Process development of a SARS-CoV-2 nanoparticle vaccine.

One of the outcomes from the global COVID-19 pandemic caused by SARS-CoV-2 has been an acceleration of development timelines to provide treatments in a timely manner. For example, it has recently been demonstrated that the development of monoclonal antibody therapeutics from vector construction to IND submission can be achieved in five to six months rather than the traditional ten-to-twelve-month timeline using CHO cells [1], [2]. This timeline is predicated on leveraging existing, robust platforms for upstream and downstream processes, analytical methods, and formulation. These platforms also reduce; the requirement for ancillary studies such as cell line stability, or long-term product stability studies. Timeline duration was further reduced by employing a transient cell line for early material supply and using a stable cell pool to manufacture toxicology study materials. The development of non-antibody biologics utilizing traditional biomanufacturing processes in CHO cells within a similar timeline presents additional challenges, such as the lack of platform processes and additional analytical assay development. In this manuscript, we describe the rapid development of a robust and reproducible process for a two-component self-assembling protein nanoparticle vaccine for SARS-CoV-2. Our work has demonstrated a successful academia-industry partnership model that responded to the COVID-19 global pandemic quickly and efficiently and could improve our preparedness for future pandemic threats.

Zwitterionic carboxybetaine polymers extend the shelf-life of human platelets.

The shelf-life of human platelets preserved in vitro for therapeutic transfusion is limited because of bacterial contamination and platelet storage lesion (PSL). The PSL is the predominant factor and limiting unfavorable interactions between the platelets and the non-biocompatible storage bag surfaces is the key to alleviate PSL. Here we describe a surface modification method for biocompatible platelet storage bags that dramatically extends platelet shelf-life beyond the current US Food and Drug Administration (FDA) standards of 5 days. The surface coating of the bags can be achieved through a simple yet effective dip-coating and light-irradiation method using a biocompatible polymer. The biocompatible polymers with tunable functional groups can be routinely fabricated at any scale and impart super-hydrophilicity and non-fouling capability on commercial hydrophobic platelet storage bags. As critical parameters reflecting the platelets quality, the activation level and binding affinity with von Willebrand factor (VWF) of the platelets stored in the biocompatible platelet bags at 8 days are comparable with those in the commercial bags at 5 days. This technique also demonstrates promise for a wide range of medical and engineering applications requiring biocompatible surfaces. STATEMENT OF SIGNIFICANCE: Current standard platelet preservation techniques agitate platelets at room temperature (20-24 °C) inside a hydrophobic (e.g., polyvinyl chloride (PVC)) storage bag, thereby allowing preservation of platelets only for 5 days. A key factor leading to quality loss is the unfavorable interaction between the platelets and the non-biocompatible storage bag surfaces. Here, a surface modification method for biocompatible platelet storage bags has been created to dramatically extend platelet shelf-life beyond the current FDA standards of 5 days. The surface coating of the bags can be achieved via a simple yet effective dip-coating and light-irradiation method using a carboxybetaine polymer. This technique is also applicable to many other applications requiring biocompatible surfaces.

Nanoscavenger provides long-term prophylactic protection against nerve agents in rodents.

Nerve agents are a class of organophosphorus compounds (OPs) that blocks communication between nerves and organs. Because of their acute neurotoxicity, it is extremely difficult to rescue the victims after exposure. Numerous efforts have been devoted to search for an effective prophylactic nerve agent bioscavenger to prevent the deleterious effects of these compounds. However, low scavenging efficiency, unfavorable pharmacokinetics, and immunological problems have hampered the development of effective drugs. Here, we report the development and testing of a nanoparticle-based nerve agent bioscavenger (nanoscavenger) that showed long-term protection against OP intoxication in rodents. The nanoscavenger, which catalytically breaks down toxic OP compounds, showed a good pharmacokinetic profile and negligible immune response in a rat model of OP intoxication. In vivo administration of the nanoscavenger before or after OP exposure in animal models demonstrated protective and therapeutic efficacy. In a guinea pig model, a single prophylactic administration of the nanoscavenger effectively prevented lethality after multiple sarin exposures over a 1-week period. Our results suggest that the prophylactic administration of the nanoscavenger might be effective in preventing the toxic effects of OP exposure in humans.

Expressing a Monomeric Organophosphate Hydrolase as an EK Fusion Protein.

Organophosphate hydrolase (OPH) is a bacterial paraoxonase that demonstrates wide substrate affinity against a wide range of organophosphate (OP) compounds. OPH is expressed as a stable dimeric protein in prokaryotic hosts. We demonstrate, to the best of our knowledge, the first example of a stable OPH monomeric unit by expressing a fusion protein containing alternating glutamic acid and lysine sequences (EK) at the C-terminus. This method was able to disrupt formation of the dimer interface found in OPH due to the highly hydrated and nonfouling properties of EK. This OPH-EK fusion protein demonstrated a 70% increase in catalytic activity per active site and increased substrate affinity by reducing Km by approximately 70%. In addition, stability conferred by EK was able to overcome the stability loss caused by the elimination of the dimer interface. This strategy can potentially be used to aid in expressing prokaryotic proteins in eukaryotic hosts.

A Coating-Free Nonfouling Polymeric Elastomer.

Medical devices face nonspecific biofouling from proteins, cells, and microorganisms, which significantly contributes to complications and device failure. Imparting these devices with nonfouling capabilities remains a major challenge, particularly for those made from elastomeric polymers. Current strategies, including surface coating and copolymerization/physical blending, necessitate compromise among nonfouling properties, durability, and mechanical strength. Here, a new strategy is reported to achieve both high bulk mechanical strength and excellent surface nonfouling properties, which are typically contradictory, in one material. This is realized through a nonfouling polymeric elastomer based on zwitterionic polycarboxybetaine derivatives. By hiding both charged moieties of the zwitterionic compounds with hydrocarbon ester and tertiary amine groups, the bulk polymer itself is elastomeric and hydrophobic while its superhydrophilic surface properties are restored upon hydrolysis. This coating-free nonfouling elastomer is a highly promising biomaterial for biomedical and engineering applications.

EKylation: Addition of an Alternating-Charge Peptide Stabilizes Proteins.

For nearly 40 years, therapeutic proteins have been stabilized by chemical conjugation of polyethylene glycol (PEG), but recently zwitterionic materials have proved to be a more effective substitute. In this work, we demonstrate that genetic fusion of alternating-charge extensions consisting of anionic glutamic acid (E) and cationic lysine (K) is an effective strategy for protein stabilization. This bioinspired "EKylation" method not only confers the stabilizing benefits of poly(zwitterions) but also allows for rapid biosynthesis of target constructs. Poly(EK) peptides of different predetermined lengths were appended to the C-terminus of a native ß-lactamase and its destabilized TEM-19 mutant. The EK-modified enzymes retained biological activity and exhibited increased stability to environmental stressors such as high temperature and high-salt solutions. This one-step strategy provides a broadly applicable alternative to synthetic polymer conjugation that is biocompatible and degradable.