The Diffusion of Hydrogen Peroxide Into the Liquid Product During Filling Operations Inside Vaporous Hydrogen Peroxide–Sterilized Isolators Can Be Predicted by a Mechanistic Model

Isolators are commonly used in filling operations of pharmaceutical products. To ensure an aseptic inner environment, isolators are regularly sterilized with vaporized hydrogen peroxide. However, despite extensive purging with air, some residual H₂O₂ remains within the isolator atmosphere and may thus end up in the liquid pharmaceutical drug product, which subsequently may cause oxidation and impact the product’s safety and efficacy. We aimed to evaluate the extent of this phenomenon and to model it. For that purpose, we studied the diffusion of H₂O₂ into water contained in small recipients exposed to the atmosphere of a H₂O₂-sterilized small-scale test isolator. Based on the results, a mechanistic model was proposed to estimate the quantity of H₂O₂ in the product, taking into account the time, filling volume, H₂O₂ concentration, and a configuration factor. Afterward, this model was challenged by filling water at a manufacturing scale, and we observed that the diffusion model could predict the trend of increasing H₂O₂ concentration. However, a consistent difference in H₂O₂ concentration between the model and the experimental results was observed, suggesting the contribution of another parameter. Our results can be used to predict more accurately H₂O₂ concentration in a pharmaceutical product at the manufacturing level.     

Physiologically Based Modeling of the Pharmacokinetics of “Catch-and-Release” Anti-Carcinoembryonic Antigen Monoclonal Antibodies in Colorectal Cancer Xenograft Mouse Models

Engineered monoclonal antibodies (mAbs) with pH-sensitive target release, or “catch-and-release” (CAR) binding, have shown promise in decreasing the extent of target-mediated mAb elimination, increasing mAb exposure, and increasing dose potency. This study developed a mechanistic physiologically based pharmacokinetic (PBPK) model to evaluate the effects of pH-sensitive CAR target binding on the disposition of anti-carcinoembryonic antigen (CEA) mAbs in mouse models of colorectal cancer. The PBPK model was qualified by comparing model-predicted plasma concentration-time data with data observed in tumor-bearing mice following the administration of T84.66, a “standard” anti-CEA mAb that demonstrates strong binding at pH 7.4 and 5.5. Further simulations evaluated the effects CAR pH-dependent binding, with decreasing CEA affinity with decreasing pH, on anti-CEA mAb plasma pharmacokinetics. Simulated data were compared with data observed for a novel CAR mAb, 10H6. The PBPK model provided precise parameter estimates, and excellent data characterization (median prediction error 18.4%) following fitting to T84.66 data. Simulations well predicted 10H6 data (median prediction error 21.4%). Sensitivity analyses demonstrated that key determinants of the disposition of CAR mAbs include the following: antigen binding affinity, the rate constant of mAb-CEA dissociation in acidified endosomes, antigen concentration, and the tumor vasculature reflection coefficient.     

Individualized Dosing of Therapeutic Monoclonal Antibodies—a Changing Treatment Paradigm?

The introduction of monoclonal antibodies (mAbs) to the treatment of inflammatory bowel disease (IBD) was an important medical milestone. MAbs have been demonstrated as safe and efficacious treatments of IBD. However, a large percentage of patients either fail to respond initially or lose response to therapy after a period of treatment. Although there are factors associated with poor treatment outcomes in IBD, one cause for treatment failure may be low mAb exposure. Consequently, gastroenterologists have begun using therapeutic drug monitoring (TDM) to guide dose adjustment. However, while beneficial, TDM does not provide sufficient information to effectively adjust doses. The pharmacokinetics (PK) and pharmacodynamics (PD) of mAbs are complex, with numerous factors impacting on mAb PK and PD. The concept of dashboard-guided dosing based on Bayesian PK models allows physicians to combine TDM with factors influencing mAb PK to individualize therapy more effectively. One issue with TDM has been the slow turnaround of assay results, either necessitating an additional clinic visit for a sample or reacting to TDM results at a subsequent, rather than the current, dose. New point-of-care (POC) assays for mAbs are being developed that would potentially allow physicians to determine drug concentration quickly. However, work remains to understand how to determine what target exposure is needed for an individual patient, and whether the combination of POC assays and dashboards presents a safe approach with substantial outcome benefit over the current standard of care.     

Inhibition of Benzoyl Peroxide–Induced Cutaneous Oxidative Stress,Toxicity, and Ear Edema in Mice by Nardostachys jatamansi.

