Exploring How Antibody Format Drives Clearance from the Brain.

Monoclonal antibodies (mAbs) have high binding specificity and affinity, making them attractive for treating brain diseases. However, their effectiveness is limited by poor blood-brain barrier (BBB) penetration and rapid central nervous system (CNS) clearance. Our group identified blood-brain barrier modulator (BBBM) peptides that improved mAb penetration across the BBB into the brain. In this study, we investigated the pharmacokinetics of a mAb delivered to the brain using BBBMs after intravenous (IV) administration and explored the impact of antibody format (size, neonatal Fc receptor (FcRn) binding, hyaluronic acid binding) on brain clearance following direct injection into the central nervous system (CNS) via intracerebroventricular (ICV) injection. IRDye800CW-labeled antibodies were administered into C57BL/6 mice via ICV or IV injection, and organ concentrations were measured after various time points. When a mAb was coadministered with a BBBM peptide, the permeation of mAb across the BBB was increased compared to mAb alone at early time points; however, the mAb was cleared within 2 h from the brain. ICV experiments revealed that an antibody Fab fragment had a higher brain exposure than a mAb, and that a Fab fused to a hyaluronic acid binding domain (Fab-VG1) showed remarkable improvement in brain exposure. These findings suggest that BBBMs and antibody format optimization may be promising strategies for enhancing brain retention of therapeutic antibodies.

Selective Uptake of Macromolecules to the Brain in Microfluidics and Animal Models Using the HAVN1 Peptide as a Blood-Brain Barrier Modulator.

Monoclonal antibodies (mAbs) possess favorable pharmacokinetic properties, high binding specificity and affinity, and minimal off-target effects, making them promising therapeutic agents for central nervous system (CNS) disorders. However, their development as effective therapeutic and diagnostic agents for brain disorders is hindered by their limited ability to efficiently penetrate the blood-brain barrier (BBB). Therefore, it is crucial to develop efficient delivery methods that enhance the penetration of antibodies into the brain. Previous studies have demonstrated the potential of cadherin-derived peptides (i.e., ADTC5, HAVN1 peptides) as BBB modulators (BBBMs) to increase paracellular porosities for penetration of molecules across the BBB. Here, we test the effectiveness of the leading BBBM peptide, HAVN1 (Cyclo(1,6)SHAVSS), in enhancing the permeation of various monoclonal antibodies through the BBB using both in vitro and in vivo systems. In vitro, HAVN1 has been shown to increase the permeability of fluorescently labeled macromolecules, such as a 70 kDa dextran, 50 kDa Fab1, and 150 kDa mAb1, by 4- to 9-fold in a three-dimensional blood-brain barrier (3D-BBB) microfluidics model using a human BBB endothelial cell line (i.e., hCMEC/D3). HAVN1 was selective in modulating the BBB endothelial cell, compared to the pulmonary vascular endothelial (PVE) cell barrier. Co-administration of HAVN1 significantly improved brain depositions of mAb1, mAb2, and Fab1 in C57BL/6 mice after 15 min in the systemic circulation. Furthermore, HAVN1 still significantly enhanced brain deposition of mAb2 when it was administered 24 h after the administration of the mAb. Lastly, we observed that multiple doses of HAVN1 may have a cumulative effect on the brain deposition of mAb2 within a 24-h period. These findings offer promising insights into optimizing HAVN1 and mAb dosing regimens to control or modulate mAb brain deposition for achieving desired mAb dose in the brain to provide its therapeutic effects.

Physicochemical and biological impact of metal-catalyzed oxidation of IgG1 monoclonal antibodies and antibody-drug conjugates via reactive oxygen species.

Biotherapeutics are exposed to common transition metal ions such as Cu(II) and Fe(II) during manufacturing processes and storage. IgG1 biotherapeutics are vulnerable to reactive oxygen species (ROS) generated via the metal-catalyzed oxidation reactions. Exposure to these metal ions can lead to potential changes to structure and function, ultimately influencing efficacy, potency, and potential immunogenicity of the molecules. Here, we stress four biotherapeutics of the IgG1 subclass (trastuzumab, trastuzumab emtansine, anti-NaPi2b, and anti-NaPi2b-vc-MMAE) with two common pharmaceutically relevant metal-induced oxidizing systems, Cu(II)/ ascorbic acid and Fe(II)/ H2O2, and evaluated oxidation, size distribution, carbonylation, Fc effector functions, antibody-dependent cellular cytotoxicity (ADCC) activity, cell anti-proliferation and autophaghic flux. Our study demonstrates that the extent of oxidation was metal ion-dependent and site-specific, leading to decreased FcγRIIIa and FcRn receptor binding and subsequently potentially reduced bioactivity, though antigen binding was not affected to a great extent. In general, the monoclonal antibody (mAb) and corresponding antibody-drug conjugate (ADC) showed similar impacts to product quality when exposed to the same metal ion, either Cu(II) or Fe(II). Our study clearly demonstrates that transition metal ion binding to therapeutic IgG1 mAbs and ADCs is not random and that oxidation products show unique structural and functional ramifications. A critical outcome from this study is our highlighting of key process parameters, route of degradation, especially oxidation (metal catalyzed or via ROS), on the CH1 and Fc region of full-length mAbs and ADCs.Abbreviations: DNPH 2,4-dinitrophenylhydrazine; ADC Antibody drug conjugate; ADCC Antibody-dependent cellular cytotoxicity; CDR Complementary determining region; DTT Dithiothreitol; HMWF high molecular weight form; LC-MS Liquid chromatography-mass spectrometry; LMWF low molecular weight forms; MOA Mechanism of action; MCO Metal-catalyzed oxidation; MetO Methionine sulfoxide; mAbs Monoclonal antibodies; MyBPC Myosin binding protein C; ROS Reactive oxygen species; SEC Size exclusion chromatography.

