Understanding Loss of Soluble High Molecular Weight Species during Filtration of Low Concentration Therapeutic Monoclonal Antibodies

Sterile filtration is an integral step in the manufacturing process of biological therapeutics. Protein adsorption to the surface of the filter is an unfortunate, common occurrence that can result in manufacturing difficulties, such as filter fouling or product loss. Although many filters have surface modifications to minimize adsorption, under certain conditions binding can still occur. We observed the loss of high molecular weight species (HMWS) during sterile filtration of eight different therapeutic monoclonal antibodies formulated at low protein concentrations across a commonly used hydrophilic polyvinylidene fluoride or polyvinylidene difluoride (PVDF) filter membrane. The protein absorption was specific to HMWS, and each antibody exhibited different degrees of filter adsorption. Debye screening length parameters of the solution (e.g. ionic strength) were adjusted, and influenced the amount of HMWS lost during filtration. Additionally, HMWS of a representative antibody (mAb1) were observed to be more positively charged than other size variants by ion-exchange chromatography. From these results, it is concluded that this HMWS loss is due to electrostatic interactions between HMWS and the filter surface. This adsorption can be reduced by increasing the ionic strength of the buffer matrix, demonstrating the influence of the Debye screening length in the filtration of low concentration proteins.     

Application of a High Throughput and Automated Workflow to Therapeutic Protein Formulation Development

Rapid and efficient formulation development is critical to successfully bringing therapeutic protein drug products into a competitive market under increasingly aggressive timelines. Conventional application of high throughput techniques for formulation development have been limited to lower protein concentrations, which are not applicable to late stage development of high concentration therapeutics. In this work, we present a high throughput (HT) formulation workflow that enables screening at representative concentrations via integration of a micro-buffer exchange system with automated analytical instruments. The operational recommendations associated with the use of such HT systems as well as the efficiencies gained (reduction in hands-on time and run time by over 70% and 30%, respectively), which enable practical characterization of an expanded formulation design space, are discussed. To demonstrate that the workflow is fit for purpose, the formulation properties and stability profiles (SEC and CEX) from samples generated by the HT workflow were compared to those processed by ultrafiltration/diafiltration, and the results were shown to be in good agreement. This approach was further applied to two case studies, one focused on a formulation screen that studied the effects of pH and excipient on viscosity and stability, and the other focused on selection of an appropriate viscosity mimic solution for a protein product.     

Variance Between Different Light Obscuration and Flow Imaging Microscopy Instruments and the Impact of Instrument Calibration

Subvisible particles (SVPs) are an obligatory critical quality attribute of the product, and yet, they are found in all biopharmaceutical products intended for infusion or injection. Light obscuration (LO) is the primary pharmacopeial method used to quantify SVPs. However, the method may not be equally sensitive toward all particles that can possibly occur. Calibration of LO instruments is usually performed using polystyrene beads suspended in water. In this study, the dependence of the sizing accuracy of LO analysis was evaluated by using a calibration suspension of lower refractive index beads made of silica suspended in sucrose solution. It was demonstrated that the sizing accuracy was strongly dependent on the reference material’s properties used for calibration. It was also demonstrated that flow imaging microscopy suffered from the same artifact, albeit to a smaller extent. We further tested different LO sensors and instruments. Interestingly, our results show that the sizing accuracy varied from instrument to instrument, strongly depending on the properties of the sensor.To summarize, sizing and counting accuracies were dependent on the material used for calibration and its optical properties as well as the calibration curve, the sensor, and the instrument supplier. Closer match of optical properties between calibration system and test system seems to improve the sensitivity of the measurement. The results of this study raise the following major practical implications: (1) LO and flow imaging microscopy are not truly orthogonal analytical methods, (2) while matching optimal properties of material used for calibration and test items increased sensitivity, this is of poor practical applicability given that analytes contain multiple particles, and (3) setting product-specific limits for SVPs require special considerations with regard to the data sets used.     

