Multi-step Manufacturing Process Research Articles

Direct Precursor Route for the Fabrication of LLZO Composite Cathodes for Solid-State Batteries.

Solid-state batteries based on Li7La3Zr2O12 (LLZO) garnet electrolyte are a robust and safe alternative to conventional lithium-ion batteries. However, the large-scale implementation of ceramic composite cathodes is still challenging due to a complex multistep manufacturing process. A new one-step route for the direct synthesis of LLZO during the manufacturing of LLZO/LiCoO2 (LCO) composite cathodes based on cheap precursors and utilizing the industrially established tape casting process is presented. It is shown that Al, Ta:LLZO can be formed directly in the presence of LCO from metal oxide precursors (LiOH, La2O3, ZrO2, Al2O3, and Ta2O5) by heating to 1050°C, eliminating the time- and energy-consuming synthesis of preformed LLZO powders. In addition, performance-optimized gradient microstructures can be produced by sequential casting of slurries with different compositions, resulting in dense and flat phase-pure cathodes without unwanted ion interdiffusion or secondary phase formation. Freestanding cathodes with a thickness of 85µm, a relative density of 95%, and an industrial relevant LCO loading of 15mg show an initial capacity of 82 mAh g-1 (63% of the theoretical capacity of LCO) in a solid-state cell with Li metal anodes, which is comparable to conventional LCO/LLZO cathodes and can be further improved in the future.

Rock-to-Pharma: Characterization of Shale Oil-Based Nonbiological Complex Drugs along the Production Process by High-Resolution Mass Spectrometry.

Antibiotic resistance has become a primary concern in medicine because of the overuse and misuse of classical pharmaceuticals. Recently, nonbiological complex drugs (NBCDs) have gained interest for their complex pharmacological profiles. Bituminosulfonates, which have lately been tentatively allocated toward NBCDs, are pharmacologically well-studied and show low potential in resistance development. However, molecular composition knowledge is limited. With this work, we present a comprehensive approach to investigate the manufacturing process of complex pharmaceuticals like bituminosulfonates on a molecular level via Fourier-transform ion cyclotron resonance mass spectrometry. The application of various hyphenations and ionization techniques comprehensively covers the entire mass and polarity range of the matrix, and the high sensitivity enables the identification of significant and minor chemical alterations caused by the multistep manufacturing process. The distillation of the shale crude oil eliminates highly aromatic PAH and PASH constituents. ESI(-) revealed strong PAH- and PASH-sulfonate formation after reacting the shale oil distillate with sulfuric acid. Increasing alkylation reduced the sulfonation yield, instead causing oligomerization side reactions, as observed by APPI analysis. Furthermore, multidimensional gas chromatography coupled with high-resolution mass spectrometry verified core structural motifs. With this work, we demonstrate the high potential of FT-ICR MS in NBCD process analysis. The results also give valuable information for future pharmacological investigations focusing on specific compound classes or properties.

Elaborating the potential of Artificial Intelligence in automated CAR-T cell manufacturing.

This paper discusses the challenges of producing CAR-T cells for cancer treatment and the potential for Artificial Intelligence (AI) for its improvement. CAR-T cell therapy was approved in 2018 as the first Advanced Therapy Medicinal Product (ATMP) for treating acute leukemia and lymphoma. ATMPs are cell- and gene-based therapies that show great promise for treating various cancers and hereditary diseases. While some new ATMPs have been approved, ongoing clinical trials are expected to lead to the approval of many more. However, the production of CAR-T cells presents a significant challenge due to the high costs associated with the manufacturing process, making the therapy very expensive (approx. $400,000). Furthermore, autologous CAR-T therapy is limited to a make-to-order approach, which makes scaling economical production difficult. First attempts are being made to automate this multi-step manufacturing process, which will not only directly reduce the high manufacturing costs but will also enable comprehensive data collection. AI technologies have the ability to analyze this data and convert it into knowledge and insights. In order to exploit these opportunities, this paper analyses the data potential in the automated CAR-T production process and creates a mapping to the capabilities of AI applications. The paper explores the possible use of AI in analyzing the data generated during the automated process and its capabilities to further improve the efficiency and cost-effectiveness of CAR-T cell production.

