Development of generic metabolic Raman calibration models using solution titration in aqueous phase and data augmentation for in-line cell culture analysis.

This study presents a novel approach for developing generic metabolic Raman calibration models for in-line cell culture analysis using glucose and lactate stock solution titration in an aqueous phase and data augmentation techniques. First, a successful set-up of the titration method was achieved by adding glucose or lactate solution at several different constant rates into the aqueous phase of a bench-top bioreactor. Subsequently, the in-line glucose and lactate concentration were calculated and interpolated based on the rate of glucose and lactate addition, enabling data augmentation and enhancing the robustness of the metabolic calibration model. Nine different combinations of spectra pretreatment, wavenumber range selection, and number of latent variables were evaluated and optimized using aqueous titration data as training set and a historical cell culture data set as validation and prediction set. Finally, Raman spectroscopy data collected from 11 historical cell culture batches (spanning four culture modes and scales ranging from 3 to 200 L) were utilized to predict the corresponding glucose and lactate values. The results demonstrated a high prediction accuracy, with an average root mean square errors of prediction of 0.65 g/L for glucose, and 0.48 g/L for lactate. This innovative method establishes a generic metabolic calibration model, and its applicability can be extended to other metabolites, reducing the cost of deploying real-time cell culture monitoring using Raman spectroscopy in bioprocesses.

Developing an ultra-intensified fed-batch cell culture process with greatly improved performance and productivity.

Intensified fed-batch (IFB), a popular cell culture intensification strategy, has been widely used for productivity improvement through high density inoculation followed by fed-batch cultivation. However, such an intensification strategy may counterproductively induce rapidly progressing cell apoptosis and difficult-to-sustain productivity. To improve culture performance, we developed a novel cell culture process intermittent-perfusion fed-batch (IPFB) which incorporates one single or multiple cycles of intermittent perfusion during an IFB process for better sustained cellular and metabolic behaviors and notably improved productivity. Unlike continuous perfusion or other semi-continuous processes such as hybrid perfusion fed-batch with only early-stage perfusion, IPFB applies limited times of intermittent perfusion in the mid-to-late stage of production and still inherits bolus feedings on nonperfusion days as in a fed-batch culture. Compared to IFB, an average titer increase of ~45% was obtained in eight recombinant CHO cell lines studied. Beyond IPFB, ultra-intensified IPFB (UI-IPFB) was designed with a markedly elevated seeding density of 20-80 × 106 cell/mL, achieved through the conventional alternating tangential flow filtration (ATF) perfusion expansion followed with a cell culture concentration step using the same ATF system. With UI-IPFB, up to ~6 folds of traditional fed-batch and ~3 folds of IFB productivity were achieved. Furthermore, the application grounded in these two novel processes showed broad-based feasibility in multiple cell lines and products of interest, and was proven to be effective in cost of goods reduction and readily scalable to a larger scale in existing facilities.

Establishment of a semi-continuous scale-down clone screening model for intensified perfusion culture.

PURPOSE: Perfusion cultures have been extensively used in the biotechnology industry to achieve high yields of recombinant products, especially those with stability issue. The WuXiUP™ platform represents a novel intensified perfusion that can achieve ultra-high productivity. This study describes a representative scale-down 24-deep well plate (24-DWP) cell culture model for intensified perfusion clone screening. METHODS: Clonal cell lines were expanded and evaluated in 24-DWP semi-continuous culture. Cell were sampled and counted daily with the aid of an automated liquid handler and high-throughput cell counter. To mimic perfusion culture, 24-DWP plates were spun down and resuspended with fresh medium daily. Top clones were ranked based on growth profiles and productivities. The best performing clones were evaluated on bioreactors. RESULTS: The selected clones achieved volumetric productivity (Pv) up to 5 g/L/day when expressing a monoclonal antibody, with the accumulative harvest Pv exceeding 60 g/L in a 21-day cell culture. Product quality attributes of clones cultured in 24-DWP were comparable with those from bioreactors. A high seeding strategy further shortened the clone screening timeline. CONCLUSION: In this study, a 24-DWP semi-continuous scale-down model was successfully developed to screen for cell lines suitable for intensified perfusion culture.

Correlation between the Protein Pharmaceutical Surface Activity and Interfacial Stability.

