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.     

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.     

Continuous Twin Screw Wet Granulation and Drying—Control Strategy for Drug Product Manufacturing

The use of continuous manufacturing has been increasing within the pharmaceutical industry over the last few years. Continuous direct compression has been the focus of publications on the topic to date. The use of wet granulation can improve segregation resistance, uniformity, enhance density, and flow properties for improved tabletability, or improve stability of products that cannot be manufactured by using a direction compression process. This article focuses on development of appropriate control strategies for continuous wet granulation (especially twin screw wet granulation) through equipment design, material properties and manufacturing process along with areas where additional understanding is required. The article also discusses the use of process analytical technologies as part of the control and automation approach to ensure a higher assurance of product quality. Increased understanding of continuous wet granulation should result in increased utilization of the technique, thereby allowing for an increase in diversity of products manufactured by continuous manufacturing and the benefits that comes with a more complex process such as wet granulation compared with direct compression process.     

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.     

Contribution of Intravenous Administration Components to Subvisible and Submicron Particles Present in Administered Drug Product

Particulate matter present in drug products intended for parenteral administration to patients is typically monitored and controlled in the finished drug product to minimize potential risks to patients. In contrast to particulates found in drug products, the current study evaluated particulates representative of materials and operations typically used in the dose preparation and administration of drug products. A comprehensive assessment of intrinsic and extrinsic sources of subvisible and submicron particulates arising from materials associated with subcutaneous and intravenous dose preparation and administration was conducted. In particular, particles arising from disposable syringes, commercial sterile diluents, and intravenous supplies were quantitated using established methods for subvisible (light obscuration, flow imaging) and submicron particles (resistive pulse sensing). Each of these sources contributed varying amounts of particulates; therefore, owing to sources from materials required for administration, it is inadequate to assume that the total particulate load delivered to patients arises solely from the drug product. Careful consideration of the administration method and supplies used can improve the predictability of particulate levels present in dose preparations or administration volumes.     

PKPD Assessment of the Anti-CD20 Antibody Obinutuzumab in Cynomolgus Monkey is Feasible Despite Marked Anti-Drug Antibody Response in This Species

The pharmacokinetics (PK) of the anti-CD20 monoclonal antibody obinutuzumab was assessed after single intravenous dosing to cynomolgus monkeys. In addition, the pharmacokinetic-pharmacodynamic (PKPD) relationship for B-cell depletion was characterized. The PKPD model was used to estimate the B-cell repopulation during the recovery phase of chronic toxicology studies, thereby supporting the study design, in particular planning the recovery phase duration. Marked immunogenicity against obinutuzumab was observed approximately 10 days after single dose, leading to an up to ∼30-fold increase in obinutuzumab clearance in the affected monkeys. Despite this accelerated clearance, the PK could be characterized, either by disregarding the clearance in noncompartmental PK analysis or by capturing it explicitly as an additional time-dependent clearance process in compartmental modeling. This latter step was crucial to model the PKPD of B-cells as an indirect response to obinutuzumab exposure, showing that—without immune response—the limiting factor is obinutuzumab elimination with concentrations below 0.02 μg/mL required for initiation of B-cell recovery.Overall, the results demonstrate that despite a marked anti-drug antibody response in the nonclinical animal species, the PK and PKPD of obinutuzumab could be characterized successfully by appropriately addressing the immune-modulated clearance pathway in data analysis and modeling.     

An update on oral drug delivery via intestinal lymphatic transport

Orally administered drug entities have to survive the harsh gastrointestinal environment, penetrate the enteric epithelia and circumvent hepatic metabolism before reaching the systemic circulation. Whereas the gastrointestinal stability can be well maintained by taking proper measures, hepatic metabolism presents as a formidable barrier to drugs suffering from first-pass metabolism. The pharmaceutical academia and industries are seeking alternative pathways for drug transport to circumvent problems associated with the portal pathway. Intestinal lymphatic transport is emerging as a promising pathway to this end. In this review, we intend to provide an updated overview on the rationale, strategies, factors and applications involved in intestinal lymphatic transport. There are mainly two pathways for peroral lymphatic transport—the chylomicron and the microfold cell pathways. The underlying mechanisms are being unraveled gradually and nowadays witness increasing research input and applications.     

