Biopharmaceutical Development Research Articles

Overcoming the inconsistent cell culture performance caused by single-use bioreactors during scaling-up the manufacturing process for a therapeutic bispecific antibody

Single use technology has been widely used in modern biopharmaceutical process development and manufacturing. However, there are concerns about the adverse effects of disposable materials on bioprocess, product quality and patient safety. Recently, we observed slow cell growth, increased lactate accumulation, and decreased production titer during scaling up a bispecific antibody (BsAb) upstream process from 3L to 500L bioreactor. To investigate the phenomenon, we conducted medium incubation and leachable spike-in experiments. We found that cell culture medium incubated in CX5-14 bag caused poor cell culture performance, which was correlated with the concentration of bis(2,4-di-tert-butylphenyl)-phosphate (bDtBPP), a breakdown product of the antioxidant Irgafos®168 in polyethylene film. In addition, when the media were spiked with 0.15μg/ml or a greater concentration of bDtBPP, slow cell growth and reduced BsAb expression level were observed. In contrast, Aegis5-14 film produced bDtBPP at a level of below detection during medium incubation. Thus, the bispecific-expressing CHO cell line restores normal cell culture performance and production titer when Aegis5-14 single-use bioreactor was employed in large-scale manufacturing. Our results reveal the importance of evaluating leachable profiles from disposable materials during process development of bispecifics and other novel therapeutic modalities.

Quantifying public and private investment in European biopharmaceutical research and development.

Robust biopharmaceutical research and development (R&D) ecosystems require investment from both the public and private sectors. In Europe, there is an interest in growing biopharmaceutical R&D given its contribution to public health and the economy, which requires an understanding of current public and private investment. In addition, recent European draft legislation has focused on the public sector's contributions to biopharmaceutical R&D to inform pharmaceutical prices. However, there is little empirical evidence on the specifics of public and private funding for medicine R&D in Europe. This paper performs aggregative data collection to quantify 2019 investment in biopharmaceutical R&D by the public and private sectors in 6 countries: Belgium, France, Germany, Norway, Poland, and the United Kingdom. We find that, across these countries, the private sector accounts for just under two-thirds of investment. We contrast results to those obtained using high-level R&D indicators from the Organization for Economic Co-operation and Development (OECD) and contextualize differences. We then provide 2013-2019 estimates for Belgium, France, Germany, and the United Kingdom (countries with data to support such analysis), and show that total spending grew over those years, although proportions attributable to each sector remained stable. These findings should provide further evidence for debates on policies to effectively grow the biopharmaceutical R&D sector.

Unlocking the combined action of Mentha pulegium L. essential oil and Thym honey: In vitro pharmacological activities, molecular docking, and in vivo anti-inflammatory effect

Mentha pulegium L., a plant widely embraced for its therapeutic properties by populations worldwide, including Morocco, has long been recognized for its potential in treating various ailments. This study aims to comprehensively evaluate the antioxidant, anti-inflammatory, and dermatoprotective properties of essential oil derived from M. pulegium, and thyme honey as well as their combined effects. To unravel the chemical composition, a rigorous GC-MS analysis was conducted. Subsequently, we examined their antioxidant potential through three distinct assays: DPPH●, hydrogen peroxide assay, and xanthine oxidase assay. The anti-inflammatory properties were scrutinized through both in vitro and in vivo experiments. Simultaneously, the dermatoprotective efficacy was investigated in vitro by evaluating tyrosinase inhibition. Our findings revealed that pulegone constitutes the predominant compound in M. pulegium essential oil (MPEO), constituting a remarkable 74.82 % of the composition. Significantly, when the essential oil was combined with thym honey, it exhibited superior anti-inflammatory and dermatoprotective effects across all in vivo and in vitro tests. Moreover, our in silico molecular docking analysis hinted at the potential role of cyclohexanone, 3-methyl, an element found in the MPEO, in contributing to the observed outcomes. While this study has unveiled promising results regarding the combined in vitro, in vivo and in silico biological activities of the essential oil and honey, it is imperative to delve further into the underlying mechanisms through additional experimentation and alternative experimental methods. Understanding these mechanisms in greater detail will not only enhance our comprehension of the therapeutic potential but also pave the way for the development of innovative treatments and applications rooted in the synergy of these natural compounds. Furthermore, it would be advantageous to test different possible combinations using experimental design model. Moreover, it would be better to test the effect of single compounds of MPEO to clearly elucidate their efficiency. MPEO alone or combined with thyme honey may be a useful for the development of novel biopharmaceuticals.