Integrated solar-rechargeable supercapacitors with a dual-functional-layered electrode

Early integrated solar-rechargeable supercapacitor (ISRS) systems with four physically distinguishable electrodes are cumbersome and require multistep manufacturing process with application disadvantages. Therefore, a stacked ISRS with a three-electrode mode that uses a dual-functional-layered (shared) electrode, which is particularly important to integrate two main active components, is one of the target configurations for a compact, safe and efficient power supplier in advanced soft electronics. This work reports the effect of various fabrication methods on the surface morphology profile of a graphene oxide-incorporated poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (m-PHGO) as a shared electrode; this process exhibited a significant impact on the charge/discharge rate and ISRS efficiency. By controlling the deposition method, the shared electrode fabricated by a modified spin/spray-coating technique using an m-PHGO dispersion features an interface that meets all of the requirements of a dual-functional layer. It is effective for hole charge filtration, transfer, and storage in the stacked ISRS. Under 1-sun illumination, the supercapacitor moiety is charged entirely by the organic solar cell unit within 5 s to build a fusion ISRS capable of approaching a high charging voltage of ∼0.6 V. The as-developed ISRS (with a thickness of ∼2.6 μm and the substrate) shows an impressive energy-storage efficiency of ∼81%. This study provides insights about fabricating high-performance and easy-to-use compact electronic devices.

Digital transformation of CAR-T cell therapy – challenges and potential for Industry 4.0

With the approval of CAR-T cell therapy as a treatment for acute leukemia and lymphoma in 2018, the first advanced therapy medicinal product (ATMP) came onto the market. ATMPs are cell- and gene-based products and very promising therapies for the successful treatment of various hereditary diseases and cancers. In recent years, more ATMPs have been approved and a strong increase is expected considering current clinical trial numbers. However, CAR-T cell production still poses great challenges. The CAR-T treatment is very costly for approved products (approx. 400.000 $). Most of the associated costs are due to the expensive manufacturing processes. Furthermore, autologous CAR-T cell therapy is limited in its scalability by a patient-specific make-to-order approach which makes economic production difficult. To make CAR-T cell products and other ATMP available to a large number of patients, the complex multi-step manufacturing process must be further developed and the potential for automation and digitalization exploited. Therefore, this article provides an analysis of CAR-T cell production focusing on characteristics, challenges, and optimization potential. For strategic guidance in the digital transformation, a systematic approach, based on the Industry 4.0 maturity index, is introduced categorizing challenges and use cases into six stages.

Translation of hyaluronic acid-based vitreous substitutes towards current regulations for medical devices.

Hydrogel-based vitreous substitutes have the potential to overcome the limitations of current clinically used endotamponades. With the goal of entering clinical trials, the present study aimed to (I) transfer the material synthesis of hyaluronic acid-based hydrogels into a routine, pharmaceutical-appropriate production and (II) evaluate the properties of the vitreous substitutes in terms of the current regulations for medical devices (MDR/ISO standards). The multistep manufacturing process of the vitreous substitutes, including the modification of hyaluronic acid with glycidyl methacrylate, photocopolymerization with N-vinylpyrrolidone, and successive hydrogel purification, was developed under laboratory conditions, characterized using 1 H-NMR, FT-IR and UV/Vis spectroscopies and HPLC, and transferred towards a pharmaceutical production environment considering GMP standards. The optical and viscoelastic characteristics of the hyaluronic acid-based hydrogels were compared with those of extracted human vitreous and silicone oil. The effect of the hydrogels on the metabolic activity, proliferation and apoptosis of fibroblast (MRC-5, BJ, L929), retinal pigment epithelial (ARPE-19, hiPSC-derived RPE) and photoreceptor cells (661W) was studied as well as their mucosal tolerance via a HET-CAM assay. Hyaluronic acid-based hydrogels having a suitable purity, sterility, high transparency (>90%), appropriate refractive index (1.3365) and viscoelasticity (G' > G″) were prepared in a standardized manner under controlled process conditions. The metabolic activity, proliferation and apoptosis of various cell types as well as egg choroid were unaffected by the hyaluronic acid-based vitreous substitutes, demonstrating their biocompatibility. The present study demonstrates the successful transferability of the crucial synthesis steps of hyaluronic acid-based hydrogels into a routine, GMP-compliant production process while achieving the optical and viscoelastic properties, biocompatibility and purity required for their clinical use as vitreous substitutes.