The interaction of protein drugs with the air-liquid interface plays a crucial role in the overall stability in aqueous formulations, particularly when the adsorbed proteins are subjected to the surface flow. Nonionic surfactants are usually added into the formulation solutions to address this issue. A diversity of studies have been focused on the usage of surfactants, the stability mechanism of surfactants, or seeking new pharmaceutical surfactants. However, the real protagonist, the basic properties of protein drugs, was neglected, which may play a vital role in the stability of protein drugs. Herein, we aim to clarify the correlation between the surface behavior of proteins and the interfacial stability. A force tensiometer is used to track the surface tension reduction and the competition between surfactants and proteins at the surface. We find that the surface behaviors of proteins vary with storage temperature and protein types including monoclonal antibodies (mAb), bispecific monoclonal antibodies (BsAb), and antibody-drug conjugates (ADCs). Especially for the protein stored at 5 °C, the surface activity of proteins is better than that of surfactants. It indicates that the ability of proteins to adsorb at the interface should not be ignored compared to surfactants. The significant difference in the interfacial stability of protein pharmaceuticals formulated in the same buffer and excipients as well as the surfactants with the same concentration further confirms the interfacial adsorption capacity of proteins that should not be ignored. These findings provide a new angle and valuable insights into the correlation between the surface activity of the proteins and interfacial stability, which may pave the way for future preformulation studies on therapeutic proteins and broaden the thoughts of formulation development.

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.

Improving an intensified and integrated continuous bioprocess platform for biologics manufacturing.

The WuXi Biologics' Ultra-high Productivity platform (WuXiUP) technology is an innovative and integrated platform of continuous biomanufacturing. Through process intensification, the platform enables continuous manufacturing of almost any type of biologics and delivers processes with ultra-high productivity. In this paper, a new case study producing a monoclonal antibody (mAb) via the WuXiUP process was further optimized. Key process parameters like culture temperature, basal media, and perfusion rate were evaluated to ensure an enhanced and robust process. To improve process efficiency for downstream processing, a continuous dual-pore size hollow fiber cell separation and product harvest system were also designed to complement the increased harvest volume from upstream production. In this case study, a significant protein concentration increase and harvest volume reduction were achieved by the application of the new WuXiUP platform.

Optimization of hydrogen-ion storage performance of tungsten trioxide nanowires by niobium doping.

The transport and storage of ions within solid state structures is a fundamental limitation for fabricate more advanced electrochemical energy storage, memristor, and electrochromic devices. Crystallographic shear structure can be induced in the tungsten bronze structures composed of corner-sharing WO6octahedra by the addition of edge-sharing NbO6octahedra, which might provide more storage sites and more convenient transport channels for external ions such as hydrogen ions and alkali metal ions. Here, we show that Nb2O5·15WO3nanowires (Nb/W = 0.008) with long length-diameter ratio, smooth surface, and uniform diameter have been successfully synthesized by a simple hydrothermal method. The Nb2O5·15WO3nanowires do exhibit more advantages over h-WO3nanowires in electrochemical hydrogen ion storage such as smaller polarization, larger capacity (71 mAh g-1, at 10C, 1C = 100 mA g-1), better cycle performance (remain at 99% of the initial capacity after 200 cycles at 100C) and faster H+ions diffusion kinetics. It might be the crystallographic shear structure induced by Nb doping that does result in the marked improvement in the hydrogen-ion storage performance of WO3. Therefore, complex niobium tungsten oxide nanowires might offer great promise for the next generation of electrochemical energy and information storage devices.

Optimized process operations reduce product retention and column clogging in ATF-based perfusion cell cultures.