Titer and charge-based heterogeneity multiattribute monitoring of mAbs in cell culture harvest using 2D ProA CEX MS

Robust monitoring of heterogeneity in biopharmaceutical development is crucial for producing safe and efficacious biotherapeutic products. Multiattribute monitoring (MAM) has emerged as an efficient tool for monitoring of mAb heterogeneities like deamidation, sialylation, glycosylation, and oxidation. Conventional biopharma analysis during mAb development relies on use of one-dimensional methods for monitoring titer and charge-based heterogeneity using non-volatile solvents without direct coupling with mass spectrometry (MS). This approach requires analysis of mAb harvest by ProA for titer estimation followed by separate cation exchange chromatography (CEX) analysis of the purified sample for estimating charge-based heterogeneity. This can take up to 60–90 min due to the required fraction collection and buffer exchange steps. In this work, a native two-dimensional liquid chromatography (2DLC) mass spectrometry method has been developed with Protein A chromatography in the first dimension for titer estimation and cation exchange chromatography (CEX) in the second dimension for charge variant analysis. The method uses volatile salts for both dimensions and enables easy coupling to MS. The proposed 2DLC method exhibits a charge variant profile that is similar to that observed via the traditional methods and takes only 15 min for mass identification of each variant. A total of six charge variants were separated by the CEX analysis after titer estimation, including linearity assessment from 5 μg to 160 μg of injected mAb sample. The proposed method successfully estimated charge variants for the mAb innovator and 4 of its biosimilars, showcasing its applicability for biosimilarity exercises. Hence, the 2D ProA CEX MS method allows direct titer and charge variant estimation of mAbs in a single workflow.

Development of a high throughput oxidation profiling strategy for monoclonal antibody products

Oxidation is one of the most common degradation pathways of biopharmaceutics, potentially leading to altered product stability, pharmacokinetics, reduced biological activity and/or an increased immunogenicity. However, it is often insufficiently assessed in early development stages, leaving potential molecule liabilities undiscovered. Aim of the present work was the development of a high throughput oxidation profiling strategy, applicable throughout various stages of biopharmaceutical development. The study demonstrates that the combination of multiple stress assays, including peroxide-based, visible light, and metal-catalyzed oxidation (MCO), enables a comprehensive understanding of a mAb's oxidation susceptibility. The most effective parameters to evaluate oxidation in a high-throughput screening workflow are aggregation, tryptophan oxidation and changes in the hydrophobicity profile of the Fc and Fab subunit measured via Size Exclusion Chromatography, Intrinsic Tryptophan Fluorescence Emission spectroscopy and Reversed-Phase Chromatography subunit analysis, respectively. This oxidation profiling approach is valuable tool to systematically characterize the oxidation susceptibility under relevant conditions, time effective and with minimal sample consumption.

An all-in-one tetrazine reagent for cysteine-selective labeling and bioorthogonal activable prodrug construction

The prodrug design strategy offers a potent solution for improving therapeutic index and expanding drug targets. However, current prodrug activation designs are mainly responsive to endogenous stimuli, resulting in unintended drug release and systemic toxicity. In this study, we introduce 3-vinyl-6-oxymethyl-tetrazine (voTz) as an all-in-one reagent for modular preparation of tetrazine-caged prodrugs and chemoselective labeling peptides to produce bioorthogonal activable peptide-prodrug conjugates. These stable prodrugs can selectively bind to target cells, facilitating cellular uptake. Subsequent bioorthogonal cleavage reactions trigger prodrug activation, significantly boosting potency against tumor cells while maintaining exceptional off-target safety for normal cells. In vivo studies demonstrate the therapeutic efficacy and safety of this prodrug design approach. Given the broad applicability of functional groups and labeling versatility with voTz, we foresee that this strategy will offer a versatile solution to enhance the therapeutic range of cytotoxic agents and facilitate the development of bioorthogonal activatable biopharmaceuticals and biomaterials.

An industry perspective approach and control strategy for implementation of ready-to-use cells in bioassays: survey outcome and recommendations.

The BioPhorum Development Group is an industry collaboration enabling the sharing of common practices for the development of biopharmaceuticals. Bioassays are an important part of an analytical control system. Utilization of ready-to-use cells can increase operational flexibility and improve efficiency by providing frozen cell banks uniform stock while removing challenges associated with maintaining cultured cells. The BioPhorum Development Group-Bioassay workstream conducted an intercompany benchmarking survey and group discussions around the use of ready-to-use cells for bioassays. The results of the collaboration provide alignment on nomenclature, production, qualification and implementation of ready-to-use cells to support the assay life cycle.