Compression Density as an Alternative to Identify an Optimal Moisture Content for High Shear Wet Granulation as an Initial Step for Spheronisation.

Pellet production is a multi-step manufacturing process comprising granulation, extrusion and spheronisation. The first step represents a critical control point, since the quality of the granule mass highly influences subsequent process steps and, consequently, the quality of final pellets. The most important parameter of wet granulation is the liquid requirement, which can often only be quantitatively evaluated after further process steps. To identify an alternative for optimal liquid requirements, experiments were conducted with a formulation based on lactose and microcrystalline cellulose. Granules were analyzed with a Powder Vertical Shear Rig. We identified the compression density (ρpress) as the said alternative, linking information from the powder material and the moisture content (R2 = 0.995). We used ρpress to successfully predict liquid requirements for unknown formulation compositions. By means of this prediction, pellets with high quality, regarding shape and size distribution, were produced by carrying out a multi-step manufacturing process. Furthermore, the applicability of ρpress as an alternative quality parameter to other placebo formulations and to formulations containing active pharmaceutical ingredients (APIs) was demonstrated.

Novel Method for Failure Modes Detection in UV-Cured Clear Coated Polymer for Automotive Interior Mechatronic Devices.

Plastic parts used in automotive interior are difficult to coat, due to their low surface energies as well as their sensitivity to temperature and solvents, rendering the development of coating systems for such substrates challenging. Automotive customer requirements are explicit and clear, mainly focused on functional and surface defects. A new failure modes detection methodology of UV clear coated polymers for automotive interior, obtained by a multi-step manufacturing process, is proposed. The polymer complex parts analyzed in this paper are manufactured in various steps as follows: two components plastic injection molding, primer coating, laser engraving, and UV-cured clear coating. The failure modes detection methodology of the parts within each process step is investigated using different tests and analyses as follows: surface tension test, painting adhesion test, optical 3D measuring, energy dispersive X-ray analysis (EDX), and microscopy. A design of the experiments (DoE) based on the Taguchi technique with the aim to detect the influence of the main factors that lead to surface defects was performed. The proposed methodology is validated by a case study. The results showed that the mold temperature and the laser engraving current have a significant influence on the surface defect occurrence. Additionally, a possible contamination of the molding tool can generate the defects. A solution to reduce the occurrence of the failures was proposed, reducing the defect rate from 50% to 0.9%.

A low-cost and simple-fabricated epidermal sweat patch based on “cut-and-paste” manufacture

Continuous, non-invasive, and multi-parametric sensing of sweat is noted to be appealing to many applications such as personalized medicine, athletic performance, and military readiness. Microfluidic sensing patches are widely reported to perform sweat collection, transportation, and multi-parameters analysis; however, time-consuming and multi-step manufacturing process actually prevents the large-scale application. In this study, a “cut-and-paste (CAP)” method is devised to pattern and transfer polydimethylsiloxane (PDMS) and polyethylene terephthalate (PET) film to fabricate the microfluidic patch. The “bear like” patch, composed of four polymer-based layer, are completely carved automatically within 2 min by the desktop cutting machine. The sweat glucose and lactate are monitored in real time with newly proposed wearable sweat patches. The concentration ratio between blood glucose and iontophoresis-induced sweat glucose was over 512.37 as calculated. And the latter lagged behind the former over 70 min about peak of concentration. Also, using the fabricated sensors, we have analyzed the levels of glucose and lactate and compared them in both exercise- and iontophoresis-induced sweat. These results indicate the CAP method has great potential for the microfluidic integrated wearable platforms with low-cost, versatility, and mechanically flexibility, which would promote the development of wearable sweat microfluidic devices and application in individualized health monitoring.