Liquid Oil Marbles: Increasing the Bioavailability of Poorly Water-Soluble Drugs

Many new therapeutic candidates and active pharmaceutical ingredients (APIs) are poorly soluble in an aqueous environment, resulting in their reduced bioavailability. A promising way of enhancing the release of an API and, thus, its bioavailability seems to be the use of liquid oil marbles (LOMs). An LOM system behaves as a solid form but consists of an oil droplet in which an already dissolved API is encapsulated by a powder. This study aims to optimize the oil/powder combination for the development of such systems. LOMs were successfully prepared for 15 oil/powder combinations, and the following properties were investigated: particle mass fraction, dissolution time, and mechanical stability. Furthermore, the release of API from both LOMs and LOMs encapsulated into gelatine capsules was studied.     

A Rapid High-Sensitivity Reversed–Phase Ultra High Performance Liquid Chromatography Mass Spectrometry Method for Assessing Polysorbate 20 Degradation in Protein Therapeutics

Polysorbate 20 (PS20), a widely used surfactant in protein therapeutics, has been reported to undergo hydrolytic degradation during product storage, causing the release of free fatty acids. The accumulation of free fatty acids in protein therapeutics was found to result in the formation of particles due to their limited aqueous solubility at 2°C-8°C. Quantitation of free fatty acids originating from PS20 degradation is thus important during bioprocess optimization and stability testing in formulation development to ensure optimum PS20 stability as well as product and process consistency in final drug products. This work reports the development of a simple and robust, high-throughput, reversed-phase ultra high performance liquid chromatography mass spectrometry method for high-sensitivity quantitation of lauric acid and myristic acid by using isotope-labeled fatty acid internal standards. The high sensitivity (<100 ng/mL for lauric acid) and suitable precision (intermediate precision relative standard deviation of 11%) of this method enable accurate detection of lauric acid produced from the degradation of less than 1% of PS20 in a 0.2-mg/mL formulation. Using accelerated thermal stability testing, this method identifies processes that exhibit fast PS20 degradation within only days and consequently allows faster iterative optimization of the process.     

Anticancer drug discovery by targeting cullin neddylation

Protein neddylation is a post-translational modification which transfers the ubiquitin-like protein NEDD8 to a lysine residue of the target substrate through a three-step enzymatic cascade. The best-known substrates of neddylation are cullin family proteins, which are the core component of Cullin–RING E3 ubiquitin ligases (CRLs). Given that cullin neddylation is required for CRL activity, and CRLs control the turn-over of a variety of key signal proteins and are often abnormally activated in cancers, targeting neddylation becomes a promising approach for discovery of novel anti-cancer therapeutics. In the past decade, we have witnessed significant progress in the field of protein neddylation from preclinical target validation, to drug screening, then to the clinical trials of neddylation inhibitors. In this review, we first briefly introduced the nature of protein neddylation and the regulation of neddylation cascade, followed by a summary of all reported chemical inhibitors of neddylation enzymes. We then discussed the structure-based targeting of protein–protein interaction in neddylation cascade, and finally the available approaches for the discovery of new neddylation inhibitors. This review will provide a focused, up-to-date and yet comprehensive overview on the discovery effort of neddylation inhibitors.     

Intranasal delivery of biotechnology-based therapeutics

Biotechnology-based therapeutics include a wide range of products, such as recombinant hormones, stem cells, therapeutic enzymes, monoclonal antibodies, genes, vaccines, among others. The administration of these macromolecules has been studied via various routes. The nasal route is one of the promising routes of administration for biotechnology products owing to its easy delivery, the rich vascularity of the nasal mucosa, high absorption and targeted action. Several preclinical studies have been reported for nasal delivery of these products and many are at the clinical stage. This review focuses on biotechnology-based therapeutics administered via the intranasal route for treating various diseases.     