Interacting with α7 nAChR is a new mechanism for AChE to enhance the inflammatory response in macrophages

Acetylcholine (ACh) regulates inflammation via α7 nicotinic acetylcholine receptor (α7 nAChR). Acetylcholinesterase (AChE), an enzyme hydrolyzing ACh, is expressed in immune cells suggesting non-classical function in inflammatory responses. Here, the expression of PRiMA-linked G4 AChE was identified on the surface of macrophages. In lipopolysaccharide-induced inflammatory processes, AChE was upregulated by the binding of NF-κB onto the ACHE promotor. Conversely, the overexpression of G4 AChE inhibited ACh-suppressed cytokine release and cell migration, which was in contrast to that of applied AChE inhibitors. AChEmt, a DNA construct without enzymatic activity, was adopted to identify the protein role of AChE in immune system. Overexpression of G4 AChEmt induced cell migration and inhibited ACh-suppressed cell migration. The co-localization of α7 nAChR and AChE was found in macrophages, suggesting the potential interaction of α7 nAChR and AChE. Besides, immunoprecipitation showed a close association of α7 nAChR and AChE protein in cell membrane. Hence, the novel function of AChE in macrophage by interacting with α7 nAChR was determined. Together with hydrolysis of ACh, AChE plays a direct role in the regulation of inflammatory response. As such, AChE could serve as a novel target to treat age-related diseases by anti-inflammatory responses.     

An Industry Perspective on Compatibility Assessment of Closed System Drug-Transfer Devices for Biologics

The Formulation Workstream of the BioPhorum Development Group (BPDG), an industry-wide consortium, has identified the increased use of closed system drug-transfer devices (CSTDs) with biologics, without an associated compatibility assessment, to be of significant concern. The use of CSTDs has increased significantly in recent years due to the recommendations by NIOSH and USP that they be used during preparation and administration of hazardous drugs. While CSTDs are valuable in the healthcare setting to reduce occupational exposure to hazardous compounds, these devices may present particular risks that must be adequately assessed prior to use to ensure their compatibility with specific types of drug products, such as biologic drugs, which may be sensitive. The responsibility of ensuring quality of biologic products through preparation and administration to the patient lies with the drug product sponsor. Due to the significant number of marketed CSTD systems, and the large variety of components offered for each system, a strategic, risk-based approach to assessing compatibility is recommended herein. In addition to traditional material compatibility, assessment of CSTD compatibility with biologics should consider additional parameters to address specific CSTD-related risks. The BPDG Formulation Workstream has proposed a systematic risk-based evaluation approach as well as a mitigation strategy for establishing suitability of CSTDs for use.     

The carboxyl-terminal di-lysine motif is essential for catalytic activity of UDP-glucuronosyltransferase 1A9

UDP-Glucuronosyltransferase (UGT) is a type I membrane protein localized to the endoplasmic reticulum (ER). UGT has a di-lysine motif (KKXX/KXKXX) in its cytoplasmic domain, which is defined as an ER retention signal. However, our previous study has revealed that UGT2B7, one of the major UGT isoform in human, localizes to the ER in a manner that is independent of this motif. In this study, we focused on another UGT isoform, UGT1A9, and investigated the role of the di-lysine motif in its ER localization, glucuronidation activity, and homo-oligomer formation. Immunofluorescence microscopy indicated that the cytoplasmic domain of UGT1A9 functioned as an ER retention signal in a chimeric protein with CD4, but UGT1A9 itself could localize to the ER in a di-lysine motif-independent manner. In addition, UGT1A9 formed homo-oligomers in the absence of the motif. However, deletion of the di-lysine motif or substitution of lysines in the motif for alanines, severely impaired glucuronidation activity of UGT1A9. This is the first study that re-defines the cytoplasmic di-lysine motif of UGT as an essential peptide for retaining glucuronidation capacity.     

