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.

Investigation of a failed DNA assay leads to identification of an unexpected contaminant in a commonly used chromatography buffer.

For mammalian cell-derived recombinant biotherapeutics, controlling host cell DNA levels below a threshold is a regulatory requirement to ensure patient safety. DNA removal during drug substance manufacture is accomplished by a series of chromatography-based purification steps and a qPCR-based analytical method is most used to measure DNA content in the purified drug substance to enable material disposition. While the qPCR approach is mature and its application to DNA measurement is widespread in the industry, it is susceptible to trace levels of process-related contaminants that are carried forward. In this study, we observed failures in spike recovery studies that are an integral component of the qPCR-based DNA testing, suggesting the presence of an inhibitory compound in the sample matrix. We generated hypotheses around the origin of the inhibitory compound and generated multiple sample matrices and deployed a suite of analytical techniques including Raman and NMR spectroscopy to determine the origin and identity of the inhibitory compound. The caustic wash step and depth filter extractables were ruled out as root causes after extensive experimentation and DNA testing. Subsequently, 2-(N-morpholino)ethanesulfonic acid (MES), a buffer used in the chromatography unit operations, was identified as the source of the contaminant. A 500-fold concentration followed by Raman and NMR spectroscopy analysis revealed the identity of the inhibitory compound as polyvinyl sulfone (PVS), an impurity that originates in the MES manufacturing process. We have implemented PVS concentration controls for incoming MES raw material, and our work highlights the need for rigor in raw material qualification and control.

Amino Acid Misincorporation Propensities Revealed through Systematic Amino Acid Starvation.

Elevated amino acid misincorporation levels during protein translation can cause disease and adversely impact biopharmaceutical product quality. Our previous work, along with that of others, identified numerous low-level unintended sequence variants. However, because of the limited analytical detection efficiency, we believed that these observations represented only a fraction of biologically relevant outcomes. Because amino acid misincorporation can be exacerbated by amino acid starvation, we believed that a more comprehensive set of sequence variants could be derived through systematic starvation. Our goals for this study were therefore (1) to systematically characterize misincorporation patterns under amino acid starvation and (2) to elucidate the major misincorporation mechanisms and propensities for cultured mammalian cells. To the best of our knowledge, this is the first study to use controlled systematic starvation to maximize the observation of unique sequence variants to provide a more holistic perspective of amino acid misincorporation. Our findings bridge the two prevailing lines of research and propose that both base mismatches during codon recognition (especially G/U and wobble mismatches) and misacylation are common and major amino acid misincorporation mechanisms. This proposal is also supported by the observation of mechanistic additivity between the base mismatch and misacylation mechanisms. In addition, we observed significant overlap in misincorporation mechanisms and propensities among cell lines and organisms. Lastly, we explored factors that can lead to codon-associated misincorporation behavior.

Amino acid misincorporation in recombinant proteins.

Proteins provide the molecular basis for cellular structure, catalytic activity, signal transduction, and molecular transport in biological systems. Recombinant protein expression is widely used to prepare and manufacture novel proteins that serve as the foundation of many biopharmaceutical products. However, protein translation bioprocesses are inherently prone to low-level errors. These sequence variants caused by amino acid misincorporation have been observed in both native and recombinant proteins. Protein sequence variants impact product quality, and their presence can be exacerbated through cellular stress, overexpression, and nutrient starvation. Therefore, the cell line selection process, which is used in the biopharmaceutical industry, is not only directed towards maximizing productivity, but also focuses on selecting clones which yield low sequence variant levels, thereby proactively avoiding potentially inauspicious patient safety and efficacy outcomes. Here, we summarize a number of hallmark studies aimed at understanding the mechanisms of amino acid misincorporation, as well as exacerbating factors, and mitigation strategies. We also describe key advances in analytical technologies in the identification and quantification of sequence variants, and some practical considerations when using LC-MS/MS for detecting sequence variants.

Effects of Non-Natural Amino Acid Incorporation into the Enzyme Core Region on Enzyme Structure and Function.

Techniques to incorporate non-natural amino acids (NNAAs) have enabled biosynthesis of proteins containing new building blocks with unique structures, chemistry, and reactivity that are not found in natural amino acids. It is crucial to understand how incorporation of NNAAs affects protein function because NNAA incorporation may perturb critical function of a target protein. This study investigates how the site-specific incorporation of NNAAs affects catalytic properties of an enzyme. A NNAA with a hydrophobic and bulky sidechain, 3-(2-naphthyl)-alanine (2Nal), was site-specifically incorporated at six different positions in the hydrophobic core of a model enzyme, murine dihydrofolate reductase (mDHFR). The mDHFR variants with a greater change in van der Waals volume upon 2Nal incorporation exhibited a greater reduction in the catalytic efficiency. Similarly, the steric incompatibility calculated using RosettaDesign, a protein stability calculation program, correlated with the changes in the catalytic efficiency.