High yield sterile filtration process for highly concentrated lentiviral vectors.

The development and manufacture of biopharmaceuticals are subject to strict regulations that specify the required minimum quality of the products. A key measure to meet these quality requirements is the integration of a sterile filtration step into the commercial manufacturing process. Whereas common procedures for most biologics exist, this is challenging for lentiviral vector (LVV) production for ex vivo gene therapy. LVVs nominal size is more than half the pore size (0.2µm) of filters used for sterile filtration. Hence, highly concentrated virus solutions are prone to filter clogging if aggregation of viruses occurs or impurities attach to the viruses. Several filters were screened aiming to identify those which allow filtering highly concentrated stocks of LVVs of up to 1E+9 transducing unitsmL-1 , which corresponds to 4.5E+12 particlesmL-1 . In addition, the effect of endonuclease treatment upstream of the purification process on filter performance was studied. In summary, three suitable filters were identified in a small-scale study (<15mL) with virus yields >80% and the process was successfully scaled-up to a final scale of 100mL LVV stock solution.

BIOTECHNOLOGY AND GENETIC ENGINEERING: CURRENT ADVANCEMENTS, EMERGING CHALLENGES, AND FUTURE HORIZONS IN MODERN SCIENCE

Biotechnology is the term given to the branch of science that employs knowledge, techniques, and genetic engineering methods for creating various products using living organisms as raw material. Although this term has been recently adopted, humans have long been manipulating biological processes for their own benefit. Biotechnology is multidisciplinary and encompasses various fields of knowledge, with significant progress achieved over the years, particularly in the medical and cosmetic sectors. Through genetic engineering, it has become possible to edit genes more precisely, revolutionizing the study and manipulation of DNA and allowing the proposal and use of strategies that were once unthinkable in the scientific field to address contemporary issues. Among its most recent contributions are the development of new drugs and biopharmaceuticals, vaccines, cellular, genetic, and protein therapies, as well as the production of biocosmetics. However, there are still challenges to be faced in the extensive use of genetic engineering, ranging from production costs and the success of the technique at the molecular level to ethical issues surrounding the application of this technology. Thus, this literature review aims to discuss the most recent advances, applications, and future perspectives of biotechnology and genetic engineering in the scientific field.

Evolving Paradigms of Recombinant Protein Production in Pharmaceutical Industry: A Rigorous Review

Many beneficial proteins have limited natural availability, which often restricts their supply and thereby reduces their potential for therapeutic or industrial usage. The advent of recombinant DNA (rDNA) technology enables the utilization of different microbes as surrogate hosts to facilitate the production of these proteins. This microbial technology continues to evolve and integrate with modern innovations to develop more effective approaches for increasing the production of recombinant biopharmaceuticals. These strategies encompass fermentation technology, metabolic engineering, the deployment of strong promoters, novel vector elements such as inducers and enhancers, protein tags, secretion signals, synthetic biology, high-throughput devices for cloning, and process screening. This appraisal commences with a general overview regarding the manufacture of recombinant proteins by microbes and the production of biopharmaceuticals, their trends towards the development of biopharmaceuticals, and then discusses the approaches adopted for accomplishing this. The design of the upstream process, which also involves host selection, vector design, and promoter design, is a crucial component of production strategies. On the other hand, the downstream process focuses on extraction and purification techniques. Additionally, the review covers the most modern tools and resources, methods for overcoming low expression, the cost of producing biopharmaceuticals in microbes, and readily available recombinant protein products.

Teaching biologics design using molecular modeling and simulations.

Teaching chemistry and biology students about biologics design remains challenging despite its increasing importance in pharmaceutical development. Monoclonal antibodies, commonly called mAbs, are the most popular biologics. They have been developed into drugs to treat various diseases in the past decades. Multiple challenges exist for designing proper formulations to stabilize mAbs, such as preventing aggregation and mitigating viscosity. Molecular modeling and simulations can improve pharmaceutical products by examining the interactions between mAbs and other compounds, such as excipients. To introduce students to biopharmaceuticals, eight students at the Stevens Institute of Technology participated in a semester-long course to learn the challenges of pharmaceutical development and different computational skills to study biologics design. The students started with a limited background in this field. Throughout one semester, they were introduced to various literature and software tools for modeling antibodies and studying their interactions with excipients. This paper aims to develop a course structure to be replicated at other universities and institutions to teach biopharmaceutical development to students.