A comparison of different hardening rules on a multi-step global manufacturing process modeling

The main difficulty presented by the simulation of a global process that includes different forming stages is the correct characterization of the material state at the end of each of these stages, which in turn, are the initial point of the following process. Hardening variables are capable of characterizing the state of the material, which, after a plastic transformation, varies according to the direction of the solicitation and its intensity. The present work carries out an analysis of the influence in the election of the hardening rule used in the behavior law, comparing the most used approach. For a work piece solicited by combined efforts in multiple stages, results are obtained by numerical simulation. A correct choice will allow obtaining reliable predictions, not the solicitations but also to the final geometry and the dissipated energy in the global process, allowing an eventual optimization of such process.

Photoluminescent surface-functionalized graphene quantum dots for spontaneous interfacial homeotropic orientation of liquid crystals

An interfacial molecular array of octadecylamine-functionalized graphene quantum dots (GQDs) was fabricated to simultaneously realize photoluminescence characteristics and molecular orientation for liquid crystals (LCs). The functionalized GQDs were synthesized using a simple bottom-up method by first grafting them with octadecylamine. The subsequent interfacial self-assembly of these functionalized GQDs in an LC medium formed a nanostructured molecular array on an indium tin oxide (ITO) electrode. Experiments revealed that the hydrophobic GQDs spontaneously covered the hydrophilic ITO surface through polar intermolecular interactions, and the resulting GQD layer induced the uniform vertical orientation of the LC molecules at the ITO interface owing to van der Waals interactions of long side chains. In addition, the unique photoluminescence of pure GQDs in the blue region was maintained even in an LC mixture with a low concentration of functionalized GQDs. A new type of homeotropically oriented LC device based on the nanostructured layer of functionalized GQDs exhibited superior electro-optical properties and a 375% improvement in response time for turn-off switching of the device relative to those of a conventional polymer layer. This functionalized GQD-based device simultaneously achieved the spontaneous alignment of the LCs, blue photoluminescence emission, and high-speed response performance for turn-off switching of the device. In addition, our method is cost-effective and encompasses a simple fabrication process for developing functional controllable surfaces without requiring a tedious multi-step manufacturing process.

Towards a flexible process-independent meta-model for production data

Data integration is a considerable challenge when investigating information sources from a multi-step manufacturing process. The interpretation of the process data profoundly depends on the incurred meta-data. However, during most data aggregating processes along the production chain, accompanying meta-information of vital importance is lost. To address this shortcoming, we propose a flexible and process-independent meta-model for efficient data integration for multi-step manufacturing processes.The product-oriented model unites process- and meta-data to reflect their mutual relationships within the manufacturing process. The context provided by the meta-information enables automatic data analysis for Predictive Quality applications in a cross-company setting.

Multi-scale modelling approach to homogenise the mechanical properties of polymeric closed-cell bead foams

The complex mechanical deformation behaviour of closed-cell foams is governed by morphological and physical properties such as cell structure and crystallinity. In this study, the micro-, meso- and macroscopic scale of commercially available bead foam was analysed. Statistical distribution of the cell structure including cell size, wall thickness and shape was determined using optical microscopy and micro computed-tomography. Local crystallinity was investigated by DSC scans and X-ray scattering. The results confirm the important influence of the multi-step manufacturing process on the physical properties of bead foams. Mesoscopic and macroscopic numerical analyses of the mechanical behaviour of polymeric closed-cell bead foams are performed. With regard to the manufacturing influence on local physical properties of bead foams, the suggested approach takes into account the density and crystallinity-specific material properties and the generation of associated material cards, using virtual test methods. To represent the mesoscopic foam morphology considered here, statistical volume elements (SVE) are generated using the Laguerre tessellation method. Crystallinity-dependent base material properties are used in SVE material cards to investigate tensile and compressive behaviour. For validation of the suggested approach, four-point bending tests are conducted on macroscopic scale and compared with the numerically predicted results. The paper shows the advanced forecast capability of locally resolved modelling of closed-cell bead foam structures and underlines the huge potential of the multi-scale modelling approach.

