A High Threshold of Biotherapeutic Aggregate Numbers is Needed to Induce an Immunogenic Response In Vitro, In Vivo, and in the Clinic.

BACKGROUND AND PURPOSE: There is concern that subvisible aggregates in biotherapeutic drug products pose a risk to patient safety. We investigated the threshold of biotherapeutic aggregates needed to induce immunogenic responses. METHODS AND RESULTS: Highly aggregated samples were tested in cell-based assays and induced cellular responses in a manner that depended on the number of particles. The threshold of immune activation varied by disease state (cancer, rheumatoid arthritis, allergy), concomitant therapies, and particle number. Compared to healthy donors, disease state patients showed an equal or lower response at the late phase (7 days), suggesting they may not have a higher risk of responding to aggregates. Xeno-het mice were used to assess the threshold of immune activation in vivo. Although highly aggregated samples (~ 1,600,000 particles/mL) induced a weak and transient immunogenic response in mice, a 100-fold dilution of this sample (~ 16,000 particles/mL) did not induce immunogenicity. To confirm this result, subvisible particles (up to ~ 18,000 particles/mL, containing aggregates and silicone oil droplets) produced under representative administration practices (created upon infusion of a drug product through an IV catheter) did not induce a response in cell-based assays or appear to increase the rate of adverse events or immunogenicity during phase 3 clinical trials. CONCLUSION: The ability of biotherapeutic aggregates to elicit an immune response in vitro, in vivo, and in the clinic depends on high numbers of particles. This suggests that there is a high threshold for aggregates to induce an immunogenic response which is well beyond that seen in standard biotherapeutic drug products.

Development of in silico models to predict viscosity and mouse clearance using a comprehensive analytical data set collected on 83 scaffold-consistent monoclonal antibodies.

Biologic drug discovery pipelines are designed to deliver protein therapeutics that have exquisite functional potency and selectivity while also manifesting biophysical characteristics suitable for manufacturing, storage, and convenient administration to patients. The ability to use computational methods to predict biophysical properties from protein sequence, potentially in combination with high throughput assays, could decrease timelines and increase the success rates for therapeutic developability engineering by eliminating lengthy and expensive cycles of recombinant protein production and testing. To support development of high-quality predictive models for antibody developability, we designed a sequence-diverse panel of 83 effector functionless IgG1 antibodies displaying a range of biophysical properties, produced and formulated each protein under standard platform conditions, and collected a comprehensive package of analytical data, including in vitro assays and in vivo mouse pharmacokinetics. We used this robust training data set to build machine learning classifier models that can predict complex protein behavior from these data and features derived from predicted and/or experimental structures. Our models predict with 87% accuracy whether viscosity at 150 mg/mL is above or below a threshold of 15 centipoise (cP) and with 75% accuracy whether the area under the plasma drug concentration-time curve (AUC0-672 h) in normal mouse is above or below a threshold of 3.9 × 106 h x ng/mL.

Physicochemical changes and chemical degradation of gliclazide during twin-screw melt granulation.

Using a miscible model formulation consisting of 80% gliclazide (GLZ) and 20% hydroxypropyl cellulose, we investigate how the twin-screw melt granulation process affects the chemical stability and process-induced physicochemical changes of the drug. No degradation was observed in the conveying section that leads to kneading element. Approximately 1/3 of the GLZ degradant was generated at the kneading section, while the remaining 2/3 was generated in the conveying section post-kneading and during cooling outside the barrel. A strong correlation was observed between the overall degradation and the temperature of the granules at the barrel exit. In the kneading section, the degradant content correlates best with the specific mechanical energy. With higher specific mechanical energies, the size of the GLZ crystals was reduced further, resulting in more surface defects. In the section post-kneading element, GLZ degradation correlates best with the granule temperature measured at the kneading section. This knowledge of drug degradation during twin-screw melt granulation can be used to develop processing strategies to maintain drug stability during and post processing.

Influence of Plasdone™ S630 Ultra-an Improved Copovidone on the Processability and Oxidative Degradation of Quetiapine Fumarate Amorphous Solid Dispersions Prepared via Hot-Melt Extrusion Technique.