"A Most Equitable Drug": How the Clinical Studies of Convalescent Plasma as a Treatment for SARS-CoV-2 Might Usefully Inform Post-Pandemic Public Sector Approaches to Drug Development.

Interventional clinical studies of convalescent plasma to treat COVID-19 were predominantly funded and led by public sector actors, including blood services operators. We aimed to analyze the processes of clinical studies of convalescent plasma to understand alternatives to pharmaceutical industry biopharmaceutical research and development, particularly where public sector actors play a dominant role. We conducted a qualitative, critical case study of purposively sampled prominent and impactful clinical studies of convalescent plasma during 2020-2021.

XYZ Biotech: A Fictional Business Development Case Utilizing Quantitative Tools to Evaluate a New Antibody-Drug-Conjugate Opportunity for Metastatic Breast Cancer

Multiple considerations must be evaluated with a new acquisition or license in biopharmaceutical business development. This case utilizes a fictional company, XYZ Biotech, to illustrate the quantitative considerations in evaluating an antibody-drug combination product in the metastatic breast cancer (mBC) space. The steps shown in this case also include the development of a market model to define the potential size of the market opportunity, a weighted benefit pricing model to evaluate the present price, and a net present value model to estimate projections from the product. The case also engages multiple qualitative considerations as part of the discussion section. It concludes by offering several nuanced questions at the end to stimulate discussion around the analysis, probe additional considerations needed for a business case, and analyze what other elements or issues would drive a decision. In the appendix is a short case summary of key facts for participants to develop solutions and share in class before reviewing the case narrative.

An Industry Perspective on the use of Forced Degradation Studies to Assess Comparability of Biopharmaceuticals

Forced degradation, also known as stress testing, is used throughout pharmaceutical development for many purposes including assessing the comparability of biopharmaceutical products according to ICH Guideline Q5E. These formal comparability studies, the results of which are submitted to health authorities, investigate potential impacts of manufacturing process changes on the quality, safety, and efficacy of the drug. Despite the wide use of forced degradation in comparability assessments, detailed guidance on the design and interpretation of such studies is scarce. The BioPhorum Development Group is an industry-wide consortium enabling networking and sharing of common practices for the development of biopharmaceuticals. The BioPhorum Development Group Forced Degradation Workstream recently conducted several group discussions and a benchmarking survey to understand current industry approaches for the use of forced degradation studies to assess comparability of protein-based biopharmaceuticals. The results provide insight into the design of forced degradation studies, analytical characterization and testing strategies, data evaluation criteria, as well as some considerations and differences for non-platform modalities (e.g., non-traditional mAbs). This article presents survey responses from several global companies of various sizes and provides an industry perspective and experience regarding the practicalities of using forced degradation to assess comparability.

Optimization of a Bacterial Cultivation Medium via a Design-of-Experiment Approach in a Sartorius Ambr® 15 Fermentation Microbioreactor System

In the evolving landscape of sustainable biopharmaceutical process development, the utilization of bacteria in the production of various compounds via fermentation has attracted extensive attention from scientists. A successful fermentation process and the release of its associated products hinge on the synergy between an efficient bacterial strain and the formulation of a suitable growth medium. Balancing all nutrient levels of a growth medium to maximize microbial growth and the product quality is quite an intricate task. In this context, significant advancements have been achieved via the strategic implementation of design-of-experiment (DOE) methodologies and the utilization of parallel microbioreactor systems. This work presents a case study of the fermentation growth medium optimization of a Gram-negative bacterium of the Neisseriaceae family that releases outer membrane vesicles (OMVs), which represent a potential vaccine platform. To achieve this, the ability of Sartorius MODDE®13 DOE software to explore multiple variables and their interactions was combined with the functionality of a Sartorius Ambr® 15F parallel microbioreactor system. The findings reported in this study have led to the design of a well-suited fermentation medium for a Gram-negative bacterium and an improvement in the quality of the OMVs produced from it.

