mAb Molecules Research Articles

Nuclear Magnetic Resonance Study of Monoclonal Antibodies Near an Oil-Water Interface.

Monoclonal antibodies (mAb) represent an important class of biologic therapeutics that can treat a variety of diseases including cancer, autoimmune disorders or respiratory conditions (e.g. COVID-19). However, throughout their development, mAb are exposed to air-water or oil-water interfaces that may trigger mAb partial unfolding that can lead to the formation of proteinaceous aggregates. Using a combination of dynamic surface tensiometry and spatially resolved 1D 1H NMR spectroscopy, this study investigates if adsorption of a model IgG2a-κ mAb to the oil-water interface affects its structure. Localized NMR spectroscopy was performed using voxels of 375 µm, incrementally approaching the oil-water interface. Dynamic interfacial tension progressively decreases at the oil-water interface over time, confirming mAb adsorption to the interface. Localized NMR spectroscopy results indicate that, while the number of mAb-related chemical resonances and chemical shift frequencies remain unaffected, spectral line broadening is observed as voxels incrementally approach the oil-water interface. Moreover, the spin-spin (T2) relaxation of the mAb molecule was measured for a voxel centered at the interface and shown to be affected differentially across the mAb resonances, indicating a rotational restriction for mAb molecules due to presence of the interface. Finally, the apparent diffusion coefficient of the mAb for the voxel centered at the interface is lower than the bulk mAb. These results suggest that this specific mAb interacts with and may be in exchange with bulk mAb phase in the vicinity of the interface. As such, these localized NMR techniques offer the potential to probe and quantify alterations of mAb properties near interfacial layers.

Immunoliposomes for Neuroblastoma: Review of the Past Experience and Design of a Novel Nanoparticle.

High-risk/refractory neuro-blastoma (NBL) treatments include anti-GD2-monoclonal antibodies (mAbs). Several immunoliposomes (ILs) covered with anti-GD2-mAbs (GD2-ILs) have been tested pre-clinically. We aimed to review literature on GD2-IL for characteristics of nanoparticles/payloads, conjugation of mAb/fragments and preclinical data, as well as to explore the feasibility of a recently proposed GD2-IL loaded with the antimetabolite oxamate. Initial PubMed search was generalized for immunoliposomes in cancer. Further search was focused on papers for GD2-IL [keywords: "Immunoliposomes and cancer (or neuroblastoma)"]. There were 811 results on "immunoliposomes"; >50% were on "immunoliposomes, cancer" (n=439, June 2024). Seventeen items resulted from "immunoliposomes, neuroblastoma" (one was "publishers' correction"). Sixteen GD2-IL references were reviewed (1993-current). The mean±SD GD2-ILs size was 124.8±31 nm (range=86-171). Six papers described GD2-ILs with DNA-damaging agents [doxorubicin (n=4), etoposide (n=1), irinotecan+HDAC inhibitor (n=1)]. Other payloads included: fenretinide (n=4 papers), C-myb antisense (n=2), survivin inhibitor (n=1), tyrosine kinase inhibitor (n=1), IL15 (n=1), and oxamate (n=1). These 9 drug-loads included both hydrophilic and hydrophobic molecules. Except for IL15 and C-myb antisense with high molecular weights (MWs), and oxamate with low MW, the remaining compounds had comparable MWs (496±100 g/mol, range=349-588.6). The overall encapsulation efficiency was 66.2±25.6%. There were 17-30 mAb molecules attached to an IL with PEGylation. Experiments with GD2-positive/GD2-negative cells demonstrated selective efficacy/tropism of GD2-ILs. Mouse models confirmed efficacy, GD2-specific tumor accumulation, decreased toxicity, and improved pharmacokinetic-pharmacodynamics. PEGylated anti-GD2-IL may allow NBL tropism. A size of approximately 100 nm could allow vascular permeability and packaging of oxamate in amounts needed for profound/selective lactate dehydrogenase-A inhibition. Thus, oxamate-loaded GD2-ILs may allow exploring the great translational potential of Warburg effect inhibition in GD2-positive cancers.

Charge Detection Mass Spectrometry Reveals Conformational Heterogeneity in Megadalton-Sized Monoclonal Antibody Aggregates.