In a formulation, traces of peroxides in copovidone can impact the stability of drug substances that are prone to oxidation. The present study aimed to investigate the impact of peroxides in novel Plasdone™ S630 Ultra and compare it with regular Plasdone™ S630 on the oxidative degradation of quetiapine fumarate amorphous solid dispersions prepared via hot-melt extrusion technique. The miscibility of copovidones with drug was determined using the Hansen solubility parameter, and the results indicated a miscible drug-polymer system. Melt viscosity as a function of temperature was determined for the drug-polymer physical mixture to identify the suitable hot-melt extrusion processing temperature. The binary drug and polymer (30:70 weight ratio) amorphous solid dispersions were prepared at a processing temperature of 160°C. Differential scanning calorimetry and Fourier transform infrared spectroscopy studies of amorphous solid dispersions revealed the formation of a single-phase amorphous system with intermolecular hydrogen bonding between the drug and polymer. The milled extrudates were compressed into tablets by using extragranular components and evaluated for tabletability. Stability studies of the milled extrudates and tablet formulations were performed to monitor the oxidative degradation impurity (N-oxide). The N-oxide impurity levels in the quetiapine fumarate - Plasdone™ S630 Ultra milled extrudates and tablet formulations were reduced by 2- and 3-folds, respectively, compared to those in quetiapine fumarate - Plasdone™ S630. The reduced oxidative degradation and improved hot-melt extrusion processability of Plasdone™ S630 Ultra make it a better choice for oxidation-labile drugs over Plasdone™ S630 copovidone.

How Does the Dissimilarity of Screw Geometry Impact Twin-screw Melt Granulation?

Using a model formulation of 80% gabapentin and 20% hydroxypropyl cellulose (KlucelTM), we investigate how differences in the geometry of mixing elements in the Leistritz Nano-16 and Micro-18 extruders affect granulation mechanisms and the properties of the resulting granules. Two extruders, Leistritz Nano-16 and Micro-18, commonly used in development and manufacturing, respectively, were used. The kneading blocks of the Nano-16 extruder are less efficient in dispersive mixing than the kneading blocks of the Micro-18 due to the thinner discs (2.5 mm wide) of the Nano-16. Therefore, our model formulation could be granulated only under a higher degree of fill (DF) by enhancing the axial compaction and heating of the barrel. In contrast, the thicker (5 mm wide) kneading blocks of the Micro-18 extruder provide efficient dispersive mixing that enables granulation without axial compaction and barrel heating. The higher specific mechanical energy (SME) achieved at higher screw speeds and lower feed rates led to more granulation. Because of the difference in granulation mechanisms between the two extruders, critical processing parameters also differed. Tabletability and degradant content of granules correlated positively with DF for the Nano-16 but with SME for the Micro-18 extruder.

Stable amorphous solid dispersions of fenofibrate using hot melt extrusion technology: Effect of formulation and process parameters for a low glass transition temperature drug.

Development of stable amorphous solid dispersions (ASDs) for a low glass transition temperature (Tg) drug is a challenging task. The physico-chemical properties of the drug and excipients play a critical role in developing stable ASDs. In this study, ASDs of poorly soluble fenofibrate, a drug with a low Tg, were formulated using hydroxy propyl methylcellulose acetate succinate (HPMCAS) via hot melt extrusion (HME). The feasible processing conditions were established at varying drug loads and processing temperatures. The prepared ASDs were characterized for crystallinity using differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD). Fourier transform-infrared spectroscopy was performed to study the potential interactions. DSC and PXRD studies confirmed the amorphous state of fenofibrate in the prepared ASDs. A discriminative in vitro dissolution method was established to study the impact of HPMCAS grades on dissolution profile. The dissolution parameters such as dissolution efficiency, initial dissolution rate and mean dissolution rate, suggested improved dissolution characteristics compared to pure fenofibrate. Accelerated stability studies at 40 °C/75% RH showed preservation of the amorphous nature of fenofibrate in formulations with 15% drug load and in vitro drug release studies indicated similar release profiles (f2 >50). This study provides an insight into the formulation and processing of ASDs for poorly soluble drugs with low Tg.

Effect of Hydroxypropyl Cellulose Level on Twin-Screw Melt Granulation of Acetaminophen.

This study investigated the effect of binder level on the physicochemical changes and tabletability of acetaminophen (APAP)-hydroxypropyl cellulose (HPC) granulated using twin-screw melt granulation. Even at 5% HPC level, the tablet tensile strength achieved up to 3.5 MPa. A minimum of 10% HPC was required for the process robustness. However, 20% HPC led to tabletability loss, attributable to the high mechanical strength of APAP granules. The over-granulated APAP granules had thick connected HPC scaffold and low porosity. Consequently, these granules were so strong that they underwent a lower degree of fracture under compression and higher elastic recovery during decompression. HPC was enriched on the surface of APAP extrudates at all HPC levels. Amorphous APAP was also observed on the extrudate surface at 20% HPC level, and it recrystallized within 24 h storage. To achieve a robust process and optimal improvement in APAP tabletability, the preferred HPC level was 10 to 15%.