Halogenation generates effective modulators of amyloid-Beta aggregation and neurotoxicity.

Halogenation of organic compounds plays diverse roles in biochemistry, including selective chemical modification of proteins and improved oral absorption/blood-brain barrier permeability of drug candidates. Moreover, halogenation of aromatic molecules greatly affects aromatic interaction-mediated self-assembly processes, including amyloid fibril formation. Perturbation of the aromatic interaction caused by halogenation of peptide building blocks is known to affect the morphology and other physical properties of the fibrillar structure. Consequently, in this article, we investigated the ability of halogenated ligands to modulate the self-assembly of amyloidogenic peptide/protein. As a model system, we chose amyloid-beta peptide (Aß), which is implicated in Alzheimer's disease, and a novel modulator of Aß aggregation, erythrosine B (ERB). Considering that four halogen atoms are attached to the xanthene benzoate group in ERB, we hypothesized that halogenation of the xanthene benzoate plays a critical role in modulating Aß aggregation and cytotoxicity. Therefore, we evaluated the modulating capacities of four ERB analogs containing different types and numbers of halogen atoms as well as fluorescein as a negative control. We found that fluorescein is not an effective modulator of Aß aggregation and cytotoxicity. However, halogenation of either the xanthenes or benzoate ring of fluorescein substantially enhanced the inhibitory capacity on Aß aggregation. Such Aß aggregation inhibition by ERB analogs except rose bengal correlated well to the inhibition of Aß cytotoxicity. To our knowledge, this is the first report demonstrating that halogenation of aromatic rings substantially enhance inhibitory capacities of small molecules on Aß-associated neurotoxicity via Aß aggregation modulation.

Different fates of Alzheimer's disease amyloid-ß fibrils remodeled by biocompatible small molecules.

Amyloid fibrils implicated in numerous human diseases are thermodynamically very stable. Stringent conditions that would not be possible in a physiological environment are often required to disrupt the stable fibrils. Recently, there is increasing evidence that small molecules can remodel amyloid fibrils in a physiologically relevant manner. In order to investigate possible fibril remodeling mechanisms using this approach, we performed comparative studies on the structural features of the different amyloid-ß (Aß) aggregates remodeled from Aß fibrils by three biocompatible small molecules: methylene blue; brilliant blue G; and erythrosine B. Combined with circular dichroism (CD), immuno-blotting, transmission electron microscopy (TEM), and atomic force microscopy (AFM) results, it was found that brilliant blue G- and erythrosine B-treatment generate fragmented Aß fibrils and protofibrils, respectively. In contrast, incubation of the Aß fibrils with methylene blue perturbs fibrillar structure, leading to amorphous Aß aggregates. Our findings provide insights on the molecular mechanism of amyloid fibril formation and remodeling and also illustrate the possibility of controlled changes in biomolecule nanostructures.

Simplifying and streamlining Escherichia coli-based cell-free protein synthesis.

Escherichia coli cell-free protein synthesis (CFPS) uses E. coli extracts to make active proteins in vitro. The basic CFPS reaction mixture is comprised of four main reagent components: (1) energy source and CFPS chemicals, (2) DNA encoding the protein of interest, (3) T7 RNA Polymerase (RNAP) for transcription, and (4) cell extract for translation. In this work, we have simplified and shortened the protocols for preparing the CFPS chemical mixture, cell extract, and T7 RNAP. First, we streamlined the workflow for preparing the CFPS chemical solutions by combining all the chemicals into a single reagent mixture, which we call Premix. We showed that productive cell extracts could be made from cells grown in simple shake flasks, and we also truncated the preparation protocol. Finally, we discovered that T7 RNAP purification was not necessary for CFPS. Crude lysate from cells over-expressing T7 RNAP could be used without deleteriously affecting protein production. Using chloramphenicol acetyltransferase (CAT) as a model protein, we showed that these streamlined protocols still support high-yielding CFPS. These simplified procedures save time and offer greater accessibility to our laboratory's CFPS technology.

Xanthene food dye, as a modulator of Alzheimer's disease amyloid-beta peptide aggregation and the associated impaired neuronal cell function.

