanoparticles with anti-oxidant polymer cores and shells comprised of scavenger receptor targeting amphiphilic macromolecules (AMs). Specifically, we designed ferulic acid-based poly(anhydride-ester) nanoparticles to counteract the uptake of high levels of oxLDL and regulate reactive oxygen species generation (ROS) in human monocyte derived macrophages (HMDMs). Compared to all compositions examined, nanoparticles with core ferulic acid-based polymers linked by diglycolic acid (PFAG) showed the greatest inhibition of oxLDL uptake. At high oxLDL concentrations, the ferulic acid diacids and polymer nanoparticles displayed similar oxLDL uptake. Treatment with the PFAG nanoparticles downregulated the expression of macrophage scavenger receptors, CD-36, MSR-1, and LOX-1 by about 20-50%, one of the causal factors for the decrease in oxLDL uptake. The PFAG nanoparticle lowered ROS production by HMDMs, which is important for maintaining macrophage growth and prevention of apoptosis. Based on these results, we propose that ferulic acid-based poly(anhydride ester) nanoparticles may offer an integrative strategy for the localized passivation of the early stages of the atheroinflammatory cascade in cardiovascular disease.

Parkinson's Disease is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta, the extracellular accumulation of toxic α-synuclein (αSYN) aggregates, and neuroinflammation. Microglia, resident macrophages of the brain, are one of the critical cell types involved in neuroinflammation. Upon sensing extracellular stimuli or experiencing oxidative stress, microglia become activated, which further exacerbates neuroinflammation. In addition, as the first line of defense in the central nervous system, microglia play a critical role in αSYN clearance and degradation. While the role of microglia in neurodegenerative pathologies is widely recognized, few therapeutic approaches have been designed to target both microglial activation and αSYN aggregation. Here, we designed nanoparticles (NPs) to deliver aggregationinhibiting antioxidants to ameliorate αSYN aggregation and attenuate activation of a pro-inflammatory microglial phenotype. Ferulic acid diacid with an adipic acid linker (FAA) and tannic acid (TA) were used as shell and core molecules to form NPs via flash nanoprecipitation. These NPs showed a strong inhibitory effect on αSYN fibrillization, significantly diminishing αSYN fibrillization in vitro compared to untreated αSYN using a Thioflavin T assay. Treating microglia with NPs decreased overall αSYN internalization and intracellular αSYN oligomer formation. NP treatment additionally lowered the in vitro secretion of pro-inflammatory cytokines TNF-α and IL-6, and also attenuated nitric oxide and reactive oxygen species production induced by αSYN. NP treatment also significantly decreased Iba-1 expression in αSYN-challenged microglia and suppressed nuclear translocation of nuclear factor kappa B (NF-κB). Overall, this work lays the foundation for an antioxidant-based nanotherapeutic candidate to target pathological protein aggregation and neuroinflammation in neurodegenerative diseases.

Neuroinflammation, a common neuropathologic feature of neurodegenerative disorders including Parkinson disease (PD), is frequently exacerbated by microglial activation. The extracellular protein α-synuclein (ASYN), whose aggregation is characteristic of PD, remains a key therapeutic target, but the control of synuclein trafficking and aggregation within microglia has been challenging. First, we established that microglial internalization of monomeric ASYN was mediated by scavenger receptors (SR), CD36 and SRA1, and was rapidly accompanied by the formation of ASYN oligomers. Next, we designed a nanotechnology approach to regulate SR-mediated intracellular ASYN trafficking within microglia. We synthesized mucic acid-derivatized sugar-based amphiphilic molecules (AM) with optimal stereochemistry, rigidity, and charge for enhanced dual binding affinity to SRs and fabricated serum-stable nanoparticles via flash nanoprecipitation comprising hydrophobe cores and amphiphile shells. Treatment of microglia with AM nanoparticles decreased monomeric ASYN internalization and intracellular ASYN oligomer formation. We then engineered composite deactivating NPs with dual character, namely shell-based SR-binding amphiphiles, and core-based antioxidant poly (ferrulic acid), to investigate concerted inhibition of oxidative activation. In ASYN-challenged microglia treated with NPs, we observed decreased ASYN-mediated acute microglial activation and diminished microglial neurotoxicity caused by exposure to aggregated ASYN. When the composite NPs were administered in vivo within the substantia nigra of fibrillar ASYN-challenged wild type mice, there was marked attenuation of activated microglia. Overall, SR-targeting AM nanotechnology represents a novel paradigm in alleviating microglial activation in the context of synucleinopathies like PD and other neurodegenerative diseases.

Process mass intensity (PMI) is a benchmarking metric to evaluate the efficiency of a manufacturing process, which is indicative of the environmental impact of the process. Although this metric is commonly applied for small molecule manufacturing processes, it is less commonly applied to biologics. In this study, an Excel based tool developed by the ACS GCI Pharmaceutical Roundtable was used to calculate PMI of different manufacturing processes for a monoclonal antibody (mAb). For the upstream process, three different versions were compared: fed‐batch, fed‐batch with N‐1 perfusion, and perfusion in the N‐stage bioreactor. For each upstream process version, an appropriate downstream operational mode was evaluated from the following: a column chromatography process utilizing Protein A and anion exchange (AEX) resin, a Protein A column and an AEX membrane, and a three‐column periodic counter‐current (3C PCC) chromatography process for Protein A and an AEX membrane. The impact of these different process variations on PMI was evaluated. Of all the process inputs, water contributes about 92–94% of the overall PMI. Additionally, the upstream processes and the chromatography steps account for 32–47 and 34–54% of the overall PMI, respectively. Sensitivity analysis was performed to identify opportunities for further reducing PMI. These data indicate that a semicontinuous manufacturing process (perfusion, 3C PCC, and AEX membrane) is the most efficient process, resulting in a 23% reduction of PMI when compared with the fed batch and two‐column chromatography process. Together, PMI can be used to guide the development of efficient and environmentally sustainable mAb manufacturing processes. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1566–1573, 2018

Clearance of aggregates during protein purification is increasingly paramount as protein aggregates represent one of the major impurities in biopharmaceutical products. Aggregates, especially dimer species, represent a significant challenge for purification processing since aggregate separation coupled with high purity protein recovery can be difficult to accomplish. Biochemical characterization of the aggregate species from the hydrophobic interaction and cation exchange chromatography elution peaks revealed two different charged populations, i.e. heterogeneous charged aggregates, which led to further challenges for chromatographic removal. This paper compares multimodal versus conventional cation exchange or hydrophobic chromatography methodologies to remove heterogeneous aggregates. A full, mixed level factorial design of experiment strategy together with high throughput experimentation was employed to rapidly evaluate chromatographic parameters such as pH, conductivity, and loading. A variety of operating conditions were identified for the multimodal chromatography step, which lead to effective removal of two different charged populations of aggregate species. This multimodal chromatography step was incorporated into a monoclonal antibody purification process and successfully implemented at commercial manufacturing scale.