Mass-customization of oral tablets via the combination of 3D printing and injection molding

The new frontier of medicine is the personalization of treatment to match a patient's individual needs. Fused-filament fabrication (FFF) offers a platform for the personalization of drug dosage forms, but one of its chief shortcomings compared to other tablet production methods such as dry compression and wet granulation is relatively low throughput. Conversely, injection molding (IM) is a manufacturing technique for the high-volume production of parts, but in which individual part customization is both expensive and slow requiring the modification of expensive mold tooling. Mass-customization is the manufacture of custom products that match the needs of individual consumers but which are produced at the low unit cost associated with high-volume production. We successfully integrated for the first time FFF with IM in a multi-step manufacturing process for the production of custom bilayer tablets loaded with two active pharmaceutical ingredients used in the treatment of cardiovascular disease. The FFF layer was loaded with the diuretic hydrochlorothiazide, while the IM layer was loaded with lovastatin. Infill percentage was varied for the FFF layer as a means to modify drug release. The IM injection pressure was evaluated for its effect on drug release and layer-layer adhesion. The bilayer tablets obtained offered different combinations of drug release profiles, which were governed by a combination of factors, including surface area to volume ratio; IM injection volume penetration into the FFF layer; FFF infill percentage; layer tortuosity and porosity. These different parameters could be utilized to modify the individual release of both drugs from the bilayer tablet. Thus for the first time, we have demonstrated a viable method for the mass-customization of oral tablets which could hasten the rollout of personalized medicine.

Protransduzin™ - A novel transduction enhancer to accelerate en gros CAR-T production

Background & Aim The advent of cellular immunotherapies using genetically reprogrammed immune cells such as T-lymphocytes with chimeric antigen receptor (CAR-Ts) or engineered T-cell receptors (TCR-Ts) has led to promising new treatments for hematological cancers. However, due to the complex multi-step manufacturing process, these therapies are still very costly and currently approved therapies exceed 400k US$ per patient. In order to make CAR-T immunotherapy more affordable, a reduction of the production-related cost of goods and process optimization is highly desirable. Methods, Results & Conclusion A crucial manufacturing step is the actual gene transfer of CARs and TCRs which to date mostly relies on lenti- or retroviral vectors and often results in low transduction rates. To date a range of genetic transfer enhancers is being used to improve transduction with variable success. These include amphiphilic macromolecules, cationic polymers, and adhesion molecules. Until now the coating of cultureware with recombinant fibronectin (or a fragment thereof) has been shown to provide fair enhancement and is widely accepted. However, coating procedure is cumbersome with time consuming liquid handling steps in a cleanroom environment. Here we describe Protransduzin™, a novel peptide that can enhance transduction of CARs in human leukapheresis-derived CD3 lymphocytes by up to 24% over control dependent on culture system and multiplicities of infection. A side-by-side comparison of Protransduzin™ with recombinant fibronectin and an additional, commercially available histidine-rich amphipathic peptide showed that Protransduzin™ is at least as potent as the adhesion molecule but more potent than the other peptide. Since Protransduzin™ can be added directly with the viral particles it can be used to streamline CAR-T production. Especially in connection with mini bio-reactors it is possible to do an en gros production by allowing transduction and expansion in one vessel. Thanks to our optimized protocol shown here hands-on time can be reduced along with the contamination risk arising from repeated liquid handling steps.