Product retention in hollow fibers is a common issue in ATF-based cell culture system. In this study, the effects of four major process factors on product (therapeutic antibody/recombinant protein) retention were investigated using Chinese hamster ovary cell. Hollow fibers made of polysulfone presented a product retention rate from 15% ± 8 to 43% ± 18% higher than those made of polyether sulfone varying with specific processes. Higher harvest flowrate and ATF exchange rate increased product retention by 13% ± 10% and up to 31% ± 13%, respectively. Hollow fibers with larger pore sizes (0.65 µm) appeared to have increased product retention by 38% ± 7% compared with smaller ones (0.2 µm) in this study. Further investigation revealed that the effects of pore size on retention could be correlated to the particle size distribution in the cell culture broth. A hollow fiber with a larger pore size (>0.5 µm) may reduce protein retention when small particles (approximately 0.01-0.2 µm in diameter) are dominant in the culture. However, if majority of the particles are larger than 0.2 µm in diameter, hollow fiber with smaller pore sizes (0.2 µm) could be a solution to reducing product retention. Alternatively, process optimization may modulate particle size distribution towards reduced production retention with selected ATF hollow fibers. This study for the first time highlights the importance of matching proper pore sizes of hollow fibers with the cell culture particles distribution and offers methods to reducing product retention and ATF column clogging in perfusion cell cultures. KEY POINTS: The material of ATF column could impact product retention during perfusion culture. Higher harvest flowrate and ATF exchange rate increased product retention. Matching culture particle size and ATF pore size is critical for retention modulation.

Highly Robust Non-Noble Alkaline Hydrogen-Evolving Electrocatalyst from Se-Doped Molybdenum Disulfide Particles on Interwoven CoSe2 Nanowire Arrays.

Developing efficient non-noble and earth-abundant hydrogen-evolving electrocatalysts is highly desirable for improving the energy efficiency of water splitting in base. Molybdenum disulfide (MoS2 ) is a promising candidate, but its catalytic activity is kinetically retarded in alkaline media due to the unfavorable water adsorption and dissociation feature. A heterogeneous electrocatalyst is reported that is constructed by selenium-doped MoS2 (Se-MoS2 ) particles on 3D interwoven cobalt diselenide (CoSe2 ) nanowire arrays that drives the hydrogen evolution reaction (HER) with fast reaction kinetics in base. The resultant Se-MoS2 /CoSe2 hybrid exhibits an outstanding catalytic HER performance with extremely low overpotentials of 30 and 93 mV at 10 and 100 mA cm-2 in base, respectively, which outperforms most of the inexpensive alkaline HER catalysts, and is among the best alkaline catalytic activity reported so far. Moreover, this hybrid catalyst shows exceptional catalytic performance with very low overpotentials of 84 and 95 mV at 10 mA cm-2 in acidic and neutral electrolytes, respectively, implying robust pH universality of this hybrid catalyst. This work may provide new inspirations for the development of high-performance MoS2 -based HER electrocatalysts in unfavorable basic media for promising catalytic applications.

Improved surface-enhanced Raman scattering (SERS) sensitivity to molybdenum oxide nanosheets via the lightning rod effect with application in detecting methylene blue.

MoO2 nanomaterials show a superior surface-enhanced Raman scattering (SERS) property due to their high concentration of free electrons and low resistivity. However, the physical process of semiconductor-based SERS is still elusive because there are many factors that affect the local electromagnetic field intensity and the subsequent Raman intensity of the molecules in close proximity to the semiconductor nanomaterials. Herein, we investigate the important contribution of surface morphology to molybdenum oxide SERS. The MoO3/MoO2 nanosheets (NSs) are synthesized by oxidizing MoO2 NS, and the surface roughness of MoO3 can be controlled through adjusting the oxidization time. Compared with the MoO2 NS before oxidization, the MoO3/MoO2 NSs exhibit a much stronger SERS signal, which favors their application as a SERS substrate to detect trace amounts of methylene blue molecules. The minimum detectable concentration is up to 10-9 M and the maximum enhancement factor is about 1.4 × 105. Meanwhile, excellent signal reproducibility is also observed using the MoO3/MoO2 NSs as the SERS substrate. A simulated electric field distribution shows that a stronger electric field enhancement is formed due to the lightning rod effect in the gap of corrugated MoO3 NSs. These results demonstrate that the surface topography of molybdenum oxide may play a more important role than their oxidation state in SERS signal enhancement.

Surface polarons and optical micro-cavity modulated broad range multi-mode emission of Te-doped CdS nanowires.