Forced Degradation of Monoclonal Antibodies After Compounding: Impact on Routine Hospital Quality Control

Compounded therapeutic mAbs used in a hospital require quality control (QC). In our hospital, analytical QC process intended to mAbs identification and quantification is based on flow injection analysis associated with second-derivative UV spectroscopy and matching method algorithm. We studied the influence of degraded mAbs after compounding on this validated QC. Three forced stress conditions including mechanical, thermal, and freeze-thawing stresses were studied to yield degraded mAbs from 2 model compounds, that is, bevacizumab (IgG1) and nivolumab (IgG4). Different degraded mAbs were generated and were analyzed in terms of turbidity, the percentage of aggregation, size distribution, and changes in tertiary structure. Stresses showed to be mAb-dependent in terms of aggregation. Tertiary structural changes were observed in most of the stressed samples by principal component analysis of the UV second-derivative data. The structural and physicochemical modifications conducted to mismatch depending on the nature of the stress. The mismatch ranged from 17% to 72% for the mAbs, except for freeze-thawed bevacizumab for which a perfect match (100%) was reached. The quantification with an unfulfilled relative error of the concentration (i.e., > ±15%) was detected only for mechanically stressed mAbs. In conclusion, the study revealed that the influence of the mAbs and the type of stress impact on the QC of compounded mAbs.     

Design,synthesis and pharmacological evaluation of 4-(3-chloro-4-(3-cyclopropylthioureido)-2-fluorophenoxy)-7-methoxyquinoline-6-carboxamide (WXFL-152): a novel triple angiokinase inhibitor for cancer therapy

Angiokinases, such as vascular endothelial-, fibroblast- and platelet-derived growth factor receptors (VEGFRs, FGFRs and PDGFRs) play crucial roles in tumor angiogenesis. Anti-angiogenesis therapy using multi-angiokinase inhibitor has achieved great success in recent years. In this study, we presented the design, synthesis, target identification, molecular mechanism, pharmacodynamics (PD) and pharmacokinetics (PK) research of a novel triple-angiokinase inhibitor WXFL-152. WXFL-152, identified from a series of 4-oxyquinoline derivatives based on a structure–activity relationship study, inhibited the proliferation of vascular endothelial cells (ECs) and pericytes by blocking the angiokinase signals VEGF/VEGFR2, FGF/FGFRs and PDGF/PDGFRβ simultaneously in vitro. Significant anticancer effects of WXFL-152 were confirmed in multiple preclinical tumor xenograft models, including a patient-derived tumor xenograft (PDX) model. Pharmacokinetic studies of WXFL-152 demonstrated high favourable bioavailability with single-dose and continuous multi-dose by oral administration in rats and beagles. In conclusion, WXFL-152, which is currently in phase Ib clinical trials, is a novel and effective triple-angiokinase inhibitor with clear PD and PK in tumor therapy.     

PEP06 polypeptide 30 is a novel cluster-dissociating agent inhibiting αv integrin/FAK/Src signaling in oral squamous cell carcinoma cells

Collectively migrating tumor cells have been recently implicated in enhanced metastasis of epithelial malignancies. In oral squamous cell carcinoma (OSCC), αv integrin is a crucial mediator of multicellular clustering and collective movement in vitro; however, its contribution to metastatic spread remains to be addressed. According to the emerging therapeutic concept, dissociation of tumor clusters into single cells could significantly suppress metastasis-seeding ability of carcinomas. This study aimed to investigate the anti-OSCC potential of novel endostatin-derived polypeptide PEP06 as a cluster-dissociating therapeutic agent in vitro. Firstly, we found marked enrichment of αv integrin in collectively invading multicellular clusters in human OSCCs. Our study revealed that metastatic progression of OSCC was associated with augmented immunostaining of αv integrin in cancerous lesions. Following PEP06 treatment, cell clustering on fibronectin, migration, multicellular aggregation, anchorage-independent survival and colony formation of OSCC were significantly inhibited. Moreover, PEP06 suppressed αv integrin/FAK/Src signaling in OSCC cells. PEP06-induced loss of active Src and E-cadherin from cell–cell contacts contributed to diminished collective migration of OSCC in vitro. Overall, these results suggest that PEP06 polypeptide 30 inhibiting αv integrin/FAK/Src signaling and disrupting E-cadherin-based intercellular junctions possesses anti-metastatic potential in OSCC by acting as a cluster-dissociating therapeutic agent.     