Epoxy resin mold and PDMS microfluidic devices through photopolymer flexographic printing plate

Photopolymer flexographic printing plate is a new photopolymeric material used for microdevices fabrication. This work demonstrates that a photopolymer flexographic master mold can be used for the fabrication of PDMS (polydimethylsiloxane) microdevices by a multi-step manufacturing process. The methodology entails three main fabrication steps: (1) a photopolymer flexographic printing plate mold (FMold) is generated by UV exposure through a transparent film, (2) an epoxy resin mold (ERmold) is fabricated by transferring the features of the photopolymer mold and (3) a PDMS microdevice is manufactured from the epoxy resin mold. The characterization of the manufactured PDMS microdevices was performed using scanning electron microscopy (SEM) and profilometry. Results showed high accuracy in the replication of the profiles. To show the feasibility of the fabrication process a microdevice for microdroplet generation was designed, manufactured and tested. Hence, the manufacturing process described in this work provides an easy, robust, and low-cost strategy that facilitates the scaling-up of microfluidic devices without requiring any sophisticated equipment.

Comparing Survival for Different CAR Ts: Need for Addressing Bias Due to Differences in the Pre-Infusion Period

▪IntroductionPatients with relapsed/refractory large B-cell lymphoma (RR-LBCL) treated with salvage chemotherapy have a poor prognosis, with a median survival of 6.3 months (Crump 2017). Given the recent approval of chimeric antigen receptor T-cell therapies (CAR T) for patients with RR-LBCL, efforts to demonstrate the comparative effectiveness of different CAR Ts are imminent. In the absence of head-to-head trials, such an assessment will likely be based on indirect or between-trial comparisons of reported survival estimates. Due to differences in the multi-step manufacturing process preceding the infusion of CAR T cells, the duration of the pre-infusion period, which accounts for time from leukapheresis to infusion, may vary across CAR Ts. This variability may be associated with differences in the prognostic characteristics of patients who endure the pre-infusion period. As such, comparisons of reported survival among these selected patients with the different CAR Ts evaluated in different trials may be biased. Estimating the comparative effects of CAR T therapies on survival requires careful attention to these potential differences and the use of thoughtful analytic approaches to address bias. We aimed to review current methodological approaches used to address bias related to time-to-treatment initiation and assess their potential utility in comparative analyses of CAR T based on between-trial comparisons.MethodsA targeted literature review was performed to identify studies that used methods to adjust for bias related to time-to-treatment initiation when estimating relative treatment effects regarding survival outcomes between interventions in oncology. Relevant studies published in English from 2008 onwards were identified by searching the Medical Literature Analysis and Retrieval System Online (MEDLINE) and Excerpta Medica dataBASE (EMBASE). In addition, manual searches of the reference lists of the identified studies were performed. Studies that provided the most recent application of a proposed method were selected for inclusion.ResultsWe identified several manuscripts that utilized methods to address survival or survival treatment selection bias (STSB) in observational studies where the classification of “treated” individuals required them to have survived until treatment initiation, and individuals who died early without the opportunity to get treatment were classified as “untreated”. The identified methods to adjust for the over-estimation of the treatment effect include landmark survival and time-dependent exposure assignment. The landmark method restricts the analysis sample to patients who are event-free and alive at a given point (i.e, landmark) during follow-up; patients are assigned to risk groups based on their status at the landmark and survival outcomes are estimated from that point. Time-dependent exposure assignment addresses STSB by accounting for changes in patient exposure or treatment status during the course of follow-up. All patients begin in the same risk group; when exposure changes, patients and person-time are switched to the applicable risk group at that point. However, these methods do not address bias resulting from a comparison of reported survival estimates between different CAR Ts for a follow-up starting at the successful infusion of the CAR T cells. When comparing CAR Ts, the differences in prognostic patient characteristics due to differences in the pre-infusion phase are similar to channeling bias or confounding-by-indication. These concepts are well known, and established methods are available. However, those methods have limited applicability without access to individual patient data for the CAR Ts of interest.ConclusionComparing survival upon successful infusion with different CAR Ts reported in separate studies is likely to be biased due to differences in the duration of the pre-infusion period. Methods previously used to address bias related to differences in time-to-treatment initiation may not be relevant or applicable, particularly in the absence of patient-level data. A between-trial comparison of reported survival estimates with the different CAR Ts might benefit from incorporating external evidence to inform the mortality during the pre-infusion periods. DisclosuresKatz:Amgen: Other: Former employee and stockholder; Kite, a GILEAD Company: Consultancy. Lin:Kite/Gilead: Employment. Blaylock:Kite, A Gilead Company: Consultancy. Jansen:Kite, A Gilead Company: Consultancy.