A homogenous nanoporous pulmonary drug delivery system based on metal-organic frameworks with fine aerosolization performance and good compatibility

Pulmonary drug delivery has attracted increasing attention in biomedicine, and porous particles can effectively enhance the aerosolization performance and bioavailability of drugs. However, the existing methods for preparing porous particles using porogens have several drawbacks, such as the inhomogeneous and uncontrollable pores, drug leakage, and high risk of fragmentation. In this study, a series of cyclodextrin-based metal-organic framework (CD-MOF) particles containing homogenous nanopores were delicately engineered without porogens. Compared with commercial inhalation carrier, CD-MOF showed excellent aerosolization performance because of the homogenous nanoporous structure. The great biocompatibility of CD-MOF in pulmonary delivery was also confirmed by a series of experiments, including cytotoxicity assay, hemolysis ratio test, lung function evaluation, in vivo lung injury markers measurement, and histological analysis. The results of ex vivo fluorescence imaging showed the high deposition rate of CD-MOF in lungs. Therefore, all results demonstrated that CD-MOF was a promising carrier for pulmonary drug delivery. This study may throw light on the nanoporous particles for effective pulmonary administration.     

Toward Biotherapeutics Formulation Composition Engineering using Site-Identification by Ligand Competitive Saturation (SILCS)

Formulation of protein-based therapeutics employ advanced formulation and analytical technologies for screening various parameters such as buffer, pH, and excipients. At a molecular level, physico-chemical properties of a protein formulation depend on self-interaction between protein molecules, protein-solvent and protein-excipient interactions. This work describes a novel in silico approach, SILCS-Biologics, for structure-based modeling of protein formulations. SILCS Biologics is based on the Site-Identification by Ligand Competitive Saturation (SILCS) technology and enables modeling of interactions among different components of a formulation at an atomistic level while accounting for protein flexibility. It predicts potential hotspot regions on the protein surface for protein-protein and protein-excipient interactions. Here we apply SILCS-Biologics on a Fab domain of a monoclonal antibody (mAbN) to model Fab-Fab interactions and interactions with three amino acid excipients, namely, arginine HCl, proline and lysine HCl. Experiments on 100 mg/ml formulations of mAbN showed that arginine increased, lysine reduced, and proline did not impact viscosity. We use SILCS-Biologics modeling to explore a structure-based hypothesis for the viscosity modulating effect of these excipients. Current efforts are aimed at further validation of this novel computational framework and expanding the scope to model full mAb and other protein therapeutics.     

Extended Characterization and Impact of Visible Fatty Acid Particles - A Case Study With a mAb Product

In recent years, there has been increased scrutiny on the presence and formation of product-related particles in biopharmaceutical formulations. These types of particles, originating from the degradation of the active pharmaceutical ingredient or the excipients, can be challenging to identify and characterize due to their fragility. Additionally, the mechanisms of their formation as well as the impact of their presence on drug product safety can be complicated to elucidate. In this work, a case study is presented in which multiple batches of one formulated monoclonal antibody (mAb-A) were analyzed at different batch ages to better understand the formation of visible particles resulting from degradation of the surfactant polysorbate 20. The particle identity was determined by Raman spectroscopy as free fatty acid (FFA) and the particle composition over time was monitored by mass spectrometry. Further experimental work includes the counts and morphologies of subvisible particles by flow imaging microscopy. Finally, we evaluated the consequences of saline and human plasma exposure to the visible particles to better understand their fate upon dilution and/or administration which is routinely performed in the clinical setting. The experiments performed in this work can be used to support risk assessments of visible product-related particles.     

Comparison of Three Processes for Parenteral Nanoemulsion Production: Ultrasounds,Microfluidizer, and Premix Membrane Emulsification

Nanoemulsions are of great interest for pharmaceutical applications, including parenteral dosage forms. However, their production is still limited and requires more efficient and adaptive technologies. The more common systems are high-shear homogenization such as microfludizers at industrial scale and ultrasounds at research scale, both based on high energy, limiting their application for sensitive drugs. Recently a process based on premix membrane emulsification (PME) was developed to produce nanoemulsions. These 3 processes have been compared for the production of a model parenteral nanoemulsion containing all-trans retinoic acid, a thermolabile molecule that is used in the treatment of acute promyelocytic leukemia in a parenteral form. Droplet size and active integrity were studied because of their major interest for efficacy and safety assessment. Regarding droplet size, PME produced monodispersed droplets of 335 nm compared with the other processes that produced nanoemulsions of around 150 nm but with the presence of micron-size droplets detected by laser diffraction and optical microscopy. No real difference between the 3 processes was observed on active degradation during emulsifcation. However regarding stability, especially at 40°C, nanoemulsions obtained with the microfluidizer showed a greater molecule degradation and unstable nanoemulsion with a 4-times droplet size increase under stress conditions.     