Aggregation of protein-based therapeutics can occur during development, production, or storage and can lead to loss of efficacy and potential toxicity. Native mass spectrometry of a covalently linked pentameric monoclonal antibody complex with a mass of ∼800 kDa reveals several distinct conformations, smaller complexes, and abundant higher-order aggregates of the pentameric species. Charge detection mass spectrometry (CDMS) reveals individual oligomers up to the pentamer mAb trimer (15 individual mAb molecules; ∼2.4 MDa) whereas intermediate aggregates composed of 6-9 mAb molecules and aggregates larger than the pentameric dimer (1.6 MDa) were not detected/resolved by standard mass spectrometry, size exclusion chromatography (SEC), capillary electrophoresis (CE-SDS), or by mass photometry. Conventional quadrupole time-of-flight mass spectrometry (QTOF MS), mass photometry, SEC, and CE-SDS did not resolve partially or more fully unfolded conformations of each oligomer that were readily identified using CDMS by their significantly higher extents of charging. Trends in the charge-state distributions of individual oligomers provides detailed insight into how the structures of compact and elongated mAb aggregates change as a function of aggregate size. These results demonstrate the advantages of CDMS for obtaining accurate masses and information about the conformations of large antibody aggregates despite extensive overlapping m/z values. These results open up the ability to investigate structural changes that occur in small, soluble oligomers during the earliest stages of aggregation for antibodies or other proteins.

The silicone depletion in combination products induced by biologics

Silicone oil (SO) migration into the drug product of combination products for biopharmaceuticals during storage is a common challenge. As the inner barrel surface is depleted of SO the extrusion forces can increase compromising the container functionality. In this context we investigated the impact of different formulations on the increase in gliding forces in a spray-on siliconized pre-filled syringe upon storage at 2–8 °C, 25 °C and 40 °C for up to 6 months. We tested the formulation factors such as surfactant type, pH, and ionic strength in the presence of one monoclonal antibody (mAb) as well as compared three mAbs in one formulation. After 1 month at 40 °C, the extrusion forces were significantly increased due to SO detachment dependent on the fill medium. The storage at 40 °C enhanced the SO migration process but it could also be observed at lower storage temperatures. Regarding the formulation factors the tendency for SO migration was predominantly dependent on the presence and type of surfactant. Interestingly, when varying the mAb molecules, one of the proteins showed a rather stabilizing effect on the SO layer resulting into higher container stability. In contrast to the formulation factors, those different stability outcomes could not be explained by interfacial tension (IFT) measurements at the SO interface. Further characterization of the mAb molecules regarding interfacial rheology and conformational stability were not adequately able to explain the observed difference. Solely a hydrophobicity ranking of the molecules correlated to the stability outcome. Further investigations are needed to clarify the role of the protein in the SO detachment process and to understand the cause for the stabilization. However, the study clearly demonstrated that the protein itself plays a critical role in the SO detachment process and underlined the importance to include verum for container stability.

Analytical Quality by Design as applied to the development of a SEC-HPLC platform procedure for the determination of monoclonal antibody purity without mobile phase additives

This work presents the application of AQbD principles to the development of a size exclusion chromatography (SEC) HPLC procedure for the determination of monoclonal antibody (mAb) product purity using state-of-the-art column technology available via the Waters™ XBridge Premier Protein SEC column. Analytical Quality by Design (AQbD) emphasizes a systematic, risk-based lifecycle approach to analytical procedure development based on sound statistical methodologies. It has recently become increasingly recommended by regulatory agencies as a response to the need for greater efficiency, improved reliability, and increased robustness among modern analytical procedures in the pharmaceutical industry. Use of an Analytical Target Profile (ATP) and formal risk assessments informed the application of Design of Experiments (DoE) to optimize this analytical procedure, as well as assess its robustness and ruggedness. Importantly, our ruggedness results demonstrated the transferability of this procedure between two laboratories within the Catalent Biologics Global Network. Application of this analytical procedure as a platform approach for evaluating mAb purity is expected to support expedited, first-in-human timelines of mAb molecules by enabling great quantitative performance with simple mobile phase buffer compositions. Taken together, this case study demonstrates the utility of adopting AQbD principles in analytical procedure development.

Correlating Surface Activity with Interface-Induced Aggregation in a High-Concentration mAb Solution.