Hypromellose acetate succinate based amorphous solid dispersions via hot melt extrusion: Effect of drug physicochemical properties.

In this study, the impact of drug and hydroxypropyl methylcellulose acetate succinate (HPMCAS) grades physicochemical properties on extrusion process, dissolution and stability of the hot melt extruded amorphous solid dispersions (ASDs) of nifedipine and efavirenz was investigated. Incorporation of drugs affected the extrusion temperature required for solid dispersion preparation. Differential scanning calorimetry and powder X-ray diffraction studies confirmed the amorphous conversion of the drugs in the prepared formulations. The amorphous nature of ASDs was unchanged after 3 months of stability testing at 40 °C and 75% relative humidity. The dissolution efficiency of the ASDs was dependent on the log P of the drug. The inhibitory effect of HPMCAS on drug precipitation was dependent on the hydrophobic interactions between drug and polymer, polymer grade, and dose of the drug. The dissolution efficiency and dissolution rate of the ASDs were dependent on the log P of the drug and solubility and hydrophilicity of the polymer grade respectively. The inhibitory effect of HPMCAS on drug precipitation was dependent on the hydrophobic interactions between drug and polymer, polymer grade, and the dissolution dose of the drug.

Pharmaceutical Applications of Cellulose Ethers and Cellulose Ether Esters.

Cellulose ethers have proven to be highly useful natural-based polymers, finding application in areas including food, personal care products, oil field chemicals, construction, paper, adhesives, and textiles. They have particular value in pharmaceutical applications due to characteristics including high glass transition temperatures, high chemical and photochemical stability, solubility, limited crystallinity, hydrogen bonding capability, and low toxicity. With regard to toxicity, cellulose ethers have essentially no ability to permeate through gastrointestinal enterocytes and many are already in formulations approved by the U.S. Food and Drug Administration. We review pharmaceutical applications of these valuable polymers from a structure-property-function perspective, discussing each important commercial cellulose ether class; carboxymethyl cellulose, methyl cellulose, hydroxypropylcellulose, hydroxypropyl methyl cellulose, and ethyl cellulose, and cellulose ether esters including hydroxypropyl methyl cellulose acetate succinate and carboxymethyl cellulose acetate butyrate. We also summarize their syntheses, basic material properties, and key pharmaceutical applications.

Influence of pressurized carbon dioxide on ketoprofen-incorporated hot-melt extruded low molecular weight hydroxypropylcellulose.

OBJECTIVES: The aim of the current research project was to investigate the effect of pressurized carbon dioxide (P-CO2) on the physico-mechanical properties of ketoprofen (KTP)-incorporated hydroxypropylcellulose (HPC) (Klucel™ ELF, EF, and LF) produced using hot-melt extrusion (HME) techniques and to assess the plasticization effect of P-CO2 on the various polymers tested. METHODS: The physico-mechanical properties of extrudates with and without injection of P-CO2 were examined and compared with extrudates with the addition of 5% liquid plasticizer of propylene glycol (PG). The extrudates were milled and compressed into tablets. Tablet characteristics of the extrudates with and without injection of P-CO2 were evaluated. RESULTS AND CONCLUSION: P-CO2 acted as a plasticizer for tested polymers, which allowed for the reduction in extrusion processing temperature. The microscopic morphology of the extrudates was changed to a foam-like structure due to the expansion of the CO2 at the extrusion die. The foamy extrudates demonstrated enhanced KTP release compared with the extrudates processed without P-CO2 due to the increase of porosity and surface area of those extrudates. Furthermore, the hardness of the tablets prepared by foamy extrudates was increased and the percent friability was decreased. Thus, the good binding properties and compressibility of the extrudates were positively influenced by utilizing P-CO2 processing.

Development of taste masked caffeine citrate formulations utilizing hot melt extrusion technology and in vitro-in vivo evaluations.

The objective of this study was to develop caffeine citrate orally disintegrating tablet (ODT) formulations utilizing hot-melt extrusion technology and evaluate the ability of the formulation composition to mask the unpleasant bitter taste of the drug using in vitro and in vivo methods. Ethylcellulose, along with a suitable plasticizer, was used as a polymeric carrier. Pore forming agents were incorporated into the extruded matrix to enhance drug release. A modified screw configuration was applied to improve the extrusion processability and to preserve the crystallinity of the API. The milled extrudates were subjected to dissolution testing in an artificial salivary fluid and investigations using e-tongue, to assess the extent of masking of bitter taste of the API. There was an insignificant amount of drug released from the formulation in the salivary medium while over 80% of drug released within 30 min in 0.1N HCl. ODTs were also developed with the extrudate mixed with mannitol and crospovidone. The quality properties such as friability and disintegration time of the ODTs met the USP specifications. The lead extrudate formulations and the ODTs prepared using this formulation were subjected to human gustatory evaluation. The formulations were found to mask the unpleasant taste of caffeine citrate significantly.