Receptor-mediated targeted drug delivery systems for treatment of inflammatory bowel disease: Opportunities and emerging strategies

Inflammatory bowel disease (IBD) is a chronic intestinal disease with painful clinical manifestations and high risks of cancerization. With no curative therapy for IBD at present, the development of effective therapeutics is highly advocated. Drug delivery systems have been extensively studied to transmit therapeutics to inflamed colon sites through the enhanced permeability and retention (EPR) effect caused by the inflammation. However, the drug still could not achieve effective concentration value that merely utilized on EPR effect and display better therapeutic efficacy in the inflamed region because of nontargeted drug release. Substantial researches have shown that some specific receptors and cell adhesion molecules highly expresses on the surface of colonic endothelial and/or immune cells when IBD occurs, ligand-modified drug delivery systems targeting such receptors and cell adhesion molecules can specifically deliver drug into inflamed sites and obtain great curative effects. This review introduces the overexpressed receptors and cell adhesion molecules in inflamed colon sites and retrospects the drug delivery systems functionalized by related ligands. Finally, challenges and future directions in this field are presented to advance the development of the receptor-mediated targeted drug delivery systems for the therapy of IBD.     

Precision medicine for rare diseases: The times they are A-Changin'

The greatest challenge of current biomedicine is to identify curative therapies for every disease in a personalized way so that every individual gets benefit. To that end, however, we need fully understand mechanisms of disease that will drive the design of novel therapies and innovative approaches.For rare diseases (RDs) which individually affect low numbers of people (< 1:2000), but together, affect 300 million (∼10% of the world population) the constraints are greater. This is because: 1) there is limited knowledge on RD physiopathology; 2) the low number of patients strongly limits clinical trials; 3) there is low commercial interest by pharma; 4) when specific drugs reach the market, their high cost precludes their reaching all those who need them.Several possibilities that can help mitigate these barriers are discussed here, including orphan drug designation, drug repurposing, break-down into theratypes (as currently in place for Cystic Fibrosis), or novel precision-medicine-based approaches.     

Impact of Precipitation of Antibody Therapeutics After Subcutaneous Injection on Pharmacokinetics and Immunogenicity

Antibody therapeutics with poor solubility in the subcutaneous matrix may carry unintended risks when administered to patients. The objective of this work was to estimate the risk of antibodies that precipitate in vitro at neutral pH by determining the impact of poor solubility on distribution of the drug from the injection site as well as immunogenicity in vivo. Using fluorescence imaging in a mouse model, we show that one such precipitation-prone antibody is retained at the injection site in the subcutaneous space longer than a control antibody. In addition, we demonstrate that retention at the injection site through aggregation is concentration-dependent and leads to macrophage association and germinal center localization. Although there was delayed disposition of the aggregated antibody to draining lymph nodes, no overall impact on the immune response in lymph nodes, systemic exposure of the antibody, or enhancement of the anti-drug antibody response was evident. Unexpectedly, retention of the precipitated antibody in the subcutaneous space delayed the onset of the immune response and led to an immune suppressive response. Thus, we conclude that precipitation due to poor solubility of high doses of antibody formulations delivered subcutaneously may not be of special concern in terms of exposure or immunogenicity.     

Evaluation of Prediction Accuracy for Volume of Distribution in Rat and Human Using In Vitro,In Vivo,PBPK and QSAR Methods