Interface-induced aggregation resulting in protein particle formation is an issue during the manufacturing and storage of protein-based therapeutics. High-concentration formulations of therapeutic proteins are even more prone to protein particle formation due to increased protein-protein interactions. However, the dependence of interface-induced protein particle formation on bulk protein concentration is not understood. Furthermore, the formation of protein particles is often mitigated by the addition of polysorbate-based surfactants. However, the details of surfactant-protein interactions that prevent protein particle formation at high concentrations remain unclear. In this work, a tensiometer technique was used to evaluate the surface pressure of an industrially relevant mAb at different bulk concentrations, and in the absence and presence of a polysorbate-based surfactant, polysorbate 20 (PS20). The adsorption kinetics was correlated with subvisible protein particle formation at the air-water interface and in the bulk protein solution using a microflow imaging technique. Our results showed that, in the absence of any surfactant, the number of subvisible particles in the bulk protein solutions increased linearly with mAb concentration, while the number of protein particles measured at the interface showed a logarithmic dependence on bulk protein concentration. In the presence of surfactants above the critical micelle concentration (CMC), our results for low-concentration mAb solutions (10 mg/mL) showed an interface that is surfactant-dominated, and particle characterization results showed that the addition of the surfactant led to reduced particle formation. In contrast, for the highest concentration (170 mg/mL), coadsorption of proteins and surfactants was observed at the air-water interface, even for surfactant formulations above CMC and the surfactant did not mitigate subvisible particle formation. Our results taken together provide evidence that the ratio between the surfactant and mAb molecules is an important consideration when formulating high-concentration mAb therapeutics to prevent unwanted aggregation.

Role of Domain-Domain Interactions on the Self-Association and Physical Stability of Monoclonal Antibodies: Effect of pH and Salt.

Monoclonal antibodies (mAbs) make up a major class of biotherapeutics with a wide range of clinical applications. Their physical stability can be affected by various environmental factors. For instance, an acidic pH can be encountered during different stages of the mAb manufacturing process, including purification and storage. Therefore, understanding the behavior of flexible mAb molecules in acidic solution environments will benefit the development of stable mAb products. This study used small-angle X-ray scattering (SAXS) and complementary biophysical characterization techniques to investigate the conformational flexibility and protein-protein interactions (PPI) of a model mAb molecule under near-neutral and acidic conditions. The study also characterized the interactions between Fab and Fc fragments under the same buffer conditions to identify domain-domain interactions. The results suggest that solution pH significantly influences mAb flexibility and thus could help mAbs remain physically stable by maximizing local electrostatic repulsions when mAbs become crowded in solution. Under acidic buffer conditions, both Fab and Fc contribute to the repulsive PPI observed among the full mAb at a low ionic strength. However, as ionic strength increases, hydrophobic interactions lead to the self-association of Fc fragments and, subsequently, could affect the aggregation state of the mAb.

Assessment of Epithelial Lining Fluid Partitioning of Systemically Administered Monoclonal Antibodies in Rats

For systemically administered monoclonal antibodies (mAbs) with pharmacological targets in the epithelial lining fluid (ELF), information on the partitioning of mAb between plasma and ELF is instrumental for dose predictions. Bronchoalveolar lavage (BAL) combined with measurements of urea as indicator of sample dilution is often used to estimate ELF concentrations of a drug. However, unbalanced extraction of mAb and urea could potentially lead to a systematic bias in the back-calculated ELF concentration. In the present study 0.5, 1, or 4 mL phosphate-buffered saline was instilled to lungs of rats to obtain lavage samples after systemic dosing of mAb and tool small molecule (n≥4/group). Furthermore, extraction of urea, mAb and the small molecule was assessed by repeatedly lavaging the lung (n = 4). There was no statistically significant difference in the calculated partitioning into ELF between the evaluated instillation volumes. Repeated BAL demonstrated that urea and the small molecule were extracted from other sources than the ELF. In contrast, there was limited to none in-flow of mAb into the lavage fluid. The unbalanced extraction of urea and mAb could theoretically result in underestimated ELF concentrations and the calculated partitioning of 0.17±0.062 might therefore constitute a lower boundary for the true partitioning.