Evaluation of the recrystallization kinetics of hot-melt extruded polymeric solid dispersions using an improved Avrami equation.

The recrystallization of an amorphous drug in a solid dispersion system could lead to a loss in the drug solubility and bioavailability. The primary objective of the current research was to use an improved kinetic model to evaluate the recrystallization kinetics of amorphous structures and to further understand the factors influencing the physical stability of amorphous solid dispersions. Amorphous solid dispersions of fenofibrate with different molecular weights of hydroxypropylcellulose, HPC (Klucel™ LF, EF, ELF) were prepared utilizing hot-melt extrusion technology. Differential scanning calorimetry was utilized to quantitatively analyze the extent of recrystallization in the samples stored at different temperatures and relative humidity (RH) conditions. The experimental data were fitted into the improved kinetics model of a modified Avrami equation to calculate the recrystallization rate constants. Klucel LF, the largest molecular weight among the HPCs used, demonstrated the greatest inhibition of fenofibrate recrystallization. Additionally, the recrystallization rate (k) decreased with increasing polymer content, however exponentially increased with higher temperature. Also k increased linearly rather than exponentially over the range of RH studied.

Expression of ORAII, a plasma membrane resident subunit of the CRAC channel, in rodent and non-rodent species.

We determined the expression of ORAI1 protein in rodent and non-rodent tissues using a monoclonal antibody directed against an extracellular loop of the protein. Previous reports using antibodies directed at the C-terminus of ORAI1 have not detected central nervous system (CNS) expression. Our results demonstrate broad tissue expression that includes the CNS using a unique monoclonal antibody specific to an extracellular loop of ORAI1. In addition, we present in situ hybridization (ISH) results using a probe within the middle of the mouse coding region showing CNS expression of Orai1 RNA. We contrast the patterns of rodent and human tissue expression and conclude that rodents have similar expression of ORAI1 in most tissue types when compared to primates, with an important exception being the male reproductive system, where human-specific expression is observed.

Mouse monoclonal antibodies to transient receptor potential ankyrin 1 act as antagonists of multiple modes of channel activation.

The transient receptor potential ankyrin 1 (TRPA1) channel has been implicated in different pathophysiologies that include asthma, cough, itch, and inflammatory pain. Agonists of TRPA1 such as mustard oil and its key component allyl isothiocyanate (AITC) cause pain and neurogenic inflammation in humans and pain behaviors in rodents. Hence, TRPA1 antagonists are being pursued as potential therapeutics. With the goal of generating monoclonal antibodies (mAbs) to human TRPA1 that could act as selective antagonists, we immunized mice with a variety of antigens expressing TRPA1 channels. After generation of hybridomas, the hybridoma conditioned media were screened to identify the mAbs that bind TRPA1 channels by a flow cytometry assay utilizing U2OS or Chinese hamster ovary (CHO) cells stably expressing TRPA1. The purified IgGs from the hybridomas that showed selective binding to TRPA1 were evaluated for antagonism in agonist-induced (45)Ca(2+) uptake assays using CHO-TRPA1 cells. Several of the mAbs showed concentration-dependent inhibition of AITC and cold (4°C) activation of TRPA1. The most potent mAb, 2B10, had IC50 values of approximately 260 and 90 nM in the two assays, respectively. These antagonist mAbs also blocked osmotically activated TRPA1 as well as activation by an endogenous agonist (4-oxo-2-nonenal). In summary, we generated mouse mAbs against TRPA1 that act as antagonists of multiple modes of TRPA1 activation.

Development of a human antibody tolerant mouse model to assess the immunogenicity risk due to aggregated biotherapeutics.

We describe a novel human immunoglobulin G2 (IgG2 )-tolerant and immune-competent heterozygous mouse model (Xeno-het) developed by crossbreeding a human Ig-tolerized XenoMouse® with a C57BL/6J wild-type mouse. The Xeno-het mouse expresses both mouse and human immunoglobulin G (IgG) genes, resulting in B-cells expressing human and mouse IgG, and secretion of human and mouse Ig into serum. This model was utilized to evaluate the immunogenicity risk of aggregated and chemically modified human antibodies. The mice were tested for their ability to break tolerance to self-tolerant monomeric antibodies. Aggregates made by mechanical stirring elicited an anti-drug antibody (ADA) response, but did not induce a robust and long-term memory B and T-cell response. Chemically modified antibodies made by oxidation were only weak and transient inducers of an immune response, as measured by a lack of both an ADA response and a B-cell antigen-specific response. Aggregate size was an important characteristic, as specific-sized protein-coated beads were able to elicit an immune response. We propose the use of this model to identify risk factors such as aggregation during manufacturing at early development for an increased potential immunogenicity risk.