Challenges of Using Closed System Transfer Devices With Biological Drug Products: An Industry Perspective.

Hazardous drug is a common term used by the National Institute of Occupational Health and Safety (NIOSH) to classify medications that may induce adverse mutagenic and reproductive responses in health care personnel. NIOSH publishes a list of drugs it defines as hazardous where it may be appropriate for health care workers to take protective measures to reduce the potential for occupational exposure. Recent updates and proposed updates to this list have included large molecule biological products with oncology indications. Both NIOSH and USP <800> recommend the use of closed system transfer devices (CSTDs) during compounding. CSTDs are required for administration of prepared solution in NIOSH. However, USP has suggested that the principles of <800> are broadly applicable to hazardous drug handling activities across all facility types. USP encourages the widespread adoption and use of <800> across all health care settings, which many health care workers have interpreted beyond compounding to include administration and preparation of conventionally manufactured sterile products per approved labeling. Although the use of CSTDs may reduce exposure of health care personnel to chemotherapy agents in health care setting, the impact of CSTDs on quality of biologic drug products, including monoclonal antibodies and other proteins, is not fully understood. To complicate this issue further, there are several commercially available CSTDs in the market which have different fluid paths and material of construction that comes in contact with the drug. Testing every combination of CSTD and drug product for potential incompatibilities can be a labor intensive and impractical approach and cause delay in getting essential drugs to patients. A panel discussion was held at a recent American Association of Pharmaceutical Scientists 2018 PharmSci 360 conference to discuss the impact of CSTDs on biologics. Impact on subvisible and visible particulates and impact to other product quality attributes such as high molecular weight species formation upon contact with CSTDs were reported in American Association of Pharmaceutical Scientists meeting. Impact to deliverable dose, holdup volumes of various CSTDs, and stopper coring were also reported that has significant impact to patient safety. Given the fact that USP chapter <800> will be implemented in December 2019, feedback from health authorities regarding the use of CSTDs for biological drug products is needed to provide an appropriate risk/benefit balance to ensure patient safety and quality of the biologic drug product while also protecting the health care worker and the environment. The purpose of this commentary is to provide an industry perspective on the challenges during the use of CSTDs for biologic drug products and is intended to raise caution and awareness on the benefits and shortcomings of these devices.

Photodisruption of the Structurally Conserved Cys-Cys-Trp Triads Leads to Reduction-Resistant Scrambled Intrachain Disulfides in an IgG1 Monoclonal Antibody.

Photostability conditions as prescribed by ICH guidelines induced highly reduction-resistant scrambled disulfides that contribute to the population of apparent nonreducible aggregates in an IgG1 mAb. Photoinduced cross-linked species were isolated under reducing conditions using an organic phase size exclusion chromatography (OP-SEC) method, followed by O18-labeling tryptic mapping to identify cross-linked peptides. Disulfide scrambling was observed within the IgG1 structurally conserved-intrachain cysteine-cysteine-tryptophan triads (Cys-Cys-Trp), and correlated with Trp-to-kynurenine (Kyn) photodegradation within these triads. We hypothesize that intrachain disulfides protect the proximal Trp within the Cys-Cys-Trp triads from photodegradation by enabling dissipation of Trp-absorbed UV energy via electron transfer to the disulfide bond. Finally, we propose three distinct mechanisms of photochemical degradation of monoclonal antibodies mediated by Trp residues.

Photo-oxidation of IgG1 and Model Peptides: Detection and Analysis of Triply Oxidized His and Trp Side Chain Cleavage Products.

