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.

Perfusion CHO cell culture applied to lower aggregation and increase volumetric productivity for a bispecific recombinant protein.

Secreted recombinant proteins can aggregate during cell culture. We studied a poorly-behaved bispecific scaffold that increasingly aggregated (up to 62% high molecular weight species, HMW) as a function of culture time in a fed-batch and intensified cell culture processes. We identified that protein aggregates increased with accumulated protein concentration inside the bioreactor. Furthermore, results indicated that a maximum product concentration was reached beyond which no additional soluble protein accumulated in culture even when doubling the integrated viable cell density with the intensified process, suggesting additional secreted protein was precipitating. To overcome this limitation and maintain the cell-specific productivity (qp) in culture, we explored a perfusion process where recombinant protein was continuously removed from the bioreactor to maintain low product concentration and consequently, minimize protein aggregation. We studied different viable cell densities (VCDs) inside the bioreactor (one to five-fold) and found a corresponding two-fold modulation of monomer levels. In all VCD conditions, qp was maintained. On the contrary, the previous intensified process showed an "apparent" 2.5-fold decrease in qp at the end of culture because of the presumed limited protein solubility at higher concentrations. The combination of lower aggregate levels and constant qp resulted in up to four to five-fold increase in recoverable product (i.e., monomer) with the improved perfusion process.

A novel mammalian cell line development platform utilizing nanofluidics and optoelectro positioning technology.

Generating a highly productive cell line is resource intensive and typically involves long timelines because of the need to screen large numbers of candidates in protein production studies. This has led to miniaturization and automation strategies to allow for reductions in resources and higher throughput. Current approaches rely on the use of standard cell culture vessels and bulky liquid handling equipment. New nanofludic technologies offer novel solutions to surpass these limits, further miniaturizing cell culture volumes (105 times smaller) by growing cells on custom nanofluidic chips. Berkeley Lights' OptoElectro Positioning technology projects light patterns to activate photoconductors that gently repel cells to manipulate single cells on nanofluidic culturing chips. Using a fully integrated technology platform (Beacon), common cell culture tasks can be programmed through software, allowing maintenance and analysis of thousands of cell lines in parallel on a single chip. Here, we describe the ability to perform key cell line development work on the Beacon platform. We demonstrate that commercial production Chinese hamster ovary cell lines can be isolated, cultured, screened, and exported at high efficiency. We compare this process head to head with a FACS-enabled microtiter plate-based workflow and demonstrate generation of comparable clonal cell lines with reduced resources. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1438-1446, 2018.

Characterization of phenotypic and genotypic diversity in subclones derived from a clonal cell line.

Regulatory guidelines require the sponsors to provide assurance of clonality of the production cell line, and when such evidence is not available, additional studies are typically required to further ensure consistent long-term manufacturing of the product. One potential approach to provide such assurance of clonal derivation of a production cell line is to characterize subclones generated from the original cell line and assess their phenotypic and genotypic similarity with the hypothesis that cell lines derived from a clonal bank will share performance, productivity and product quality characteristics. In this study, a production cell line that was cloned by a validated FACS approach coupled with day 0 imaging for verification of single-cell deposition was subcloned using validated FACS and imaging methods. A total of 46 subclones were analyzed for growth, productivity, product quality, copy number, and integration site analysis. Significant diversity in cell growth, protein productivity, product quality attributes, and copy number was observed between the subclones, despite stability of the parent clone over time. The diversity in protein productivity and quality of the subclones were reproduced across time and production scales, suggesting that the resulting population post sub-cloning originating from a single cell is stable but with unique properties. Overall, this work demonstrates that the characteristics of isolated subclones are not predictive of a clonally derived parental clone. Consequently, the analysis of subclones may not be an effective approach to demonstrate clonal origin of a cell bank. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:613-623, 2018.

Analysis of Tubespins as a suitable scale-down model of bioreactors for high cell density CHO cell culture.

High cell density (HCD) culture increases recombinant protein productivity via higher biomass. Compared to traditional fed-batch cultures, HCD is achieved by increased nutrient availability and removal of undesired metabolic components via regular medium replenishment. HCD process development is usually performed in instrumented lab-scale bioreactors (BR) that require time and labor for setup and operation. To potentially minimize resources and cost during HCD experiments, we evaluated a 2-week 50-mL Tubespin (TS) simulated HCD process where daily medium exchanges mimic the medium replacement rate in BR. To best assess performance differences, we cultured 13 different CHO cell lines in simulated HCD as satellites from simultaneous BR, and compared growth, metabolism, productivity and product quality. Overall, viability, cell-specific productivity and metabolism in TS were comparable to BR, but TS cell growth and final titer were lower by 25 and 15% in average, respectively. Peak viable cell densities were lower in TS than BR as a potential consequence of lower pH, different medium exchange strategy and dissolved oxygen limitations. Product quality attributes highly dependent on intrinsic molecule or cell line characteristics (e.g., galactosylation, afucosylation, aggregation) were comparable in both scales. However, product quality attributes that can change extracellularly as a function of incubation time (e.g., deamidation, C-terminal lysine, fragmentation) were in general lower in TS because of shorter residence time than HCD BR. Our characterization results and two case studies show that TS-simulated HCD cultures can be effectively used as a simple scale-down model for relative comparisons among cell lines for growth or productivity (e.g., clone screening), and for investigating effects on protein galactosylation. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:490-499, 2017.