Exploring the neuroprotective potential of antimicrobial peptides from Dinoponera quadriceps venom against pentylenetetrazole-induced seizures in vivo

Epilepsy affects around 50 million people worldwide and 30% of patients have difficulty controlling the disease. The search for substances that can fill the existing gaps in the treatment of epilepsy is of great importance. Arthropod venoms are promising sources for this purpose due to the presence of small peptides that modulate the activity of ion channels and neuron receptors. The aim of this study was to investigate dinoponeratoxins from the Dinoponera quadriceps ant venom (M-PONTX-Dq3a, M-PONTX-Dq3b and M-PONTX-Dq3c) as potential anticonvulsants. We evaluated them in a seizure model induced by pentylenetetrazole (PTZ) in male swiss mice. Interestingly, intraperitoneal treatment with each peptide increased the time until the first seizure and the percentage of survival, with M-PONTX-Dq3b showing the best results. M-PONTX-Dq3a was discarded due to the appearance of some signs of toxicity with the increase in malondialdehyde (MDA) levels in the striatum. Both, M-PONTX-Dq3b and M-PONTX-Dq3c decreased iNOS and TNF-α in the hippocampus. Notably, M-PONTX-Dq3c treatment decreased the levels of MDA and nitrite in the cortex and hippocampus. Our results indicate that, M-PONTX-Dq3b and M-PONTX-Dq3c have anticonvulsant activity and exhibit anti-inflammatory effects in epilepsy, offering new perspectives for biopharmaceutical development.

Automated method for quantification of20aminoacids in cell culture media during biopharmaceutical development.

Herein, a step-by-step protocol for simultaneous detection of 20 amino acids commonly present incell culture media is described. The protocol facilitates detection of both primary and secondary amino acids through a two-step precolumn derivatization strategy usingortho-phthalaldehyde and 9-fluorenylmethyl chloroformate as derivatizing agents. The separation of derivatized amino acids with varying hydrophobicity is achieved through reverse-phase chromatography. The amino acids are simultaneously detected in a single workflow through the use of Variable Wavelength Detector at 338 and 262nm. The protocol is applicable for both mammalian and bacterial cell culture matrices with an option for automation of precolumn derivatization.

Process intensification in biopharmaceutical process development and production – an industrial perspective

Abstract Process intensification aims to increase productivity in biologics manufacturing. Significant progress has been made in academia, the biopharmaceutical industry, and by the regulatory guidance since the 2000s. Process intensification can include all unit operations of a drug substance manufacturing process. The applied upstream concepts have consequences on the downstream process (DSP). The DSP process must manage larger product amounts while ensuring the required quality and impurity profiles, and cope with the available time frame as per scheduling requirements in a facility. Further, intensification in DSP is not based on a single technology only but rather on various technologies. This contribution provides an industry perspective on process intensification, describing basic concepts, technical and engineering aspects as well as the impact on the manufacturing process given existing facilities and a product portfolio to be manufactured. It also covers scientific approaches that support understanding and design of intensified bioprocesses. From an implementation perspective, the technologies used for intensification must be robust, scalable, and suitable for commercial manufacturing. Specific examples for a high seeding density fed batch (using N-1 perfusion) and a continuous process are provided for Chinese hamster ovary (CHO) cells producing therapeutic antibodies. Economic and sustainability aspects are addressed as well. Process intensification in an industrial environment is complex and many factors need to be considered, ranging from characteristics of a specific molecule to its commercial manufacturing at internal or external sites for global or regional markets.

Three-Dimensional Homodyne Light Detection (3D-HLD) for High-Throughput Submicron Particle Analysis in (Highly Concentrated) Protein Biopharmaceuticals, Viral Vectors, and LNPs

During biopharmaceutical development, particle monitoring and characterization are crucial. Notably, particles can be impurities considered as critical quality attribute, or active pharmaceutical ingredient (e.g., viral vectors) or drug delivery system (e.g., lipid nanoparticles) itself. Three-dimensional homodyne light detection (3D-HLD) is a novel technique that can characterize particles in the ∼0.2 µm to 2.0 µm size range.We evaluated 3D-HLD for the analysis of high concentration protein formulations (up to 200 mg/mL), where formulation refractive index and background noise became limiting factors with increasing protein concentration. Sample viscosity however did not impact 3D-HLD results, in contrast to comparative analyses with NTA and MRPS. We also applied 3D-HLD in high-throughput screenings at high protein concentration or of lipid nanoparticle and viral vector formulations, where impurities were analyzed in the presence of a small (<0.2 µm) particulate active pharmaceutical ingredient. 3D-HLD turned out to be in good agreement with or a good complement to other state-of-the-art particle characterization techniques, including BMI, MRPS, and DLS.The main application of 3D-HLD is high-throughput particle analysis at low sample volume. Follow-up investigation of the optimized particle sizing approach and of detection settings could further improve the understanding of the method and potentially increase ease of operation.