PURPOSE: Triply oxidized histidine in an IgG1 monoclonal antibody was noticed when exposed to ICH light conditions. In order to understand the role of light source, irradiation wavelengths and primary sequence, specifically those of a nearby tryptophan, we synthesized and exposed several peptides to ICH light conditions and analyzed the products using LC-MS analysis. METHODS: Protein and peptide samples were photo-irradiated under ICH conditions as well as with monochromatic light at λ = 254 nm and analyzed using either LTQ Orbitrap or a LTQ-FT ion cyclotron resonance mass spectrometer respectively. RESULTS: A triply oxidized His residue was detected along with a second doubly oxidized His residue in an IgG1. Both of these oxidized His residues are located near Trp residues. In order to investigate the role of Trp photosensitization in His oxidation we synthesized model peptides and Ala mutants. Peptides exposed to ICH light stress conditions revealed a small percent of triply oxidized His in the Trp-containing peptide sequences but not in their corresponding Ala mutants. CONCLUSIONS: The differences in product formation under different photo-irradiation conditions underline the importance of light source, irradiation wavelengths and primary sequence in the photosensitivity of proteins.

Reactions of benzene and 3-methylpyrrole with the •OH and •OOH radicals: an assessment of contemporary density functional theory methods.

A high-level quantum chemistry investigation has been carried out for the addition and abstraction reactions by the radicals (•)OH and (•)OOH to and from the model alkenes 3-methylpyrrole and benzene. These models were chosen to reflect the functionalities contained in the side chain of the amino acid tryptophan. The W1BD procedure was used to calculate benchmark barriers and reaction energies for the smaller model system of (•)OOH addition to ethylene. It was found that the CBS-QB3 methodology compares best with the W1BD benchmark, demonstrating a mean absolute deviation (MAD) from W1BD of 3.9 kJ mol(-1). For the reactions involving the (•)OH radical and benzene or 3-methylpyrrole, addition is favored over abstraction in all cases. In particular the CBS-QB3 calculations suggest a barrierless addition reaction of the (•)OH radical to position two of 3-methylpyrrole. For the analogous addition and abstraction reactions involving the (•)OOH radical, the same order of reactivity was found, albeit with higher barriers. A number of other processes involving the addition of the (•)OOH radical were also investigated. The main findings of these studies determined that the initial (•)OOH barrier of stepwise addition to 3-methylpyrrole (+18.8 kJ mol(-1)) is significantly smaller than the concerted addition barrier (+71.5 kJ mol(-1)). This conclusion contrasts starkly with the situation for ethylene in which it is well established that the concerted process has the smaller barrier. A considerable variety of contemporary density functional theory procedures have been tested to examine their accuracy in predicting the CBS-QB3 results. It was found that the best overall performing method was UBMK with an MAD of 7.3 kJ mol(-1). A number of other functionals additionally performed well. They included UM06, RM06, UXYG3 and RXYG3, all of which have MADs of less than 8 kJ mol(-1).

Compatibility and stability of pertuzumab and trastuzumab admixtures in i.v. infusion bags for coadministration.

The physical/chemical stability and potential interactions after diluting two immunoglobulin G1 monoclonal antibodies (mAb), pertuzumab (Perjeta®) and trastuzumab (Herceptin®), in a single intravenous (i.v.) infusion bag containing 0.9% saline (NaCl) solution was evaluated. As commercial products, pertuzumab and trastuzumab are administered through i.v. infusion to patients sequentially, that is, one drug after the other. To increase convenience and minimize the in-clinic time for patients, the compatibility of coadministering pertuzumab (420 and 840 mg) mixed with either 420 or 720 mg trastuzumab, respectively, in a single 250 mL polyolefin or polyvinyl chloride i.v. bag stored for up to 24 h at 5°C or 30°C was determined. The controls (i.e., pertuzumab alone in an i.v. bag, trastuzumab alone in an i.v. bag) and the mAb mixture were assessed using color, appearance, and clarity, concentration and turbidity by ultraviolet spectroscopy, particulate analysis by light obscuration, size-exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate, analytical ultracentrifugation, and ion-exchange chromatography. Additionally, capillary zone electrophoresis, imaged capillary isoelectric focusing, and potency were utilized to measure the stability of the admixtures containing 1:1 mixtures of pertuzumab/trastuzumab and their respective controls (420 mg pertuzumab alone and 420 mg trastuzumab alone). No observable differences were detected by the above methods in the pertuzumab/trastuzumab mixtures stored up to 24 h at either 5°C or 30°C. The physicochemical methods as listed above were able to detect both molecules as well as the minor variants in the drug mixture, even though some overlap of mAb species were seen in the chromatograms and electropherograms. Furthermore, biophysical analysis also did not show any interactions between the two mAbs or any physical instability under these conditions. Additionally, the drug mixture tested by the pertuzumab-specific inhibition of cell proliferation bioassay showed comparable potency before and after storage. On the basis of these results, pertuzumab and trastuzumab admixture in a single i.v. bag is physically and chemically stable for up to 24 h at 5°C or 30°C and can be used for clinical administration.