Targeted codon optimization improves translational fidelity for an Fc fusion protein.

High levels of translational errors, both truncation and misincorporation in an Fc-fusion protein were observed. Here, we demonstrate the impact of several commercially available codon optimization services, and compare to a targeted strategy. Using the targeted strategy, only codons known to have translational errors are modified. For an Fc-fusion protein expressed in Escherichia coli, the targeted strategy, in combination with appropriate fermentation conditions, virtually eliminated misincorporation (proteins produced with a wrong amino acid sequence), and reduced the level of truncation. The use of full optimization using commercially available strategies reduced the initial errors, but introduced different misincorporations. However, truncation was higher using the targeted strategy than for most of the full optimization strategies. This targeted approach, along with monitoring of translation fidelity and careful attention to fermentation conditions is key to minimizing translational error and ensuring high-quality expression. These findings should be useful for other biopharmaceutical products, as well as any other transgenic constructs where protein quality is important.

Isolation of a human single chain antibody fragment against oligomeric alpha-synuclein that inhibits aggregation and prevents alpha-synuclein-induced toxicity.

Protein misfolding and aggregation are pathological aspects of numerous neurodegenerative diseases. Aggregates of alpha-synuclein are major components of the Lewy bodies and Lewy neurites associated with Parkinson's Disease (PD). A natively unfolded protein, alpha-synuclein can adopt different aggregated morphologies, including oligomers, protofibrils and fibrils. The small oligomeric aggregates have been shown to be particularly toxic. Antibodies that neutralize the neurotoxic aggregates without interfering with beneficial functions of monomeric alpha-synuclein can be useful therapeutics. We were able to isolate single chain antibody fragments (scFvs) from a phage displayed antibody library against the target antigen morphology using a novel biopanning technique that utilizes atomic force microscopy (AFM) to image and immobilize specific morphologies of alpha-synuclein. The scFv described here binds only to an oligomeric form of alpha-synuclein and inhibits both aggregation and toxicity of alpha-synuclein in vitro. This scFv can have potential therapeutic value in controlling misfolding and aggregation of alpha-synuclein in vivo when expressed intracellularly in dopaminergic neurons as an intrabody.

Isolating recombinant antibodies against specific protein morphologies using atomic force microscopy and phage display technologies.

Isolation of antibodies to antigens that are either unstable, exist in multiple morphologies or have very limited availability can be prohibitively difficult. Here we describe a novel technique combining the capabilities of phage display antibody technology and atomic force microscopy (AFM) that is used to isolate antibody fragments that bind to a specific morphology of the target antigen, alpha-synuclein. AFM imaging allows us to both visualize the presence and morphology of the target antigen as well as to monitor the efficiency of each step in the bio-panning process. We demonstrate that phage displayed antibodies specific to the target antigen morphology can be isolated after only two rounds of selection. The target antigen, alpha-synuclein, has been correlated with the Parkinson's disease (PD). Accumulation of alpha-synuclein fibrillar aggregates into Lewy body inclusions is a hallmark feature of PD. While alpha-synuclein can form several different aggregate morphologies including oligomers, protofibrils and fibrils, the role of these morphologies in the progression of PD is not known. The successful selection of the recombinant antibody described here can have potential therapeutic value since the single-chain fragment variable (scFv) can be expressed intracellularly to control folding and toxicity of the specific protein aggregates.

Trehalose differentially inhibits aggregation and neurotoxicity of beta-amyloid 40 and 42.

A key event in Alzheimer's disease (AD) pathogenesis is the conversion of the peptide beta-amyloid (Abeta) from its soluble monomeric form into various aggregated morphologies in the brain. Preventing aggregation of Abeta is being actively pursued as a primary therapeutic strategy for treating AD. Trehalose, a simple disaccharide, has been shown to be effective in preventing the deactivation of numerous proteins and in protecting cells against stress. Here, we show that trehalose is also effective in inhibiting aggregation of Abeta and reducing its cytotoxicity, although it shows differential effects toward Abeta40 and Abeta42. When co-incubated with Abeta40, trehalose inhibits formation of both fibrillar and oligomeric morphologies as determined by fluorescence staining and atomic force microscopy (AFM). However, when co-incubated with Abeta42, trehalose inhibits formation only of the fibrillar morphology, with significant oligomeric formation still present. When aggregated mixtures were incubated with SH-SY5Y cells, trehalose was shown to reduce the toxicity of Abeta40 mixtures, but not Abeta42. These results provide additional evidence that aggregation of Abeta into soluble oligomeric forms is a pathological step in AD and that Abeta42 in particular is more susceptible to forming these toxic oligomers than Abeta40. These results also suggest that the use of trehalose, a highly soluble, low-priced sugar, as part of a potential therapeutic cocktail to control Abeta peptide aggregation and toxicity warrants further study.