Molecular Dynamics Simulation to Uncover the Mechanisms of Protein Instability During Freezing

Freezing is a common process applied in the pharmaceutical industry to store and transport biotherapeutics. Herewith, multi-scale molecular dynamics simulations of Lactate dehydrogenase (LDH) protein in phosphate buffer with/without ice formation performed to uncover the still poorly understood mechanisms and molecular details of protein destabilization upon freezing. Both fast and slow ice growing conditions were simulated at 243 K from one or two-side of the simulation box, respectively. The rate of ice formation at all-atom simulations was crucial to LDH stability, as faster freezing rates resulted in enhanced structural stability maintained by a higher number of intramolecular hydrogen bonds, less flexible protein's residues, lower solvent accessibility and greater structural compactness. Further, protein aggregation investigated by coarse-grained simulations was verified to be initiated by extended protein structures and retained by electrostatic interactions of the salt bridges between charged residues and hydrogen bonds between polar residues of the protein. Lastly, the study of free energy of dissociation through steered molecular dynamics simulation revealed LDH was destabilized by the solvation of the hydrophobic core and the loss of hydrophobic interactions. For the first time, experimentally validated molecular simulations revealed the detailed mechanisms of LDH destabilization upon ice formation and cryoconcentration of solutes.     

Bile Duct Obstruction Leads to Increased Intestinal Expression of Breast Cancer Resistance Protein With Reduced Gastrointestinal Absorption of Imatinib

Liver dysfunction reduces systemic clearance of drugs that are primarily eliminated by the liver. However, liver dysfunction can cause a reduction in the plasma concentration profiles of certain drugs, including several tyrosine kinase inhibitors, after oral administration. The aim of the present study was to clarify the reduction in oral absorption of a tyrosine kinase inhibitor, imatinib, and the mechanisms of action involved under conditions of hepatic dysfunction, focusing on intestinal transporters. The maximum plasma concentration of imatinib after oral administration in mice subjected to bile duct ligation (BDL) was lower than that in sham-operated mice, whereas the plasma concentration profile after intravenous administration was essentially unaffected by BDL. The change in maximum plasma concentration was compatible with a reduction in small intestinal permeability of imatinib observed in the in situ closed loop. Gene expression of abcg2 was increased in BDL mice compared with that in sham-operated mice. Expression of breast cancer resistance protein and P-glycoprotein in the small intestinal brush border membrane fraction from BDL mice was also increased compared with that in sham-operated mice. In summary, the intestinal absorption and permeability of imatinib was decreased in BDL mice, and this may be attributed to the up-regulation of the efflux transporter(s).     

Chemo-photothermal immunotherapy for eradication of orthotopic tumors and inhibition of metastasis by intratumoral injection of polydopamine versatile hydrogels

Cancer remains one of the leading causes of death globally and metastasis always leads to treatment failure. Here, we develop a versatile hydrogel loading photothermal agents, chemotherapeutics, and immune-adjuvants to eradicate orthotopic tumors and inhibit metastasis by combinational therapy. Hydrogel networks were synthesized via the thiol-Michael addition of polydopamine (PDA) with thiolated hyaluronic acid. PDA acted as a cross-linking agent and endowed the hydrogel with excellent photothermal property. Meanwhile, a chemotherapeutic agent, doxorubicin (DOX), was loaded in the hydrogel via π?π stacking with PDA and an immune-adjuvant, CpG-ODN, was loaded via electrostatic interaction. The release of DOX from the hydrogel was initially slow but accelerated due to near infrared light irradiation. The hydrogels showed remarkably synergistic effect against 4T1 cancer cells and stimulated plenty of cytokines secreting from RAW264.7 cells. Moreover, the hydrogels eradicated orthotopic murine breast cancer xenografts and strongly inhibited metastasis after intratumoral injection and light irradiation. The high anticancer efficiency of this chemo-photothermal immunotherapy resulted from the strong synergistic effect of the versatile hydrogels, including the evoked host immune response. The combinational strategy of chemo-photothermal immunotherapy is promising for highly effective treatment of breast cancer.     