Development and Validation of Analytical Methods to Determine the Prolonged in-use Stability of Anticancer Monoclonal Antibodies in Clinical Practice

Background/purpose: Personalized dosing of monoclonal antibodies (mAbs) based on body weight and surface area may result in leftovers that need to be discarded due to the absence of stability data. After manufacturing, these mAbs undergo extensive quality control with a panel of methods. With some of these similar methods, these mAbs could be tested to see if stability is maintained after the opening of the pharmaceutical product vial and dilution for administration in a daily clinical setting. Prolonged in-use stability may reduce unnecessary waste and minimize the financial loss of these expensive drugs. Methods: Previously, based on extensive literature research, a complementary panel of methods was selected including visual inspection colour, apparency and, clarity (CAC), size exclusion chromatography (SEC), enzyme-linked immunosorbent assay (ELISA), dynamic light scattering (DLS), thermal denaturation (Tm), and aggregation (Tagg) determination, pH and dye ingress method. We evaluated, developed, and validated these methods with respect to the accuracy, precision, reproducibility, stability-indicating capability, carry-over, limit of quantification (LOQ) and limit of detection (LOD) for nivolumab and pembrolizumab as prototype anticancer mAbs. Results: Analytical results show that stability can be determined from a physiochemical, biological, and microbiological point of view with the selected methods. Forced degradation studies showed that even small instabilities can be detected using the panel of methods together, therefore the stability-indicating capability has been established. CAC shows mainly aggregation and discolouration. SEC shows aggregation and fragmentation/degradation, DLS gives information about turbidity and soluble aggregates, Tm and Tagg give information changes intra- and inter conformational changes, respectively, in the mAb molecule. ELISA shows potential loss in bioactivity and the pH of the sample is essential to the chemical composition of the mAb molecules in the solution. Conclusion: A panel of analytical methods was selected, enabling in-use stability determination of anticancer mAbs. This panel will be used in future studies to determine the prolonged use of leftover anticancer mAb products and infusion solutions, starting with nivolumab and pembrolizumab.

Protein-protein interactions in solutions of monoclonal antibodies probed by the dependence of the high-frequency viscosity on temperature and concentration.

Using a quartz crystal microbalance with dissipation monitoring (QCM-D), the complex high-frequency viscosity,  = η' - iη'', of concentrated solutions of a monoclonal antibody (mAb) was studied with respect to its dependence on temperature, T, and concentration, c. Lysozyme and bovine serum albumin (BSA) served as reference materials. Viscoelasticity was found for the mAb solution, while the reference materials behaved like Newtonian liquids. The QCM-D probes the solution's dynamics on the time scale of a few tens of nanoseconds. The processes of relaxation accessed with the QCM-D are not amenable to standard viscometry. The inverse loss tangent at 15 MHz (equal to η''/η' at 15 MHz, quantifying the elastic contribution to the oscillatory stress) was between 0.1 and 0.5 for the concentrated mAb solutions. It decreased with increasing temperature and decreasing pH. Activation energies of viscous flow, Ea,η, were derived from the functions η'(T). Ea,η was found to be higher for the mAb solutions than for water. No such increase was found for the reference materials. This difference evidences protein-protein interactions (PPIs) between the mAb molecules, which do not exist in the same way for lysozyme and BSA. The excipients citrate and arginine did not noticeably affect the mAb's high-frequency viscosity as determined with the QCM-D.

Monoclonal antibodies for the treatment of COVID-19 infection in children

ABSTRACT Introduction Monoclonal antibodies (mAbs) have been authorized for the treatment of COVID-19 in pediatric populations, however, there is a lack of evidence for their use in these populations. Areas covered We outline the evidence of mAbs for COVID-19, discuss their use in the treatment of COVID-19 infection for pediatric patients, and consider alternative treatment options and challenges to COVID-19 drug approvals. Expert opinion Limited evidence exists for the safety and efficacy of mAbs to treat COVID-19 in children as new variants emerge. In rare pediatric outpatient settings, such as profound immunodeficiency or severe pulmonary disease, the benefits of antiviral treatment for COVID-19 likely outweigh the relatively small risks. However, for the great majority of pediatric patients, mAb treatment is likely not indicated. Small molecule antiviral therapies are another potential treatment for COVID-19 in children in an outpatient setting, though neither mAb nor small molecule antiviral treatments have significant supporting evidence in children and developing a strong evidence base for these decisions will be challenging if not impractical. Ultimately, these decisions are likely to be made at the level of individual cases using expert opinion as the primary guiding principle.

