Analytical characterization of broadly neutralizing antibody CAP256LS heavy chain clipping during manufacturing development.

Broadly neutralizing antibody (bNAb) CAP256-VRC26.25 (abbreviated CAP256LS), a human IgGI monoclonal antibody targeting the V1V2 site of the HIV-1 envelope, has demonstrated high therapeutic potential as a broadly neutralizing monoclonal antibody against HIV-1. During the process development, a heavy chain fragmentation (clipping) was observed, that led to a relative potency reduction. In this report, we highlighted a series of process and product mitigation strategies deployed to advance this product. We have detailed how analytical characterization tools, especially the microchip reduced capillary gel electrophoresis (CGE-SDS), played a pivotal role in identifying the development issues and in providing measurements to guide implementation of mitigation strategies.

Tyrosine O-sulfation proteoforms affect HIV-1 monoclonal antibody potency.

CAP256V2LS, a broadly neutralizing monoclonal antibody (bNAb), is being pursued as a promising drug for HIV-1 prevention. The total level of tyrosine-O-sulfation, a post-translational modification, was known to play a key role for antibody biological activity. More importantly, here wedescribe for the first time the significance of the tyrosine-O-sulfation proteoforms. We developed a hydrophobic interaction chromatography (HIC) method to separate and quantify different sulfation proteoforms, which led to the direct functionality assessment of tyrosine-sulfated species. The fully sulfated (4-SO3) proteoform demonstrated the highest in vitro relative antigen binding potency and neutralization efficiency against a panel of HIV-1 viruses. Interestingly, highly variable levels of 4-SO3 were produced by different clonal CHO cell lines, which helped the bNAb process development towards production of a highly potent CAP256V2LS clinical product with high 4-SO3 proteoform. This study presents powerful insight for any biotherapeutic protein development where sulfation may play an important role in product efficacy.

Comprehensive Flow Cytometry Analysis of PEI-Based Transfections for Virus-Like Particle Production.

The generation of stable clones for biomolecule production is a common but lengthy and labor-intensive process. For complex molecules, such as viruses or virus-like particles (VLPs), the timeline becomes even more cumbersome. Thus, in the early stages of development, transient production methods serve as a reasonable alternative to stable clone construction. In this work, an investigation of a polyethylenimine- (PEI-) based transfection method for the transient production of Chikungunya (Chik) VLPs, a vaccine candidate molecule, was undertaken. This effort focuses on tracking cell population responses during transfection, understanding how process changes affect these responses, and monitoring patterns in cell performance over the culture duration. Plasmid labeling and VLP staining were employed to comprehensively track cells via flow cytometry and to draw correlations between plasmid DNA (pDNA) uptake and the resulting VLP expression. The method detected high transfection efficiency (≥97%) in all samples tested and demonstrated the capability to track kinetics of plasmid-cell binding. With varied transfection cell concentrations, the pDNA binding kinetics are altered and saturation binding is observed in the lowest cell concentration sample tested in less than 3 hours of incubation. Interestingly, in all samples, the flow cytometry analysis of relative pDNA amount versus VLP expression staining showed that cells which contained fewer pDNA complexes resulted in the highest levels of VLP stain. Finally, to determine the potential breadth of our observations, we compared daily expression patterns of ChikVLP with a reporter, monomeric GFP molecule. The similarities detected suggest the interpretations presented here to likely be more broadly informative and applicable to PEI-based transient production of additional biological products as well.

Characterization of AEBSF-antibody modifications for a protease inhibitor supplementation strategy.

Application of a protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), during the cell culture process was demonstrated to effectively reduce proteolytic activity at a specific amino acid site during the production of an HIV-1 broadly neutralizing antibody (bNAb). However, the addition of AEBSF could potentially introduce some modifications to the bNAb protein. Experimental design from sample preparation to LC-MS characterization was performed using middle-up and bottom-up approaches to identify AEBSF-modified species for the bNAb using an AEBSF supplementation in the cell culture media. Modified species along with the unmodified control sample were also subjected to binding activity assessment. The results showed that two amino acids (Tyr177 and Lys250) were susceptible to AEBSF modification in the bNAb test articles but at a negligible level and not in the CDR regions, which therefore did not reduce the in vitro binding activity of the bNAb.

Development of an alternating tangential flow (ATF) perfusion-based transient gene expression (TGE) bioprocess for universal influenza vaccine.

An alternating tangential flow (ATF) perfusion-based transient gene expression (TGE) bioprocess has been developed using human embryonic kidney (HEK) 293 cells to produce H1-ss-np, a promising candidate for a universal influenza vaccine. Two major adjustments were taken to improve the process: (1) eliminate the interference of microbubbles during gene transfection; and (2) utilize an ATF perfusion system for a prolonged culture period. As a result, a closed-operation 9-days ATF perfusion-based TGE bioprocess was developed. The TGE bioprocess showed continuous cell growth with high cell viability and prolonged cellular productivity that achieved recombinant product level of ~270 mg/L which was more than two times that of 4-days base-line TGE bioprocess. In addition, the consumables cost per milligram for ATF perfusion-based TGE bioprocess was ~70% lower than that of the base-line TGE bioprocess suggesting high cost savings potential in vaccine manufacturing. Based on the lower contamination risk, higher productivity, and cost efficiency, the ATF perfusion-based TGE bioprocess can likely provide potential benefits to many future applications in vaccine and drug manufacturing.

Quantification of residual AEBSF-related impurities by reversed-phase liquid chromatography.