Volume of distribution at steady state (V_ss) is an important pharmacokinetic parameter of a drug candidate. In this study, V_ss prediction accuracy was evaluated by using: (1) seven methods for rat with 56 compounds, (2) four methods for human with 1276 compounds, and (3) four in vivo methods and three Kp (partition coefficient) scalar methods from scaling of three preclinical species with 125 compounds. The results showed that the global QSAR models outperformed the PBPK methods. Tissue fraction unbound (f_u,t) method with adipose and muscle also provided high V_ss prediction accuracy. Overall, the high performing methods for human V_ss prediction are the global QSAR models, Øie-Tozer and equivalency methods from scaling of preclinical species, as well as PBPK methods with Kp scalar from preclinical species. Certain input parameter ranges rendered PBPK models inaccurate due to mass balance issues. These were addressed using appropriate theoretical limit checks. Prediction accuracy of tissue Kp were also examined. The f_u,t method predicted Kp values more accurately than the PBPK methods for adipose, heart and muscle. All the methods overpredicted brain Kp and underpredicted liver Kp due to transporter effects. Successful V_ss prediction involves strategic integration of in silico, in vitro and in vivo approaches.     

The Impact of Product and Process Related Critical Quality Attributes on Immunogenicity and Adverse Immunological Effects of Biotherapeutics

The pharmaceutical industry has experienced great successes with protein therapeutics in the last two decades and with novel modalities, including cell therapies and gene therapies, more recently. Biotherapeutics are complex in structure and present challenges for discovery, development, regulatory, and life cycle management. Biotherapeutics can interact with the immune system that may lead to undesired immunological responses, including immunogenicity, hypersensitivity reactions (HSR), injection site reactions (ISR), and others. Many product and process related critical quality attributes (CQAs) have the potential to trigger or augment such immunological responses to the product. Tremendous efforts, both clinically and preclinically, have been invested to understand the impact of product and process related CQAs on adverse immunological effects. The information and knowledge are critical for the implementation of Quality by Design (QbD), which requires risk assessment and establishment of specifications and control strategies for CQAs. A quality target product profile (QTPP) that identifies the key CQAs through process development can help assign severity scores based on safety, immunogenicity, pharmacokinetics (PK) and pharmacodynamics (PD) of the molecule. Gaps and future directions related to biotherapeutics and emerging novel modalities are presented.     

Spatial-resolved metabolomics reveals tissue-specific metabolic reprogramming in diabetic nephropathy by using mass spectrometry imaging

Detailed knowledge on tissue-specific metabolic reprogramming in diabetic nephropathy (DN) is vital for more accurate understanding the molecular pathological signature and developing novel therapeutic strategies. In the present study, a spatial-resolved metabolomics approach based on air flow-assisted desorption electrospray ionization (AFADESI) and matrix-assisted laser desorption ionization (MALDI) integrated mass spectrometry imaging (MSI) was proposed to investigate tissue-specific metabolic alterations in the kidneys of high-fat diet-fed and streptozotocin (STZ)-treated DN rats and the therapeutic effect of astragaloside IV, a potential anti-diabetic drug, against DN. As a result, a wide range of functional metabolites including sugars, amino acids, nucleotides and their derivatives, fatty acids, phospholipids, sphingolipids, glycerides, carnitine and its derivatives, vitamins, peptides, and metal ions associated with DN were identified and their unique distribution patterns in the rat kidney were visualized with high chemical specificity and high spatial resolution. These region-specific metabolic disturbances were ameliorated by repeated oral administration of astragaloside IV (100 mg/kg) for 12 weeks. This study provided more comprehensive and detailed information about the tissue-specific metabolic reprogramming and molecular pathological signature in the kidney of diabetic rats. These findings highlighted the promising potential of AFADESI and MALDI integrated MSI based metabolomics approach for application in metabolic kidney diseases.     

Collection of biochemical samples with brain-wide electrophysiological recordings from a freely moving rodent

Bridging accumulating insights from microscopic and macroscopic studies in neuroscience research requires monitoring of neuronal population dynamics and quantifying specific molecules or genes from the brain of identical animals. To this end, by minimizing the size and weight of an electrode array, we developed a method that records local field potential signals of multiple brain regions from one side of the hemisphere in a freely moving rodent. At the same time, extracellular cerebrospinal fluid for biochemical assays or a small part of brain tissue samples for gene expression assays are collected from the other side of the hemisphere. This method allows ongoing stable recordings and sample collections for at least two months. The methodological concept is applicable to a wide range of biological reactions at various spatiotemporal scales, allowing us to integrate an idea of physiolomics into existing omics analyses, leading to a new combination of multi-omics approaches.