Influence of ligand density variations on the two peak elution behavior of a monoclonal antibody in cation exchange chromatography

Cation exchange chromatography, as part of the monoclonal antibody purification train, is known as a mild polishing technique. However, in the last couple of years, more and more publications have shown unusual elution behavior, resulting from e.g. on-column (reversible) unfolding and aggregation of the predominantly mAb molecules. The stability of the investigated protein seems to play a significant role in this phenomenon. We have used a glycosylated IgG1 antibody as a model protein and investigated several influencing factors, including pH value and ligand density variations of three prototype Fractogel® cation exchange resins. Ligand density, pH and salt concentration are the main contributing factors in the Donnan effect, i.e. distribution of ions, between resin pore volume and bulk volume. This leads to a significantly lower pH value the protein is subjected to during the on-column hold time and therefore influences the conformational stability of our protein. Nano-DSF and kinetic SEC measurements show that the protein is destabilized at low pH values, but also, that the binding to the CEX resin and the elution with increasing salt concentration is responsible for the resulting two-peak elution behavior and partially reversible unfolding and aggregation.

Formation of transient highly-charged mAb clusters strengthens interactions with host cell proteins and results in poor clearance of host cell proteins by protein A chromatography

Protein A chromatography with a high salt wash usually leads to robust clearance of host cell proteins (HCPs) in most recombinant monoclonal antibodies (mAbs), but a small subset of recalcitrant mAbs show significant HCP copurification. In this study, we carried out systematic studies using 4 different mAbs to explore the HCP copurification mechanism. HCP identification results revealed that the 3 high-HCP mAbs had many common HCPs which do not copurify with the low-HCP mAb, suggesting a similar mechanism is at play. Through wash evaluation, surface patch analysis, chain-swapping, domain evaluation, and structure-guided mutations, several charged residues in each mAb were found which correlated with HCP copurification. Surprisingly, these residues are also critical for self-association propensity. We observed an inverse correlation between diffusion interaction parameter and HCP copurification. Each of the high-HCP mAbs could form dynamic clusters consisting of 3∼6 mAb molecules. Therefore, a mAb cluster can exhibit higher net positive charges on the order of 3 to 6, compared with the individual mAb. In Protein A chromatography, high-HCP mAbs had elution tailing which contained high level of HCPs. Addition of Arginine-HCl or point mutations preventing cluster formation effectively reduced HCP copurification and elution tailing. Based on these results, we propose a novel HCP-copurification mechanism that formation of mAb clusters strengthens charge-charge interactions with HCPs and thus compromises HCP removal by Protein A chromatography. Besides arginine, histidine under acidic pH conditions prevented cluster formulation and resulted in effective HCP removal. Finally, structure-guided protein engineering and solution screening by using cluster size as indicator are useful tools for managing mAbs with high-HCP issues.

Antibody-Based Approaches to Target Pancreatic Tumours.

Pancreatic cancer is an aggressive cancer with a dismal prognosis. This is due to the difficulty to detect the disease at an early and curable stage. In addition, only limited treatment options are available, and they are confronted by mechanisms of resistance. Monoclonal antibody (mAb) molecules are highly specific biologics that can be directly used as a blocking agent or modified to deliver a drug payload depending on the desired outcome. They are widely used to target extracellular proteins, but they can also be employed to inhibit intracellular proteins, such as oncoproteins. While mAbs are a class of therapeutics that have been successfully employed to treat many cancers, they have shown only limited efficacy in pancreatic cancer as a monotherapy so far. In this review, we will discuss the challenges, opportunities and hopes to use mAbs for pancreatic cancer treatment, diagnostics and imagery.

Effects of Monovalent Salt on Protein-Protein Interactions of Dilute and Concentrated Monoclonal Antibody Formulations.