Abstract

Recent years have shown considerable efforts to identify new chemopreventive agents that could be of clinical value. In the current study, modulatory effect of Nardostachys jatamansi. (Jones) DC on benzoyl peroxide–induced oxidative stress, toxicity, and ear edema is investigated. Pretreatment with jatamansi at doses of 2.5 and 5 mg/kg body weight in acetone prior to the application of benzoyl peroxide (20 mg/animal per 0.2 ml acetone) resulted in significant inhibition of benzoyl peroxide–induced cutaneous oxidative stress, toxicity, and ear edema in a dose-dependent manner. The cutaneous microsomal membrane lipid peroxidation and xanthine oxidase activities were significantly reduced (p < 0.05). Moreover, the depleted levels of phase II metabolizing enzymes and glutathione were recovered significantly (p < 0.05). Our findings suggest that N. jatamansi. is an effective chemopreventive agent in mouse skin with potential of ameliorating benzoyl peroxide–induced cutaneous oxidative stress, toxicity, and ear edema.     

Ionic Strength Affects Tertiary Structure and Aggregation Propensity of a Monoclonal Antibody Adsorbed to Silicone Oil–Water Interfaces

Therapeutic proteins formulated in prefilled syringes lubricated with silicone oil come in contact with silicone oil–water interfaces for their entire shelf lives. Thus, the interactions between protein and silicone oil were studied to determine the effect of silicone oil on a monoclonal antibody's stability, both at the interface and in the bulk solution. The influence of ionic strength on these interactions was also investigated through the addition of various monovalent and divalent salts to sample formulations. The tertiary structure of the antibody was perturbed when it adsorbed to the silicone oil–water interface in solutions at low ionic strength. However, the tertiary structure of the antibody at the interface was not perturbed when the ionic strength of the formulation was increased. Even at low ionic strength, the secondary structure of the antibody adsorbed to the silicone oil–water interface was retained, suggesting that at low ionic strength, the adsorbed antibody assumes a molten globule-like conformation. This partially unfolded species was aggregation-prone, especially during agitation. Silicone oil-induced aggregation of the antibody was inhibited at higher ionic strength.     

Rapid Prediction of Deamidation Rates of Proteins to Assess Their Long-Term Stability Using Hydrogen Exchange–Mass Spectrometry

Deamidation is an important degradation pathway for proteins. Estimating deamidation propensities is essential for predicting their long-term stability. However, predicting deamidation rates in folded proteins is challenging because higher-order structure has a significant and unpredictable effect on deamidation. Here, we investigated the correlation between amide hydrogen exchange (HX) and deamidation to assess the potential of using hydrogen exchange–mass spectrometry (HX-MS) to rapidly predict deamidation propensity. Maltose-binding protein and a structurally less stable mutant, W169G, were stored in the dark at pH 7.0 at 23 ± 2°C for 1 year. Deamidation at each asparagine site was measured using liquid chromatography–mass spectrometry after trypsin digestion. Deamidation rates at each deamidation site were determined based on first-order kinetics. HX rates at the deamidation sites were determined before storage using the shortest peptic peptide containing each site using conventional bottom-up HX-MS at pD 7.0 at 25°C. We observed a power law correlation between deamidation half-life and HX half-life for the NG sites with measurable kinetics. For NA sites, slow deamidation was only observed at 2 sites located in rapidly exchanging regions. Our findings demonstrate that HX-MS can be used to reliably and rapidly rank deamidation propensity in folded proteins.     

Hydrogen Bonding Potential as a Determinant of the in Vitro and in Situ Blood–Brain Barrier Permeability of Peptides