FGF23 neutralization improves chronic kidney disease-associated hyperparathyroidism yet increases mortality.

Chronic kidney disease-mineral and bone disorder (CKD-MBD) is associated with secondary hyperparathyroidism (HPT) and serum elevations in the phosphaturic hormone FGF23, which may be maladaptive and lead to increased morbidity and mortality. To determine the role of FGF23 in the pathogenesis of CKD-MBD and development of secondary HPT, we developed a monoclonal FGF23 antibody to evaluate the impact of chronic FGF23 neutralization on CKD-MBD, secondary HPT, and associated comorbidities in a rat model of CKD-MBD. CKD-MBD rats fed a high-phosphate diet were treated with low or high doses of FGF23-Ab or an isotype control antibody. Neutralization of FGF23 led to sustained reductions in secondary HPT, including decreased parathyroid hormone, increased vitamin D, increased serum calcium, and normalization of bone markers such as cancellous bone volume, trabecular number, osteoblast surface, osteoid surface, and bone-formation rate. In addition, we observed dose-dependent increases in serum phosphate and aortic calcification associated with increased risk of mortality in CKD-MBD rats treated with FGF23-Ab. Thus, mineral disturbances caused by neutralization of FGF23 limited the efficacy of FGF23-Ab and likely contributed to the increased mortality observed in this CKD-MBD rat model.

Long-term inhibition of the glucagon receptor with a monoclonal antibody in mice causes sustained improvement in glycemic control, with reversible alpha-cell hyperplasia and hyperglucagonemia.

Uncontrolled hepatic glucose output (HGO) contributes significantly to the pathological hyperglycemic state of patients with type 2 diabetes. Glucagon, through action on its receptor, stimulates HGO, thereby leading to increased glycemia. Antagonizing the glucagon signaling pathway represents an attractive therapeutic approach for the treatment of type 2 diabetes. We previously reported the generation and characterization of several high-affinity monoclonal antibodies (mAbs) targeting the glucagon receptor (GCGR). In the present study, we demonstrate that a 5-week treatment of diet-induced obese mice with mAb effectively normalized nonfasting blood glucose. Similar treatment also reduced fasting blood glucose without inducing hypoglycemia or other undesirable metabolic perturbations. In addition, no hypoglycemia was found in db/db mice that were treated with a combination of insulin and mAb. Long-term treatment with the mAb caused dose-dependent hyperglucagonemia and minimal to mild alpha-cell hyperplasia in lean mice. There was no evidence of pancreatic alpha-cell neoplastic transformation in mice treated with mAb for as long as 18 weeks. Treatment-induced hyperglucagonemia and alpha-cell hyperplasia were reversible after treatment withdrawal for periods of 4 and 10 weeks, respectively. It is noteworthy that pancreatic beta-cell function was preserved, as demonstrated by improved glucose tolerance throughout the 18-week treatment period. Our studies further support the concept that long-term inhibition of GCGR signaling by a mAb could be an effective approach for controlling diabetic hyperglycemia.

Fully human monoclonal antibodies antagonizing the glucagon receptor improve glucose homeostasis in mice and monkeys.

Antagonizing the glucagon signaling pathway represents an attractive therapeutic approach for reducing excess hepatic glucose production in patients with type 2 diabetes. Despite extensive efforts, there is currently no human therapeutic that directly inhibits the glucagon/glucagon receptor pathway. We undertook a novel approach by generating high-affinity human monoclonal antibodies (mAbs) to the human glucagon receptor (GCGR) that display potent antagonistic activity in vitro and in vivo. A single injection of a lead antibody, mAb B, at 3 mg/kg, normalized blood glucose levels in ob/ob mice for 8 days. In addition, a single injection of mAb B dose-dependently lowered fasting blood glucose levels without inducing hypoglycemia and improved glucose tolerance in normal C57BL/6 mice. In normal cynomolgus monkeys, a single injection improved glucose tolerance while increasing glucagon and active glucagon-like peptide-1 levels. Thus, the anti-GCGR mAb could represent an effective new therapeutic for the treatment of type 2 diabetes.