Rapidly accelerated development of neutralizing COVID-19 antibodies by reducing cell line and CMC development timelines.

Since the Coronavirus Disease 2019 (COVID-19) outbreak, unconventional cell line development (CLD) strategies have been taken to enable development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-neutralizing antibodies at expedited speed. We previously reported a novel chemistry, manufacturing, and control (CMC) workflow and demonstrated a much-shortened timeline of 3-6 months from DNA to investigational new drug (IND) application. Hereafter, we have incorporated this CMC strategy for many SARS-CoV-2-neutralizing antibody programs at WuXi Biologics. In this paper, we summarize the accelerated development of a total of seven antibody programs, some of which have received emergency use authorization  approval in less than 2 years. Stable pools generated under good manufacturing practice (GMP) conditions consistently exhibited similar productivity and product quality at different scales and batches, enabling rapid initiation of phase I clinical trials. Clones with comparable product quality as parental pools were subsequently screened and selected for late-stage development and manufacturing. Moreover, a preliminary stability study plan was devised to greatly reduce the time required for final clone determination and next-generation sequencing-based viral testing was implemented to support rapid conditional release of the master cell bank for GMP production. The successful execution of these COVID-19 programs relies on our robust, fit for purpose, and continuously improving CLD platform. The speed achieved for pandemic-related biologics development may innovate typical biologics development timelines and become a new standard in the industry.

Reshaping cell line development and CMC strategy for fast responses to pandemic outbreak.

The global pandemic outbreak COVID-19 (SARS-COV-2), has prompted many pharmaceutical companies to develop vaccines and therapeutic biologics for its prevention and treatment. Most of the therapeutic biologics are common human IgG antibodies, which were identified by next-generation sequencing (NGS) with the B cells from the convalescent patients. To fight against pandemic outbreaks like COVID-19, biologics development strategies need to be optimized to speed up the timeline. Since the advent of therapeutic biologics, strategies of transfection and cell line selection have been continuously improved for greater productivity and efficiency. NGS has also been implemented for accelerated cell bank testing. These recent advances enable us to rethink and reshape the chemistry, manufacturing, and controls (CMC) strategy in order to start supplying Good Manufacturing Practices (GMP) materials for clinical trials as soon as possible. We elucidated an accelerated CMC workflow for biologics, including using GMP-compliant pool materials for phase I clinical trials, selecting the final clone with product quality similar to that of phase I materials for late-stage development and commercial production.

Production of 1-Dodecanol, 1-Tetradecanol, and 1,12-Dodecanediol through Whole-Cell Biotransformation in Escherichia coli.

Medium- and long-chain 1-alkanol and α,ω-alkanediols are used in personal care products, in industrial lubricants, and as precursors for polymers synthesized for medical applications. The industrial production of α,ω-alkanediols by alkane hydroxylation primarily occurs at high temperature and pressure using heavy metal catalysts. However, bioproduction has recently emerged as a more economical and environmentally friendly alternative. Among alkane monooxygenases, CYP153A from Marinobacter aquaeolei VT8 (CYP153A M.aq ; the strain is also known as Marinobacter hydrocarbonoclasticus VT8) possesses low overoxidation activity and high regioselectivity and thus has great potential for use in terminal hydroxylation. However, the application of CYP153A M.aq is limited because it is encoded by a dysfunctional operon. In this study, we demonstrated that the operon regulator AlkR M.aq is functional, can be induced by alkanes of various lengths, and does not suffer from product inhibition. Additionally, we identified a transposon insertion in the CYP153A M.aq operon. When the transposon was removed, the expression of the operon genes could be induced by alkanes, and the alkanes could then be oxyfunctionalized by the resulting proteins. To increase the accessibility of medium- and long-chain alkanes, we coexpressed a tunable alkane facilitator (AlkL) from Pseudomonas putida GPo1. Using a recombinant Escherichia coli strain, we produced 1.5 g/liter 1-dodecanol in 20 h and 2 g/liter 1-tetradecanol in 50 h by adding dodecane and tetradecane, respectively. Furthermore, in 68 h, we generated 3.76 g/liter of 1,12-dodecanediol by adding a dodecane-1-dodecanol substrate mixture. This study reports a very efficient method of producing C12/C14 alkanols and C12 1,12-alkanediol by whole-cell biotransformation.IMPORTANCE To produce terminally hydroxylated medium- to long-chain alkane compounds by whole-cell biotransformation, substrate permeability, enzymatic activity, and the control of overoxidability should be considered. Due to difficulties in production, small amounts of 1-dodecanol, 1-tetradecanol, and 1,12-dodecanediol are typically produced. In this study, we identified an alkane-inducible monooxygenase operon that can efficiently catalyze the conversion of alkane to 1-alkanol with no detection of the overoxidation product. By coexpressing an alkane membrane facilitator, high levels of 1-dodecanol, 1-tetradecanol, and 1,12-dodecanediol could be generated. This study is significant for the bioproduction of medium- and long-chain 1-alkanol and α,ω-alkanediols.