With the exception of various central nervous system (CNS)-required nutrients for which specific, saturable transport systems exist, the passage of most water-soluble solutes through the blood–brain barrier (BBB) is believed to depend largely on the lipid solubility of the solutes. Most peptides, therefore, do not enter the CNS because of their hydrophilic character. Recently, utilizing homologous series of model peptides and Caco-2 cell monolayers as a model of the intestinal mucosa, it was concluded that the principal determinant of peptide transport across the intestinal cellular membrane is the energy required to desolvate the polar amide bonds in the peptide (P. S. Burton et al., Adv. Drug Deliv. Rev. 7:365–386, 1991). To determine whether this correlation can be extended to the BBB, the permeabilities of the same peptides were determined using an in vitro as well as an in situ BBB model. The peptides, blocked on the N- and C-terminal ends, consisted of D-phenylalanine (F) residues: AcFNH₂, AcF₂NH₂, AcF₃NH₂, AcF₂(NMeF)NH₂, AcF(NMeF)₂NH₂, Ac(NMeF)₃NH₂, and Ac(NMeF)₃NHMe. A good correlation among the permeabilities of these model peptides across the bovine brain microvessel endothelial cell (BBMEC) monolayers, an in vitro model of the BBB, and their permeabilities across the BBB in situ was observed (r = 0.928, P < 0.05). The permeabilities of these peptides did not correlate with the octanol–buffer partition coefficients of the peptides (r = 0.389 in vitro and r = 0.155 in situ; P < 0.05). However, correlations were observed between the permeabilities of these peptides and the number of potential hydrogen bonds the peptides can make with water (r = 0.837 in vitro and r = 0.906 in situ; P < 0.05), suggesting that desolvation of the polar bonds in the molecule is a determinant of permeability. Consistent with this, good correlations were found between the permeabilities of these peptides and their partition coefficients between heptane–ethylene glycol (r = 0.981 in vitro and r = 0.940 in situ ; P < 0.05) or the differences in partition coefficients between octanol–buffer and isooctane–buffer (logPC) (r = 0.961 in vitro and r = 0.962 in situ; P < 0.05), both of which are experimental estimates of hydrogen bond or desolvation potential. These results suggest that the permeability of peptides through the BBB is governed by the same physicochemical parameter (hydrogen bonding potential) as their permeability through the intestinal mucosa.     

Mechanisms of Chronic Nicotine Treatment–Induced Enhancement of the Sensitivity of Cortical Neurons to the Neuroprotective Effect of Donepezil in Cortical Neurons

We have previously shown that chronic donepezil treatment induces nicotinic acetylcholine receptor up-regulation and enhances the sensitivity of the neurons to the neuroprotective effect of donepezil. Further analyses revealed that the nicotinic receptor is involved in this enhancement. In this study, we examined whether nicotinic receptor stimulation is sufficient to make neurons more sensitive to donepezil. We treated primary cultures of rat cortical neurons with nicotine and confirmed that chronic nicotine treatment induced nicotinic receptor up-regulation and made the neurons more sensitive to the neuroprotective effects of donepezil. Analyses with receptor antagonists and kinase inhibitors revealed that the effects of chronic nicotine treatment are mediated by nicotinic receptors and their downstream effectors including phosphatidylinositol 3-kinase. In contrast to chronic donepezil treatment that enhanced the level of nicotine-induced Ca²⁺ influx, chronic nicotine treatment did not significantly alter the level of Ca²⁺ influx.     

Prediction of the disposition ofβ-lactam antibiotics in humans from pharmacokinetic parameters in animals

Various pharmacokinetic parameters (disposition half-life, total body clearance, renal clearance, hepatic clearance, volume of distribution, intrinsic clearance and volume of distribution of unbound drug) of six-lactam antibiotics were compared in mouse, rat, rabbit, dog, monkey, and human. Two methods for prediction of the disposition of the-lactam antibiotics in humans by extrapolation of the animal data were evaluated. One was the Adolph-Dedrick approach, which can be used to predict clearances in humans from the relationship between intrinsic clearances and body weight in the other five species. The volume of distribution in humans was predicted from the relationship between the volume of distribution and serum unbound fraction in the five species. The other was the Boxenbaum approach, which can be used to predict the pharmacokinetic parameters of the six-lactam antibiotics in humans by using the regression lines of log-log plots of intrinsic clearance and volume of distribution of unbound drug in a single species, in this case the monkey. The half-life calculated according to the latter method had a smaller absolute error than that calculated according to the former method, but the better method for extrapolation of animal data to humans seems to be the former method, which does not require a prioriinformation regarding structure-pharmacokinetic relationships among the antibiotics.     

Prevention of Topical Surfactant–Induced Itch-Related Responses by Chlorogenic Acid Through the Inhibition of Increased Histamine Production in the Epidermis

Effects of chlorogenic acid on surfactant-induced itching were studied in mice. Topical application of sodium laurate increased hind-paw scratching, an itch-related response, 2 h after application, which was inhibited by topical post-treatment with chlorogenic acid. Sodium laurate increased the histamine content and 53-kDa l-histidine decarboxylase in the epidermis, which were also inhibited by post-treatment with chlorogenic acid. These results suggest that topical chlorogenic acid is effective in the prevention of itching induced by anionic surfactants. The inhibitory activity of chlorogenic acid may be due to the inhibition of an increase in histamine in the epidermis.     

How Well Do Low- and High-Concentration Protein Interactions Predict Solution Viscosities of Monoclonal Antibodies?