BACKGROUND: Alzheimer's disease (AD) is the most common form of dementia. AD is a degenerative brain disorder that causes problems with memory, thinking and behavior. It has been suggested that aggregation of amyloid-beta peptide (Aß) is closely linked to the development of AD pathology. In the search for safe, effective modulators, we evaluated the modulating capabilities of erythrosine B (ER), a Food and Drug Administration (FDA)-approved red food dye, on Aß aggregation and Aß-associated impaired neuronal cell function. METHODOLOGY/PRINCIPAL FINDINGS: In order to evaluate the modulating ability of ER on Aß aggregation, we employed transmission electron microscopy (TEM), thioflavin T (ThT) fluorescence assay, and immunoassays using Aß-specific antibodies. TEM images and ThT fluorescence of Aß samples indicate that protofibrils are predominantly generated and persist for at least 3 days. The average length of the ER-induced protofibrils is inversely proportional to the concentration of ER above the stoichiometric concentration of Aß monomers. Immunoassay results using Aß-specific antibodies suggest that ER binds to the N-terminus of Aß and inhibits amyloid fibril formation. In order to evaluate Aß-associated toxicity we determined the reducing activity of SH-SY5Y neuroblastoma cells treated with Aß aggregates formed in the absence or in the presence of ER. As the concentration of ER increased above the stoichiometric concentration of Aß, cellular reducing activity increased and Aß-associated reducing activity loss was negligible at 500 µM ER. CONCLUSIONS/SIGNIFICANCE: Our findings show that ER is a novel modulator of Aß aggregation and reduces Aß-associated impaired cell function. Our findings also suggest that xanthene dye can be a new type of small molecule modulator of Aß aggregation. With demonstrated safety profiles and blood-brain permeability, ER represents a particularly attractive aggregation modulator for amyloidogenic proteins associated with neurodegenerative diseases.

A safe, blood-brain barrier permeable triphenylmethane dye inhibits amyloid-ß neurotoxicity by generating nontoxic aggregates.

Growing evidence suggests that on-pathway amyloid-ß (Aß) oligomers are primary neurotoxic species and have a direct correlation with the onset of Alzheimer's disease (AD). One promising therapeutic strategy to block AD progression is to reduce the levels of these neurotoxic Aß species using small molecules. While several compounds have been shown to modulate Aß aggregation, compounds with such activity combined with safety and high blood-brain barrier (BBB) permeability have yet to be reported. Brilliant Blue G (BBG) is a close structural analogue of a U.S. Food and Drug Administration (FDA)-approved food dye and has recently garnered prominent attention as a potential drug to treat spinal cord injury due to its neuroprotective effects along with BBB permeability and high degree of safety. In this work, we demonstrate that BBG is an effective Aß aggregation modulator, which reduces Aß-associated cytotoxicity in a dose-dependent manner by promoting the formation of off-pathway, nontoxic aggregates. Comparative studies of BBG and three structural analogues, Brilliant Blue R (BBR), Brilliant Blue FCF (BBF), and Fast Green FCF (FGF), revealed that BBG is most effective, BBR is moderately effective, and BBF and FGF are least effective in modulating Aß aggregation and cytotoxicity. Therefore, the two additional methyl groups of BBG and other structural differences between the congeners are important in the interaction of BBG with Aß leading to formation of nontoxic Aß aggregates. Our findings support the hypothesis that generating nontoxic aggregates using small molecule modulators is an effective strategy for reducing Aß cytotoxicity. Furthermore, key structural features of BBG identified through structure-function studies can open new avenues into therapeutic design for combating AD.

Cell-free production of transducible transcription factors for nuclear reprogramming.

Ectopic expression of a defined set of transcription factors chosen from Oct3/4, Sox2, c-Myc, Klf4, Nanog, and Lin28 can directly reprogram somatic cells to pluripotency. These reprogrammed cells are referred to as induced pluripotent stem cells (iPSCs). To date, iPSCs have been successfully generated using lentiviruses, retroviruses, adenoviruses, plasmids, transposons, and recombinant proteins. Nucleic acid-based approaches raise concerns about genomic instability. In contrast, a protein-based approach for iPSC generation can avoid DNA integration concerns as well as provide greater control over the concentration, timing, and sequence of transcription factor stimulation. Researchers recently demonstrated that polyarginine peptide conjugation can deliver recombinant protein reprogramming factor (RF) cargoes into cells and reprogram somatic cells into iPSCs. However, the protein-based approach requires a significant amount of protein for the reprogramming process. Producing fusion RFs in the large amounts required for this approach using traditional heterologous in vivo production methods is difficult and cumbersome since toxicity, product aggregation, and proteolysis by endogenous proteases limit yields. In this work, we show that cell-free protein synthesis (CFPS) is a viable option for producing soluble and functional transducible transcription factors for nuclear reprogramming. We used an E. coli-based CFPS system to express the above set of six human RFs as fusion proteins, each with a nona-arginine (R9) protein transduction domain. Using the flexibility offered by the CFPS platform, we successfully addressed proteolysis and protein solubility problems to produce full-length and soluble R9-RF fusions. We subsequently showed that R9-Oct3/4, R9-Sox2, and R9-Nanog exhibit cognate DNA-binding activities, R9-Nanog translocates across the plasma and nuclear membranes, and R9-Sox2 exerts transcriptional activity on a known downstream gene target.