An Approach to Bioactivity Assessment for Critical Quality Attribute Identification Based on Antibody-Antigen Complex Structure

Identification of critical quality attributes (CQAs) is an important step for development of biopharmaceuticals with intended performance. An accurate CQA assessment is needed to ensure product quality and focusing on development efforts where control is needed. The assignment of criticality is based on safety and efficacy. Efficacy is related to PK and bioactivity. Here, we developed a novel approach based on antibody-antigen complex structure and modeling as a complementary method for bioactivity assessment. To validate this approach, common product related quality attributes and mutagenesis data from several IgGs were assessed using available antibody-antigen complex structures, and results were compared with experimental data from bioactivity or binding affinity measurements. A stepwise evaluation scheme for structural based analysis is proposed; based on systematic assessment following the scheme, good correlation has been observed between structural analysis and experimental data. This demonstrates that such an approach can be applied as a complementary tool for bioactivity assessment. Main applications are 1) To decouple multiple attributes to achieve amino acid resolution for bioactivity assessment, 2) To assess bioactivity of attributes that cannot be experimentally generated, 3) To provide molecular mechanism for experimental observation and understand structure function relationship. Examples are provided to illustrate these applications.     

Discovery of the anti-angiogenesis effect of eltrombopag in breast cancer through targeting of HuR protein

HuR (human antigen R), an mRNA-binding protein responsible for poor prognosis in nearly all kinds of malignancies, is a potential anti-tumor target for drug development. While screening HuR inhibitors with a fluorescence polarization (FP) based high-throughput screening (HTS) system, the clinically used drug eltrombopag was identified. Activity of eltrombopag on molecular level was verified with FP, electrophoretic mobility shift assay (EMSA), simulation docking and surface plasmon resonance (SPR). Further, we showed that eltrombopag inhibited in vitro cell proliferation of multiple cancer cell lines and macrophages, and the in vivo anti-tumor activity was also demonstrated in a 4T1 tumor-bearing mouse model. The in vivo data showed that eltrombopag was efficient in reducing microvessels in tumor tissues. We then confirmed the HuR-dependent anti-angiogenesis effect of eltrombopag in 4T1 cells and RAW264.7 macrophages with qRT-PCR, HuR-overexpression and HuR-silencing assays, RNA stability assays, RNA immunoprecipitation and luciferase assays. Finally, we analyzed the in vitro anti-angiogenesis effect of eltrombopag on human umbilical vein endothelial cells (HUVECs) mediated by macrophages with cell scratch assay and in vitro Matrigel angiogenesis assay. With these data, we revealed the HuR-dependent anti-angiogenesis effect of eltrombopag in breast tumor, suggesting that the existing drug eltrombopag may be used as an anti-cancer drug.     

Coamorphous System of Florfenicol-Oxymatrine for Improving the Solubility and Dissolution Rate of Florfenicol: Preparation,Characterization and Molecular Dynamics Simulation

Coamorphous system has proved to be an effective approach to improve the solubility of BCSⅡ drugs. Florfenicol (FF) is a widely used veterinary antibiotic but has poor aqueous solubility. Therefore, the coamorphous system of florfenicol and oxymatrine (OMT) formulated at 1:1 and 1:2 M ratios were prepared by using solvent evaporation, followed by a series of characterization in terms of PXRD, DSC, FTIR and Raman spectroscopy. It was found that FF and OMT are miscible according to Hansen solubility parameters. The molecular electrostatic potential (MEP) and radial distribution function (RDF) analysis demonstrated the possible hydrogen bond interaction in coamorphous system, which was confirmed by FTIR and Raman spectra. The coamorphous FF-OMT (1:1) maintained stability for 60 days at 25 °C/0% RH and 30 days at 40 °C/75% RH, which may be attributed to better molecular miscibility of FF and OMT and the strong hydrogen bond of O–H (FF)?O–N (OMT) and N–H (FF)?O–N (OMT). In addition, the apparent solubility and permeability, dissolution and intrinsic dissolution rate (IDR) of the acquired coamorphous solids were obviously increased compared with crystalline FF. In conclusion, a drug-drug coamorphous formulation can be applied to improve the solubility and dissolution of crystalline FF.