Protein-protein interactions (PPI) and solution viscosities were measured at low and high protein concentrations under a range of formulation conditions for 4 different monoclonal antibodies. Static light scattering was used to quantify the osmotic second virial coefficient (B₂₂) and the zero-q limit static structure factor (S_q=0), versus protein concentration (c₂) from low to high c₂. Dynamic light scattering was used to measure the collective diffusion coefficient as a function of c₂ and to determine the protein interaction parameter (k_D). Static light scattering and dynamic light scattering were combined to determine the hydrodynamic factor (H_q=0), which accounts for changes in hydrodynamic PPI as a function of c₂. The net PPI ranged from strongly repulsive to attractive interactions, via changes in buffer pH, ionic strength, and choice of monoclonal antibodies. Multiple-particle tracking microrheology and capillary viscometery were used to measure monoclonal antibodies solution viscosities under the same solution conditions. In most cases, even large and qualitative changes in PPI did not result in significant changes in protein solution viscosity. This highlights the complex nature of PPI and how they influence protein solution viscosity and raises questions as to the validity of using experimental PPI metrics such as k_D or B₂₂ as predictors of high viscosity.     

Quality Control of Personalized Drug Products – Identity and Quantity of Monoclonal Antibodies as Active Pharmaceutical Ingredient

Deliberate underdosing occurred in personalized preparations of drugs such as monoclonal antibodies as the active pharmaceutical ingredient in the past. To ensure the required quality standard and to prevent future fraud attempts at an early stage, a HPLC-DAD-HRMS method was established. Thereby, identity and quantity of the active ingredients bevacizumab, rituximab and trastuzumab were determined. The analysis of ten samples from seven pharmacies fulfilled the quality criteria and were therefore not objectionable.     

Mechanism of Protein–PDMS Visible Particles Formation in Liquid Vial Monoclonal Antibody Formulation

Visible particles (VPs) formation in liquid monoclonal antibody formulations is a critical quality issue. Formulations that include poloxamer 188 (PX188) as a surfactant are prone to the formation of VPs comprising aggregated complexes of protein and polydimethylsiloxane (PDMS; silicone oil) derived from primary containers. However, the mechanisms through which these VPs form are complicated and remain to be fully elucidated. This study demonstrates for the first time the dominant spot and pathway of protein–PDMS VP formation in a particular liquid vial formulation. Specifically, when a vial sealed with a PDMS-coated stopper is stored in an upright position under conditions whereby the antibody solution has become well-adhered to the stopper and an air phase exists in the vicinity, protein–PDMS aggregates form on the stopper and are then desorbed into the drug solution to be detected as VPs. Here, we evaluated the effects of several factors on VP formation: adhesion of the drug solution to the stopper, storage orientation, silicone coating on the stopper, vial material, and hydrophobicity of PX188. Remarkably, we found that changing any one of the factors could significantly affect VP formation. Our findings are instructive for better understanding the mechanisms of VP formation in vial products and can provide strategies for VP mitigation in biotherapeutics.     

Stress Temperature Studies in Small Scale Hastelloy® Drug Substance Containers Lead to Increased Extent of and Increased Variability in Antibody-Drug Conjugate and Monoclonal Antibody Aggregation: Evidence for Novel Oxidation-Induced Crosslinking in Monoclonal Antibodies

Health authorities require that suitable stability of the drug substance be shown in relevant materials of construction. ICH Q1A(R2) explicitly states that “stability studies should be conducted on drug substance packaged in a container closure system that is the same as or simulates the packaging proposed for storage and distribution”. Stainless steel containers are commonly used for the long-term storage of frozen bulk drug substances (DSs). Hastelloy®-based metal containers are sometimes used due to their higher corrosion resistance and significantly lower iron content to mitigate the potential corrosion-related risks associated with high salt formulations. Despite their benefits, we have found that elevated temperature stability studies in small scale Hastelloy® containers can lead to degradation that is not representative of degradation under typical storage conditions relevant to the manufacturing process. We provide evidence for an oxidation-induced aggregation mechanism that is based on Fenton chemistry with peroxide being supplied by the autoxidation of polysorbate at stress temperatures. Further, variation in the rates of iron leaching between individual small scale containers is shown to be the cause of the variable rates of degradation through strong correlations between leached iron levels and the extents of oxidation and aggregation. The addition of a metal chelator or the removal of polysorbate from the formulation mitigates the oxidation and the non-representative behavior. Extended characterization by LC-MS and ¹⁸O labeled peptide mapping shows that a significant portion of the aggregate formed under these conditions is covalently crosslinked and that the predominant covalent species is either a dityrosine or tyrosine-tryptophan crosslink between an Fc peptide and a Fab peptide. This report is the first time either of these two crosslinks have been reported for antibodies with detailed analytical characterization. Because the behavior observed in these studies is not representative of degradation under typical storage conditions relevant to the manufacturing process, this study demonstrates that small scale stress studies in metal containers should be performed with caution and that extended incubation times can lead to non-representative degradation mechanisms.     