Optimization of Micropump Performance Utilizing a Single Membrane with an Active Check Valve.

In this study, we successfully designed and tested a new micropump that utilizes an active check valve and bottom-protruding structure to achieve sample transportation. We performed theoretical analyses and numerical simulations to determine the optimal location of the active check valve. We also experimentally analyzed variations in the generated flow rate with respect to the pneumatic frequencies, actuated air pressures, and locations of the active check valve. The experimental results indicate the optimum air pressure, driving frequency, and location of the active check valve to be 68.9 kPa, 26.0 Hz, and 2.0 mm, respectively. We obtained a maximum pumping rate of 488 µL/min and a maximum pumping efficiency of 35.4%. The proposed micropump could perform a crucial function in the transportation of microfluids and could be incorporated into micro total analysis systems.

A highly flexible platform for nanowire sensor assembly using a combination of optically induced and conventional dielectrophoresis.

The number and position of assembled nanowires cannot be controlled using most nanowire sensor assembling methods. In this paper, we demonstrate a high-yield, highly flexible platform for nanowire sensor assembly using a combination of optically induced dielectrophoresis (ODEP) and conventional dielectrophoresis (DEP). With the ODEP platform, optical images can be used as virtual electrodes to locally turn on a non-contact DEP force and manipulate a micron- or nano-scale substance suspended in fluid. Nanowires were first moved next to the previously deposited metal electrodes using optical images and, then, were attracted to and arranged in the gap between two electrodes through DEP forces generated by switching on alternating current signals to the metal electrodes. A single nanowire can be assembled within 24 seconds using this approach. In addition, the number of nanowires in a single nanowire sensor can be controlled, and the assembly of a single nanowire on each of the adjacent electrodes can also be achieved. The electrical properties of the assembled nanowires were characterized by IV curve measurement. Additionally, the contact resistance between the nanowires and electrodes and the stickiness between the nanowires and substrates were further investigated in this study.

Transcriptomic analysis reveals that reactive oxygen species and genes encoding lipid transfer protein are associated with tobacco hairy root growth and branch development.

The hairy root, a specialized plant tissue that emerges from a cell transformed with transfer DNA (T-DNA) from Agrobacterium rhizogenes, can be used to study root biology and utilized in biotechnological applications. The rol genes are known to participate in the generation of hairy roots; however, the means by which the rol genes contribute to the initiation and the maintenance of hairy roots remains largely unknown. We demonstrated that tobacco hairy roots lacking either rolB or rolC exhibited fewer branch roots and lost their growth ability in long-term subculture. Additionally, a microarray analysis revealed that the expression of several genes encoding lipid transfer proteins (LTP) and reactive oxygen species (ROS)-related genes was significantly suppressed in rolB- or rolC-deficient hairy roots. We found that hairy root clones that exhibited greater branching expressed higher levels of RolB or RolC and the genes encoding LTP identified from the microarray. When hairy roots were compared with intact roots, the expression levels of LTP-encoding genes were dramatically different. In addition, ROS were present at lower levels in rolB- and rolC-deficient hairy roots. We therefore suggest that upregulating LTP and increasing the level of ROS is important for hairy root growth.

Nucleic acid amplification using microfluidic systems.