The optical confinement and strong carrier coupling within a semiconductor nanostructure cavity are crucial for the modulation of emission properties. Fundamental understanding of the light-matter interaction in a low dimensional system is important. In this paper, we synthesized high-quality hexagonal Te-doped CdS nanowires by two-step chemical vapor deposition and investigated systematically the doping concentration, temperature, excitation power, excitation wavelength dependent Raman, photoluminescence and carrier lifetime decay. Scanning electron microscopy, energy dispersive x-ray spectrometry and x-ray diffraction confirmed Te-doping in the as-prepared samples. The strong surface optical (SO) phonon mode is observed in the micro-Raman spectra of an individual Te-CdS nanowire, which is unsuitable in large-sized structures. In situ micro-photoluminescence (µ-PL) characterization shows dominant confined defect state emission with whispering gallery mode (WGM) characteristics. The emission peak position shifts under increased excitation power, demonstrating the inelastic scattering by bound carriers. In addition, the short wavelength emission modes are dominant at a low temperature (80 K) while the long wavelength emission modes are dominant at a high temperature (300 K) due to different recombination processes contributing to the WGM resonant bands, which was also confirmed by the time-resolved PL measurement. All these results reflect strong coupling between the surface evanescent-wave in the WGM cavity and the SO phonon/polaron, which will facilitate the rational tailoring of surface/interface relevant properties for nanophotonic device applications.

Spin-exciton interaction and related micro-photoluminescence spectra of ZnSe:Mn DMS nanoribbon.

For their spintronic applications the magnetic and optical properties of diluted magnetic semiconductors (DMS) have been studied widely. However, the exact relationships between the magnetic interactions and optical emission behaviors in DMS are not well understood yet due to their complicated microstructural and compositional characters from different growth and preparation techniques. Manganese (Mn) doped ZnSe nanoribbons with high quality were obtained by using the chemical vapor deposition (CVD) method. Successful Mn ion doping in a single ZnSe nanoribbon was identified by elemental energy-dispersive x-ray spectroscopy mapping and micro-photoluminescence (PL) mapping of intrinsic d-d optical transition at 580 nm, i.e. the transition of 4 T 1(4 G) â†’ 6 A 1(6 s),. Besides the d-d transition PL peak at 580 nm, two other PL peaks related to Mn ion aggregates in the ZnSe lattice were detected at 664 nm and 530 nm, which were assigned to the d-d transitions from the Mn2+-Mn2+ pairs with ferromagnetic (FM) coupling and antiferromagnetic (AFM) coupling, respectively. Moreover, AFM pair formation goes along with strong coupling with acoustic phonon or structural defects. These arguments were supported by temperature-dependent PL spectra, power-dependent PL lifetimes, and first-principle calculations. Due to the ferromagnetic pair existence, an exciton magnetic polaron (EMP) is formed and emits at 460 nm. Defect existence favors the AFM pair, which also can account for its giant enhancement of spin-orbital coupling and the spin Hall effect observed in PRL 97, 126603(2006) and PRL 96, 196404(2006). These emission results of DMS reflect their relation to local sp-d hybridization, spin-spin magnetic coupling, exciton-spin or phonon interactions covering structural relaxations. This kind of material can be used to study the exciton-spin interaction and may find applications in spin-related photonic devices besides spintronics.

Advancement in bioprocess technology: parallels between microbial natural products and cell culture biologics.

The emergence of natural products and industrial microbiology nearly eight decades ago propelled an era of bioprocess innovation. Half a century later, recombinant protein technology spurred the tremendous growth of biologics and added mammalian cells to the forefront of industrial producing cells in terms of the value of products generated. This review highlights the process technology of natural products and protein biologics. Despite the separation in time, there is a remarkable similarity in their progression. As the new generation of therapeutics for gene and cell therapy emerges, its process technology development can take inspiration from that of natural products and biologics.

Ultrahigh sensitivity and gain white light photodetector based on GaTe/Sn:CdS nanoflake/nanowire heterostructures.