In this study, we used sodium chloride (NaCl) to extensively modulate non-specific protein-protein interactions (PPI) of a humanized anti-streptavidin monoclonal antibody class 2 molecule (ASA-IgG2). The changes in PPI with varying NaCl (CNaCl) and monoclonal antibody (mAb) concentration (CmAb) were assessed using the diffusion interaction parameter kD and second virial coefficient B22 measured from solutions with low to moderate CmAb. The effective structure factor S(q)eff measured from concentrated mAb solutions using small-angle X-ray and neutron scattering (SAXS/SANS) was also used to characterize the PPI. Our results found that the nature of net PPI changed not only with CNaCl, but also with increasing CmAb. As a result, parameters measured from dilute and concentrated mAb samples could lead to different predictions on the stability of mAb formulations. We also compared experimentally determined viscosity results with those predicted from interaction parameters, including kD and S(q)eff. The lack of a clear correlation between interaction parameters and measured viscosity values indicates that the relationship between viscosity and PPI is concentration-dependent. Collectively, the behavior of flexible mAb molecules in concentrated solutions may not be correctly predicted using models where proteins are considered to be uniform colloid particles defined by parameters derived from low CmAb.

Dipolar Interactions and Protein Hydration in Highly Concentrated Antibody Formulations.

Molecular interaction mechanisms in high-concentrated protein systems are of fundamental importance for the rational development of biopharmaceuticals such as monoclonal antibody (mAb) formulations. In such high-concentrated protein systems, the intermolecular distances between mAb molecules are reduced to the size of the protein diameter (approx. 10 nm). Thus, protein-protein interactions are more pronounced at high concentrations; so a direct extrapolation of physicochemical properties obtained from measurements at a low protein concentration of the corresponding properties at a high protein concentration is highly questionable. Besides the charge-charge interaction, the effects of molecular crowding, dipolar interaction, changes in protein hydration, and self-assembling tendency become more relevant. Here, protein hydration, protein dipole moment, and protein-protein interactions were studied in protein concentrations up to 200 mg/mL (= 1.3 mM) in different formulations for selected mAbs using dielectric relaxation spectroscopy (DRS). These data are correlated with the second virial coefficient, A2, the diffusion interaction parameter, kD, the elastic shear modulus, G', and the dynamic viscosity, η. When large contributions of dipolar protein-protein interactions were observed, the tendency of self-assembling and an increase in solution viscosity were detected. These effects were examined using specific buffer conditions. Furthermore, different types of protein-water interactions were identified via DRS, whereby the effect of high protein concentration on protein hydration was investigated for different high-concentrated liquid formulations (HCLFs).

Remodeling the Tumor Myeloid Landscape to Enhance Antitumor Antibody Immunotherapies.

Simple SummaryIn addition to cancer cells themselves, tumors consist of several cell types that either function to suppress or promote tumor growth and disease progression. Macrophages are amongst the most abundant of these cell types and possess contrasting abilities to either enhance tumor growth and spread or to destroy cancer cells. Anti-cancer therapeutics such as Rituximab, Herceptin and Cetuximab, are cornerstones of current treatment for cancer patients. These therapeutics are antibodies that directly bind to cancer cells and aid macrophages in detecting and destroying these cells, through a process known as antibody dependent cellular phagocytosis (ADCP). Although the use of anti-cancer antibodies to treat large established tumors has improved survival rates, significant numbers of patients do not respond to this type of therapy. Thus, recently there has been an intense focus on designing new therapies which alter macrophages inside tumors, so that they can more effectively eliminate cancer cells through ADCP. Several molecules on the surface of macrophages can be targeted with antibodies to improve their ability to recognize and eliminate cancer cells. Here we review the most promising of these new targets, which offer the potential to circumvent resistance to therapy in cancer patients. Among the diverse tumor resident immune cell types, tumor-associated macrophages (TAMs) are often the most abundant, possess an anti-inflammatory phenotype, orchestrate tumor immune evasion and are frequently associated with poor prognosis. However, TAMs can also be harnessed to destroy antibody-opsonized tumor cells through the process of antibody-dependent cellular phagocytosis (ADCP). Clinically important tumor-targeting monoclonal antibodies (mAb) such as Rituximab, Herceptin and Cetuximab, function, at least in part, by inducing macrophages to eliminate tumor cells via ADCP. For IgG mAb, this is mediated by antibody-binding activating Fc gamma receptors (FcγR), with resultant phagocytic activity impacted by the level of co-engagement with the single inhibitory FcγRIIb. Approaches to enhance ADCP in the tumor microenvironment include the repolarization of TAMs to proinflammatory phenotypes or the direct augmentation of ADCP by targeting so-called ‘phagocytosis checkpoints’. Here we review the most promising new strategies targeting the cell surface molecules present on TAMs, which include the inhibition of ‘don’t eat me signals’ or targeting immunostimulatory pathways with agonistic mAb and small molecules to augment tumor-targeting mAb immunotherapies and overcome therapeutic resistance.