Identification of Complex Production Systems with Using Iterative Networks

In the article the technique of identification of complex production systems using iterative networks is considered. The identification process begins with the allocation of functional blocks, which can later be described by automata working with discrete information and changing internal states only at specified times. The basic laws of the functioning of the selected subsystems are simulated. Characteristics of cells formed parameters associated with multi-step manufacturing process. As the semi-finished product passes through the processing stages under consideration, the values of the technological factors being realized are fixed in the system being formed.

752 Simultaneous gene-editing and reprogramming of epidermolysis bullosa simplex fibroblasts

Epidermolysis bullosa simplex (EBS) is an autosomal dominant skin fragility disorder caused by mutations in either Keratin (K) 5 or K14. Although current therapy for EBS is limited to wound care, advances in reprogramming somatic cells into induced pluripotent stem cells (iPSC) offer the possibility of developing new approaches for EBS treatment. The iPSC-based therapeutic approach for EBS relies on the generation of patient-specific iPSCs, which then undergo genetic editing and differentiation into skin cells suitable for transplantation. One of the main hurdles in advancing the iPSC-based therapy into the clinic is the complexity of a multi-step manufacturing process to produce genetically corrected iPSC-derived epidermal sheets. To reduce the manufacturing complexity of an iPSC-based therapy for EBS, we have combined our previously presented high-efficiency RNA-based reprogramming approach with a specific CRISPR/Cas9-mediated knock-out of the mutant allele of the K14 gene into a one-step procedure. We established a fibroblast line from an EBS patient carrying a hot-spot heterozygous C373T mutation at codon 125 in K14 and successfully generated multiple isogenic EBS iPSCs lines with a specific knock out of the mutant K14 allele (KO EBS iPSC) within 4 weeks of initiating simultaneous gene editing and reprogramming. Simultaneous reprogramming and gene correction not only reduces the complexity of the iPSC-based therapy but also improves safety of the approach by avoiding lengthy cell culture periods, drug selection, and multiple sub-cloning steps. We are currently differentiating these KO EBS iPSCs into keratinocytes to determine if these gene edited iPSC-derived keratinocytes can be used for EBS therapy by employing a mouse xenograft model.

The process defines the product: what really matters in biosimilar design and production?

Biologic drugs are highly complex molecules produced by living cells through a multistep manufacturing process. The key characteristics of these molecules, known as critical quality attributes (CQAs), can vary based on post-translational modifications that occur in the cellular environment or during the manufacturing process. The extent of the variation in each of the CQAs must be characterized for the originator molecule and systematically matched as closely as possible by the biosimilar developer to ensure bio-similarity. The close matching of the originator fingerprint is the foundation of the biosimilarity exercise, as the analytical tools designed to measure differences at the molecular level are far more sensitive and specific than tools available to physicians during clinical trials. Biosimilar development, therefore, has a greater focus on preclinical attributes compared with the development of an original biological agent. As changes in CQAs can occur at different stages of the manufacturing process, even small modifications to the process can alter biosimilar attributes beyond the point of similarity and impact clinical effectiveness and safety. The manufacturer’s ability to provide consistent production and quality control will greatly influence the acceptance of biosimilars. To this end, preventing drift from the required specifications over time and avoiding the various implications brought by product shortage will enhance biosimilar integration into daily practice. As most prescribers are not familiar with this new drug development paradigm, educational programmes will be needed so that prescribers see biosimilars as fully equivalent, efficacious and safe medicines when compared with originator products.