In the post-human-genome-project era, the development of molecular diagnostic techniques has advanced the frontiers of biomedical research. Nucleic-acid-based technology (NAT) plays an especially important role in molecular diagnosis. However, most research and clinical protocols still rely on the manual analysis of individual samples by skilled technicians which is a time-consuming and labor-intensive process. Recently, with advances in microfluidic designs, integrated micro total-analysis-systems have emerged to overcome the limitations of traditional detection assays. These microfluidic systems have the capability to rapidly perform experiments in parallel and with a high-throughput which allows a NAT analysis to be completed in a few hours or even a few minutes. These features have a significant beneficial influence on many aspects of traditional biological or biochemical research and this new technology is promising for improving molecular diagnosis. Thus, in the foreseeable future, microfluidic systems developed for molecular diagnosis using NAT will become an important tool in clinical diagnosis. One of the critical issues for NAT is nucleic acid amplification. In this review article, recent advances in nucleic acid amplification techniques using microfluidic systems will be reviewed. Different approaches for fast amplification of nucleic acids for molecular diagnosis will be highlighted.

An integrated chip capable of performing sample pretreatment and nucleic acid amplification for HIV-1 detection.

This study reports on a microfluidic system equipped with a sample pretreatment device and a nucleic acid amplification device for the rapid diagnosis of the human immunodeficiency virus-1 (HIV-1). The system analyzed proviral deoxyribonucleic acid (DNA) from an HIV-infected Jurkat T cell line. In order to ensure accurate diagnosis among other prevalent B-type strains, simultaneous detections of four conserved HIV-1 B-type DNA fragments were performed in this integrated microfluidic system. The entire protocol including cell lysis, extraction of DNA, polymerase chain reaction (PCR), and optical detection were successfully integrated in order to perform a rapid, automated diagnosis. Experimental results showed that four primer sets with conserved HIV-1 B-type sequences specific for the 167-bp RU5 promoter region, the 424-bp int, the 117-bp tat, and the 162-bp vpr coding regions were successfully amplified from the respective regions of the proviral DNA, even from a single infected cell. This accurate real-time detection was achieved within 95 min using the integrated optical system.

Sample pretreatment and nucleic acid-based detection for fast diagnosis utilizing microfluidic systems.

Recently, micro-electro-mechanical-systems (MEMS) technology and micromachining techniques have enabled miniaturization of biomedical devices and systems. Not only do these techniques facilitate the development of miniaturized instrumentation for biomedical analysis, but they also open a new era for integration of microdevices for performing accurate and sensitive diagnostic assays. A so-called "micro-total-analysis-system", which integrates sample pretreatment, transport, reaction, and detection on a small chip in an automatic format, can be realized by combining functional microfluidic components manufactured by specific MEMS technologies. Among the promising applications using microfluidic technologies, nucleic acid-based detection has shown considerable potential recently. For instance, micro-polymerase chain reaction chips for rapid DNA amplification have attracted considerable interest. In addition, microfluidic devices for rapid sample pretreatment prior to nucleic acid-based detection have also achieved significant progress in the recent years. In this review paper, microfluidic systems for sample preparation, nucleic acid amplification and detection for fast diagnosis will be reviewed. These microfluidic devices and systems have several advantages over their large-scale counterparts, including lower sample/reagent consumption, lower power consumption, compact size, faster analysis, and lower per unit cost. The development of these microfluidic devices and systems may provide a revolutionary platform technology for fast sample pretreatment and accurate, sensitive diagnosis.

An integrated microfluidic chip for non-immunological determination of urinary albumin.

This study presents an integrated microfluidic chip for non-immunologically determining the concentrations of albumin in clinical urine samples. This microchip integrates membrane-type micromixers and a fan-shaped micropump capable of simultaneously and precisely delivering assay reagents to react with 6 urine samples in one single operation. The experimental results show that the coefficient of variation in the pumping rate is 2.42%. More importantly, using this unique chip design, only 2 electromagnetic valves are required for the actuation of the micromixer and the micropump. The working range of the proposed microchip is 2-200 mg/L of albumin, which covers the range of interest for the determination of microalbuminuria. Moreover, statistical analysis show that the results obtained by the proposed microchip are in good agreement with the conventional detection method, based on immunological assays. This simple, inexpensive and microchip-based platform presents a promising alternative to conventional immunological assays for measurement of urinary albumin, and is well suited for clinical applications.

A microfabricated CE chip for DNA pre-concentration and separation utilizing a normally closed valve.