A Fully Integrated Online Platform For Real Time Monitoring Of Multiple Product Quality Attributes In Biopharmaceutical Processes For Monoclonal Antibody Therapeutics

In response to FDA's call for Quality by Design (QbD) in biopharmaceutical product development, the biopharmaceutical industry has been developing highly sensitive and specific technologies in the monitoring and controlling of product quality attributes for bioprocesses. We previously published the successful application of an off-line multi-attribute method (MAM) to monitor more than 20 critical quality attributes (CQA) with superior sensitivity for the upstream process. To further remove the hurdles of laborious process sampling and sample preparation associated with the offline method, we present here a fully integrated MAM based online platform for automated real time online process monitoring. This integrated system includes Modular Automated Sampling Technology (MAST) based aseptic sampling, multi-function Sequential Injection Analysis (SIA) sample preparation, UHPLC separation and high-resolution mass spectrometry (HRMS) analysis. Continuous automated daily monitoring of a 17-day cell culture process was successfully demonstrated for a model monoclonal antibody (mAb) molecule with similar specificity and sensitivity as we reported earlier. To the best of our knowledge, this is the first report of an end-to-end automated online MAM system, which would allow the MAM to be applied to routine bioprocess monitoring, potentially replacing multiple conventional low resolution and low sensitivity off-line methods. The online HPLC or HPLC/MS platform could be easily adapted to support other processing steps such as downstream purification with minimal software re-configuration.

Machine Learning Attempts for Predicting Human Subcutaneous Bioavailability of Monoclonal Antibodies.

One knowledge gap related to subcutaneous (SC) delivery is unpredictable and variable bioavailability. This study was aimed to develop machine learning methods to predict whether mAb's bioavailability was ≥70% or below, without completely knowing the mechanism and causality between inputs and outputs. A database of mAb SC products was built. The model training and validation were accomplished based on this database and a set of the inputs (product properties) were mapped to the output (bioavailability) using different machine learning algorithms. Dimensionality reduction was undertaken using principal component analysis (PCA). The bioavailability of the mAb products being investigated varied from 35% to 90%. The tree-based methods, including random forest (RF), Adaptive Boost (AdaBoost), and decision tree (DT) presented the best predictability and generalization power on bioavailability classification. The models based on Multi-layer perceptron (MLP), Gaussian Naïve Bayes (GaussianNB), and k nearest neighbor (kNN) algorithms also provided acceptable prediction accuracy. Machine learning could be a potential tool to predict mAb's bioavailability. Since all input features were acquired using theoretical calculations and predictions rather than experiments, the models may be particularly applicable to some early-stage research activities such as mAb molecule triage, design/optimization, mutant screening, molecule selection, and formulation design.

Evolving Antibody Therapies for the Treatment of Type 1 Diabetes.

Type 1 diabetes (T1D) is widely considered to be a T cell driven autoimmune disease resulting in reduced insulin production due to dysfunction/destruction of pancreatic β cells. Currently, there continues to be a need for immunotherapies that selectively reestablish persistent β cell-specific self-tolerance for the prevention and remission of T1D in the clinic. The utilization of monoclonal antibodies (mAb) is one strategy to target specific immune cell populations inducing autoimmune-driven pathology. Several mAb have proven to be clinically safe and exhibit varying degrees of efficacy in modulating autoimmunity, including T1D. Traditionally, mAb therapies have been used to deplete a targeted cell population regardless of antigenic specificity. However, this treatment strategy can prove detrimental resulting in the loss of acquired protective immunity. Nondepleting mAb have also been applied to modulate the function of immune effector cells. Recent studies have begun to define novel mechanisms associated with mAb-based immunotherapy that alter the function of targeted effector cell pools. These results suggest short course mAb therapies may have persistent effects for regaining and maintaining self-tolerance. Furthermore, the flexibility to manipulate mAb properties permits the development of novel strategies to target multiple antigens and/or deliver therapeutic drugs by a single mAb molecule. Here, we discuss current and potential future therapeutic mAb treatment strategies for T1D, and T cell-mediated autoimmunity.