Optoelectronic diode based on PN heterostructure is one of the most fundamental device building blocks with extensive applications. Here we reported the fabrication and optoelectronic properties of GaTe/Sn : CdS nanoflake/nanowire PN heterojunction photodetectors. With high quality contacts between metal electrodes and Sn : CdS or GaTe, the electrical measurement of GaTe/Sn : CdS hybrid heterojunction under dark condition demonstrates an excellent diode characteristic with well-defined current rectification behavior. The photocurrent increases drastically under LED white light as well as red, green, UV illumination. The on-off ratio of current is about 100 for forward bias and 3000 for reverse bias, which clearly indicates the ultrahigh sensitivity of the heterostructure photodetector to white light. The responsivity and optical gain are determined to be 607 A W(-1) and (1.06-2.16) × 10(5)%, which is higher than previous reports of single GaTe or CdS nanostructures. Combination the Ids-Vds curves under different illumination power with energy band diagrams, we assign that both the light modulation effect under forward and reverse bias and the surface molecular oxygen adsorption/desorption mechanism are dominant to the electrical transport behavior of GaTe/Sn : CdS heterojunction. This heterostructure photodetector also shows good stability and fast response speed. Both the high photosensibility and fast response time described in the present study suggest strongly that the GaTe/Sn : CdS hybrid heterostructure is a promising candidate for photodetection, optical sensing and switching devices.

Intensified perfusion culture (IPC) reduced recombinant protein fragmentation.

Mammalian cells remain the mainstay of biological production host. In industry, cultivating and harvest strategies are sorted in batch mode (e.g., batch, fed-batch, concentrated fed-batch and intensified fed-batch) and continuous mode (e.g., perfusion). To retrieve greater productivity and better product quality, especially for the sensitive products prone to fragmentation, culture modes with various modifications are innovated (e.g., intensified perfusion culture [IPC]). In our study, we demonstrated that the fragmentation of Fc-fusion product (Molecule A) is time-dependent in traditional fed-batch (TFB) culture. The fragmentation proportion increased from 3.8% to 12.4% for Clone A, 0.8% to 1.7% for Clone B and 0.9% to 2.0% for Clone C from Day 10 to Day 14. By applying a novel bioprocess, IPC, which allows continuous feeding of the fresh medium and constant removal of the spent medium without bleeding cells to maintain a defined constant viable cell density, the fragmentation was reduced to 0.3% while the productivity was increased from 2.96 g/L to 15.51 g/L for Clone A. To validate whether the fragmentation reduction is product-sensitive, plasmids carrying the DNA sequences of two other Fc-fusion molecules (Molecule B and Molecule C) were transfected into the host. The results showed consistent fragmentation reducing effect by using IPC. Furthermore, the cultivation scale was expanded to 50 L and 1000 L. A minimum fragmentation level below 0.1% was observed for Molecule C. Our study revealed the capability of IPC in reducing Fc-fusion protein fragmentation and the reproducibility when scaling up while maintaining high productivity.

Further accelerating biologics development from DNA to IND: the journey from COVID-19 to non-COVID-19 programs.

The Coronavirus Disease (COVID-19) pandemic has spurred adoption of revolutionary initiatives by regulatory agencies and pharmaceutical industry worldwide to deliver therapeutic COVID-19 antibodies to patients at unprecedented speed. Among these, timeline of chemistry, manufacturing and control (CMC), which involves process development and manufacturing activities critical for the assurance of product quality and consistency before first-in-human clinical trials, was greatly reduced from typically 12-15 months (using clonal materials) to approximately 3 months (using non-clonal materials) in multiple cases. In this perspective, we briefly review the acceleration approaches published for therapeutic COVID-19 antibodies and subsequently discuss the applicability of these approaches to achieve investigational new drug (IND) timelines of ≤10 months in over 60 COVID-19 and non-COVID-19 programs performed at WuXi Biologics. We are of the view that, with demonstrated product quality and consistency, innovative approaches used for COVID-19 can be widely applied in all disease areas for greater speed to clinic.

A comparison of different intensified upstream processes highlighting the advantage of WuXi Biologics' Ultra-high Productivity platform (WuXiUPTM) in improved product quality and purification yield.

WuXiUPTM, WuXi Biologics' Ultra-high Productivity platform, is an intensified and integrated continuous bioprocess platform developed for production of various biologics including monoclonal antibodies, fusion proteins, and bispecific antibodies. This process technology platform has manifested its remarkable capability in boosting the volumetric productivity of various biologics and has been implemented for large-scale clinical material productions. In this paper, case studies of the production of different pharmaceutical proteins using two high-producing and intensified culture modes of WuXiUPTM and the concentrated fed-batch (CFB), as well as the traditional fed-batch (TFB) are discussed from the perspectives of cell growth, productivity, and protein quality. Both WuXiUPTM and CFB outperformed TFB regarding volumetric productivity. Additionally, distinctive advantages in product quality profiles in the WuXiUPTM process, such as reduced acidic charge variants and fragmentation, are revealed. Therefore, a simplified downstream purification process with only two chromatographic steps can be developed to deliver the target product at a satisfactory purity and an extremely-high yield.