A simple, sequential DNA pre-concentration and separation method by using a micro-CE chip integrated with a normally closed valve, which is activated by pneumatic suction, has been developed. The CE chip is fabricated using PDMS. A surface treatment technique for coating a polymer bilayer with an anionic charge is applied to modify the surface of the microchannel. A normally closed valve with anionic surface charges forms a nanoscale channel that only allows the passage of electric current but traps the DNA samples so that they are pre-concentrated. After the pre-concentration step, a pneumatic suction force is applied to the normally closed valve. This presses down the valve membrane, which reconnects the channels. The DNA samples are then moved into a separation channel for further separation and analysis. Successful DNA pre-concentration and separation has been achieved. Fluorescent intensity at the pre-concentration area is increased by approximately 3570 times within 1.9 min of operation. The signals from the separation of phiX174 DNA/HaeIII markers are enhanced approximately 41 times after 100 s of pre-concentration time, as compared with the results using a traditional cross-shaped micro-CE chip. These results clearly demonstrate that successful DNA pre-concentration for signal enhancement and separation analysis can be performed by using this new micro-CE chip.

An integrated microfluidic system for C-reactive protein measurement.

This study presents a new microfluidic chip integrated with pneumatic micropumps, normally close microvalves and vortex-type micromixers for C-reactive protein (CRP) measurement. CRP is a protein produced during the inflammation process. It has been reported that CRP in serum can be used for risk assessment of cardiovascular diseases. In this study, CRP measurements were performed by using the integrated microfluidic chip incorporated with magnetic beads. The magnetic beads coated with CRP-specific DNA aptamers were used to recognize, purify and enrich the target CRP. The entire process including sample pre-treatment, and the interaction between the target CRP and anti-CRP antibody was automatically performed on a single chip. The chemiluminescence signal was measured using a luminometer to detect the concentration of CRP afterwards. The entire reaction time is less then 25 min, which is only about 20% of the time required when compared to using traditional bench-top machines (150 min). More importantly, the detection limit has been improved from 0.125 to 0.0125 mg/L with only half the amount of reagent consumption. The development of this microfluidic system is promising for fast, accurate, and sensitive detection of CRP.

Amyloid-degrading ability of nattokinase from Bacillus subtilis natto.

More than 20 unrelated proteins can form amyloid fibrils in vivo which are related to various diseases, such as Alzheimer's disease, prion disease, and systematic amyloidosis. Amyloid fibrils are an ordered protein aggregate with a lamellar cross-beta structure. Enhancing amyloid clearance is one of the targets of the therapy of these amyloid-related diseases. Although there is debate on whether the toxicity is due to amyloids or their precursors, research on the degradation of amyloids may help prevent or alleviate these diseases. In this study, we explored the amyloid-degrading ability of nattokinase, a fibrinolytic subtilisin-like serine protease, and determined the optimal conditions for amyloid hydrolysis. This ability is shared by proteinase K and subtilisin Carlsberg, but not by trypsin or plasmin.

A micro circulating PCR chip using a suction-type membrane for fluidic transport.

A new micromachined circulating polymerase chain reaction (PCR) chip is reported in this study. A novel liquid transportation mechanism utilizing a suction-type membrane and three microvalves were used to create a new microfluidic control module to rapidly transport the DNA samples and PCR reagents around three bio-reactors operating at three different temperatures. When operating at a membrane actuation frequency of 14.29 Hz and a pressure of 5 psi, the sample flow rate in the microfluidic control module can be as high as 18 microL/s. In addition, an array-type microheater was adopted to improve the temperature uniformity in the reaction chambers. Open-type reaction chambers were designed to facilitate temperature calibration. Experimental data from infrared images showed that the percentage of area inside the reaction chamber with a thermal variation of less than 1 degrees C was over 90% for a denaturing temperature of 94 degrees C. Three array-type heaters and temperature sensors were integrated into this new circulating PCR chip to modulate three specific operating temperatures for the denaturing, annealing, and extension steps of a PCR process. With this approach, the cycle numbers and reaction times of the three separate reaction steps can be individually adjusted. To verify the performance of this circulating PCR chip, a PCR process to amplify a detection gene (150 base pairs) associated with the hepatitis C virus was performed. Experimental results showed that DNA samples with concentrations ranging from 10(5) to 10(2)copies/microL can be successfully amplified. Therefore, this new circulating PCR chip may provide a useful platform for genetic identification and molecular diagnosis.