During research of a broadly neutralizing antibody (bNAb) for HIV-1 infection, site-specific clipping was observed during cell culture incubation. Protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), was supplemented to the cell culture feeding to mitigate clipping as one of the control strategies. It led to the need and development of a new assay to monitor the free AEBSF-related impurities during the purification process. In this work, a reversed-phase liquid chromatography (RPLC-UV) method was developed to measure the total concentration of AEBSF and its major degradant product, 4-(aminoethyl) benzenesulfonic acid (AEBS-OH). This quantitative approach involved hydrolysis pre-treatment to drive all AEBSF to AEBS-OH, a filtration step to remove large molecules, followed by RPLC-UV analysis. The method was qualified and shown to be capable of measuring AEBS-OH down to 0.5 µM with good accuracy and precision, which was then applied for process clearance studies. The results demonstrated that a Protein A purification step in conjunction with a mock ultrafiltration/diafiltration (UF/DF) step could remove AEBSF-related impurities below the detection level. Overall, this study is the first to provide a unique approach for monitoring the clearance of free AEBSF and its related degradant, AEBS-OH, in support of the bNAb research.

Dynamic protein assembly by programmable DNA strand displacement.

Inspired by the remarkable ability of natural protein switches to sense and respond to a wide range of environmental queues, here we report a strategy to engineer synthetic protein switches by using DNA strand displacement to dynamically organize proteins with highly diverse and complex logic gate architectures. We show that DNA strand displacement can be used to dynamically control the spatial proximity and the corresponding fluorescence resonance energy transfer between two fluorescent proteins. Performing Boolean logic operations enabled the explicit control of protein proximity using multi-input, reversible and amplification architectures. We further demonstrate the power of this technology beyond sensing by achieving dynamic control of an enzyme cascade. Finally, we establish the utility of the approach as a synthetic computing platform that drives the dynamic reconstitution of a split enzyme for targeted prodrug activation based on the sensing of cancer-specific miRNAs.

Hydrolysis-Kinetic Study of AEBSF, a Protease Inhibitor Used during Cell-Culture Processing of the HIV-1 Broadly Neutralizing Antibody CAP256-VRC25.26.

One approach to mitigate product clipping during HIV mAb CAP256-VRC26.25 cell-culture development is the addition of the protease inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) to the cell-culture media. AEBSF can undergo hydrolysis to form an inactive compound, 4-(2-aminoethyl) benzenesulfonic acid. Using mass-spectrometry detection, a kinetic profile of AEBSF hydrolysis was generated for conditions simulating those of cell culture at pH 7.0 and 37 °C. It was found that increasing the pH or the temperature could accelerate AEBSF hydrolysis. The kinetic-study results in this report provide an analytical characterization and guidance when optimizing an AEBSF-addition strategy for product-clipping control during cell-culture development and offer an alternative approach for AEBSF-related clearance studies post protein production.

Post-Translational Modification of Bionanoparticles as a Modular Platform for Biosensor Assembly.

Context driven biosensor assembly with modular targeting and detection moieties is gaining significant attentions. Although protein-based nanoparticles have emerged as an excellent platform for biosensor assembly, current strategies of decorating bionanoparticles with targeting and detection moieties often suffer from unfavorable spacing and orientation as well as bionanoparticle aggregation. Herein, we report a highly modular post-translational modification approach for biosensor assembly based on sortase A-mediated ligation. This approach enables the simultaneous modifications of the Bacillus stearothermophilus E2 nanoparticles with different functional moieties for antibody, enzyme, DNA aptamer, and dye decoration. The resulting easy-purification platform offers a high degree of targeting and detection modularity with signal amplification. This flexibility is demonstrated for the detection of both immobilized antigens and cancer cells.

Fluorescent protein-based molecular beacons by zinc finger protein-guided assembly.

Molecular beacons (MBs) are stem-loop structured oligonucleotides capable of sensitive and specific nucleic acid detection. However, large-scale usage of MBs for high throughput applications has been hindered by the many expensive and tedious chemical modifications. In this paper, we reported a new class of fluorescent protein-based molecular beacons (FP-MBs) based on zinc finger protein-guided assembly. The design consisted of a single oligonucleotide which forms a hairpin structure with an extending arm on both ends for the attachment of two different fluorescent proteins upon simple mixing. This new design allows for simplified MB assembly and modifications for a wide range of applications.

Halo-tag mediated self-labeling of fluorescent proteins to molecular beacons for nucleic acid detection.

We report here the generation of a fluorescent protein (FP)-based dual molecular beacon (MB) system for nucleic acid detection. Halo-tag mediated conjugation was used for the site-specific decoration of MBs with two different FP fusions, thereby enabling easy detection of target sequences by fluorescence resonance energy transfer or FRET. Enhanced intracellular delivery was demonstrated by simply tethering a well-known TAT peptide sequence to the N-terminus of the fusion proteins.

Detection of RNA viruses: current technologies and future perspectives.

RNA viruses constitute one of the major classes of pathogenic organisms causing human diseases, with varying degrees of severity. This review summarizes the conventional and emerging technologies that are available for the detection of these organisms. Cell culture-based techniques for viral detection have been popular since their inception and continue to be the gold standard against which all other techniques. Over many years, these techniques have undergone some radical changes, reducing the total time needed for detection and improving sensitivity, although even with their reliability and improved features they are being slowly replaced by nucleic acid-based technologies. These molecular detection techniques have revolutionized the area of viral detection by their high sensitivity, selectivity, and short detection time. The majority of nucleic acid-based techniques depend on amplifying viral RNA; however, there are some newer emerging techniques that detect viral RNA in live cells using various configurations of florescent probes. In addition, nucleic acid-based technology has made it possible for multiviral detection with either multiplex polymerase chain reaction assays or microarrays. Every technique described in this review has its own unique abilities, making them indispensable for viral detection. However, we believe that nucleic acid-based technologies will find widespread use after being standardized, limiting other technologies to very specific uses.