A Review of Recent FDA-Approved Biologic-Device Combination Products.

With the remarkably strong growth of the biopharmaceutical market, an increasing demand for self-administration and rising competitions attract substantial interest to the biologic-device combination products. The ease-of-use of biologic-device combination products can minimize dosing error, improve patient compliance and add value to the life-cycle management of biological products. As listed in the purple book issued by the U.S. Food and Drug Administration (FDA), a total of 98 brand biologic-device combination products have been approved with Biologic License Application from January 2000 to August 2023, where this review mainly focused on 63 products containing neither insulin nor vaccine. Prefilled syringes (PFS) and autoinjectors are the most widely adopted devices, whereas innovative modifications like needle safety guard and dual-chamber design and novel devices like on-body injector also emerged as promising presentations. All 16 insulin products employ pen injectors, while all 19 vaccine products are delivered by a PFS. This review provides a systematic summary of FDA-approved biologic-device combination products regarding their device configurations, routes of administration, formulations, instructions for use, etc. In addition, challenges and opportunities associated with biologic-device compatibility, regulatory complexity, and smart connected devices are also discussed. It is believed that evolving technologies will definitely move the boundaries of biologic-device combination product development even further.

Nanoparticle technology for mRNA: Delivery strategy, clinical application and developmental landscape.

The mRNA vaccine, a groundbreaking advancement in the field of immunology, has garnered international recognition by being awarded the prestigious Nobel Prize, which has emerged as a promising prophylactic and therapeutic modality for various diseases, especially in cancer, rare disease, and infectious disease such as COVID-19, wherein successful mRNA treatment can be achieved by improving the stability of mRNA and introducing a safe and effective delivery system. Nanotechnology-based delivery systems, such as lipid nanoparticles, lipoplexes, polyplexes, lipid-polymer hybrid nanoparticles and others, have attracted great interest and have been explored for mRNA delivery. Nanoscale platforms can protect mRNA from extracellular degradation while promoting endosome escape after endocytosis, hence improving the efficacy. This review provides an overview of diverse nanoplatforms utilized for mRNA delivery in preclinical and clinical stages, including formulation, preparation process, transfection efficiency, and administration route. Furthermore, the market situation and prospects of mRNA vaccines are discussed here.

In Situ Regulating Cobalt/Iron Oxide-Oxyhydroxide Exchange by Dynamic Iron Incorporation for Robust Oxygen Evolution at Large Current Density.

The key dilemma for green hydrogen production via electrocatalytic water splitting is the high overpotential required for anodic oxygen evolution reaction (OER). Co/Fe-based materials show superior catalytic OER activity to noble metal-based catalysts, but still lag far behind the state-of-the-art Ni/Fe-based catalysts probably due to undesirable side segregation of FeOOH with poor conductivity and unsatisfied structural durability under large current density. Here, a robust and durable OER catalyst affording current densities of 500 and 1000 mA cm-2 at extremely low overpotentials of 290 and 304 mV in base is reported. This catalyst evolves from amorphous bimetallic FeOOH/Co(OH)2 heterostructure microsheet arrays fabricated by a facile mechanical stirring strategy. Especially, in situ X-ray photoelectron spectroscopy (XPS) and Raman analysis decipher the rapid reconstruction of FeOOH/Co(OH)2 into dynamically stable Co1-x Fex OOH active phase through in situ iron incorporation into CoOOH, which perform as the real active sites accelerating the rate-determining step supported by density functional theory calculations. By coupling with MoNi4 /MoO2 cathode, the self-assembled alkaline electrolyzer can deliver 500 mA cm-2 at a low cell voltage of 1.613 V, better than commercial IrO2 (+) ||Pt/C(-) and most of reported transition metal-based electrolyzers. This work provides a feasible strategy for the exploration and design of industrial water-splitting catalysts for large-scale green hydrogen production.