Isomerization of Asp–Asp Motif in Model Peptides and a Monoclonal Antibody Fab Fragment

Isomerization of aspartyl (Asp or D) residues is a critical degradation route to consider for stable monoclonal antibody formulations. Among the known hotspot sequences, the DD motif is relatively understudied. To gain mechanistic insights, we used model hexapeptides, YADXFK, YADDXK, and DIDDDM, as surrogates for the hotspots in a Fab protein (YADDFK and DIDDDM), to characterize the rate-pH profile of Asp isomerization. Compared with the YADGFK peptide, isomerization of D3 (the first D in the DD pair) in YADDFK was highly pH dependent. Comparison of rate-pH profiles of YADDFK, YADNFK, and YADHFK revealed a charge effect of the n + 1 residue—isomerization rate is accelerated by the positive side chain and reduced by negative side chain at n + 1 residue. Studies on YADDFK, YADDAK, and YADDGK indicated a mutual impact of D3 and D4 on their respective isomerization rates through charge effect. Comparison of rate-pH profile of DIDDDM sequence in peptide models with that in the complementary determining region of the Fab showed a faster rate in the Fab than in peptides, presumably because of contribution from structural factors in the former. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:947–959, 2013     

An Investigation of Indomethacin–Nicotinamide Cocrystal Formation Induced by Thermal Stress in the Solid or Liquid State

The impact of thermal stress on indomethacin (IMC)–nicotinamide (NIC) cocrystal formation with or without neat cogrinding was investigated using differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) microspectroscopy, and simultaneous DSC–FTIR microspectroscopy in the solid or liquid state. Different evaporation methods for preparing IMC–NIC cocrystals were also compared. The results indicated that even after cogrinding for 40 min, the FTIR spectra for all IMC–NIC ground mixtures were superimposable on the FTIR spectra of IMC and NIC components, suggesting there was no cocrystal formation between IMC and NIC after cogrinding. However, these IMC–NIC ground mixtures appear to easily undergo cocrystal formation after the application of DSC determination. Under thermal stress induced by DSC, the amount of cocrystal formation increased with increasing cogrinding time. Moreover, simultaneous DSC–FTIR microspectroscopy was a useful one-step technique to induce and clarify the thermal-induced stepwise mechanism of IMC–NIC cocrystal formation from the ground mixture in real time. Different solvent evaporation rates induced by thermal stress significantly influenced IMC–NIC cocrystal formation in the liquid state. In particular, microwave heating may promote IMC–NIC cocrystal formation in a short time. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:2386–2395, 2014     

Which in vitro Screens Guide the Prediction of Oral Absorption and Volume of Distribution?

The development of medium to high-throughput in vitro screening of ADME (Absorption, Distribution, Metabolism, Excretion) properties has been the reply to higher demands on drug metabolism scientists to cope with progress in chemistry and biology. Two areas will be discussed here, namely screens for oral absorption and for volume of distribution. The prediction of these human pharmacokinetic parameters can be based on proper combination of simple physicochemical measurements. In the future in vitro screens most likely will be combined with in silico assessments of various ADME properties leading to the concept of in combo screening in drug discovery.     

Predicting the Crystallization Propensity of Carboxylic Acid Buffers in Frozen Systems—Relevance to Freeze-Drying

Selective crystallization of buffer components in frozen solutions is known to cause pronounced pH shifts. Our objective was to study the crystallization behavior and the consequent pH shift in frozen aqueous carboxylic acid buffers. Aqueous carboxylic acid buffers were cooled to ?25°C and the pH of the solution was measured as a function of temperature. The thermal behavior of solutions during freezing and thawing was investigated by differential scanning calorimetry. The crystallized phases in frozen solution were identified by X-ray diffractometry. The malate buffer system was robust with no evidence of buffer component crystallization and hence negligible pH shift. In the citrate and tartarate systems, at initial pH <pKa₂, only the most acidic buffer component (neutral form) crystallized on cooling, causing an increase in the freeze-concentrate pH. Carboxylic acid buffers were rank ordered based on their propensity to crystallize in frozen solutions. From the aqueous solubility values of these carboxylic acids, which have been reported over a range of temperatures, it was also possible to estimate the degree of supersaturation at the subambient temperature of interest. This enabled us to predict their crystallization propensity in frozen systems. The experimental and the predicted rank orderings were in excellent agreement.