CD33 CAR T-Cells (CD33CART) for Children and Young Adults with Relapsed/Refractory AML: Dose-Escalation Results from a Phase I/II Multicenter Trial

Introduction: Current therapies for patients with acute myeloid leukemia (AML) push the limits of chemotherapy intensity but cure only 30% of adults and 70% of children. Recently developed antibody-based therapies and molecularly-targeted agents only modestly improve outcomes for select patient subsets. Thus, new approaches are needed. Given the success of chimeric antigen receptor (CAR) T-cell therapies for acute lymphoblastic leukemia (ALL), we developed and optimized a novel CD33 CAR T-cell (CD33CART) construct for clinical testing in a multicenter Phase I/II clinical trial in children, adolescents, and young adults (AYA) with relapsed/refractory (r/r) AML. ( Qin H, et al. JITC 2021) We report interim results following completion of the dose-escalation phase. Methods: This multicenter phase I/II clinical trial (NCT03971799) was conducted as a 3+3 dose escalation study through the Pediatric Transplantation and Cell Therapy Consortium with National Marrow Donor Program sponsorship and managed by CIBMTR CRO. Autologous CD33CART product were centrally manufactured on a ClinicMACS Prodigy® by the Biopharmaceutical Development Program at the Frederick National Laboratory for Cancer Research (NCI). Eligibility criteria were r/r AML in subjects &amp;lt; 35 years old with adequate organ/performance status and an identified allogeneic stem cell transplant (SCT) donor. Bone marrow assessment was used for standard morphologic evaluation and central flow cytometric minimal residual disease (MRD) quantification. All subjects received pre-CD33CART lymphodepleting chemotherapy (LD) with fludarabine (75-120 mg/m 2) and cyclophosphamide (900-1000 mg/m 2). CD33CART doses levels (DLs) were: DL1: 3 x 10 5 CD33 CAR+ T-cells/kg; DL2: 1 x 10 6/kg; DL3: 3 x 10 6/kg and DL4: 1 x 10 7/kg. Adverse event grading used CTCAE v5 with incorporation of ASTCT consensus definitions for cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS). Dose-limiting toxicities (DLTs) included &amp;gt; grade 3 CRS, &amp;gt; grade 3 ICANS, and persistent &amp;gt; grade 3 non-hematologic toxicity that did not resolve &amp;lt; grade 2 in 72 hours. CD33CART persistence was assessed via flow cytometric evaluation of the blood and bone marrow. Data cut-off was June 1, 2023. Results: A total of 24 subjects (median age of 16 years, range 1-34 years), were enrolled, 12 (50%) of whom underwent a prior SCT. CD33CART products were successfully manufactured for 23 subjects and infused into 19. In 4 non-infused subjects with manufactured products, 1 died from progressive disease (PD) prior to LD, 2 transitioned to palliative care and 1 withdrew consent to seek alternative therapy. Manufacturing was not performed in one subject due to death from PD after enrollment. One morbidly obese subject (BMI=49.3) enrolled at DL4 but received treatment at DL3 due to weight-based manufacturing limitations. The median time from enrollment to infusion was 47 days (range, 24-242). (Table) Three subjects each were infused at DL1 and DL2 without DLTs. At DL3, 1 subject experienced DLT (grade 4 CRS) prompting expansion at this dose level, and no subsequent DLTs were observed. At DL4, 1 subject experienced a prolonged grade 3 CRS (&amp;gt; 72 hours) and grade 3 ICANS, both constituting DLT and necessitating expansion of the cohort without additional DLTs seen. Across all dose-levels, CRS was seen in 13 (68%) patients and was &amp;gt; grade 3 in 4 (21%) with onset typically within the first 24 hours post-infusion. Complete remission (CR) was only seen at DL4 and achieved in 2 subjects, both achieving an MRD negative CR alongside myeloid aplasia. One SCT naïve subject developed candidemia during aplasia but was able to proceed to an allogenic SCT, achieved engraftment, and was in remission at Day +100 post SCT. The second patient (post-two prior SCTs) declined third SCT, had spontaneous count recovery and remained in remission until day +119 (MRD+ relapse). Transient CD33CART expansion was detected in 9 (47.4%) subjects overall and in all 6 (100%) subjects at DL4. Conclusions: CD33CART manufacturing is feasible in children and AYAs with r/r AML with acceptable toxicity experienced in treated subjects. CD33CART expansion was best in subjects treated at DL4 (1 x 10 7/kg) with MRD negative CRs and transient myeloid aplasia occurring in 2 of 5 (40%) subjects evaluable for response (Table). Based on early clinical efficacy at DL4, enrollment continues in the phase 2 portion.