Development of a high throughput oxidation profiling strategy for monoclonal antibody products.

Oxidation is one of the most common degradation pathways of biopharmaceutics, potentially leading to altered product stability, pharmacokinetics, reduced biological activity and/or an increased immunogenicity. However, it is often insufficiently assessed in early development stages, leaving potential molecule liabilities undiscovered. Aim of the present work was the development of a high throughput oxidation profiling strategy, applicable throughout various stages of biopharmaceutical development. The study demonstrates that the combination of multiple stress assays, including peroxide-based, visible light, and metal-catalyzed oxidation (MCO), enables a comprehensive understanding of a mAb's oxidation susceptibility. The most effective parameters to evaluate oxidation in a high-throughput screening workflow are aggregation, tryptophan oxidation and changes in the hydrophobicity profile of the Fc and Fab subunit measured via Size Exclusion Chromatography, Intrinsic Tryptophan Fluorescence Emission spectroscopy and Reversed-Phase Chromatography subunit analysis, respectively. This oxidation profiling approach is valuable tool to systematically characterize the oxidation susceptibility under relevant conditions, time effective and with minimal sample consumption.

A High-Throughput MEMS-Based Differential Scanning Calorimeter for Direct Thermal Characterization of Antibodies.

Calorimeters, which can be used for rapid thermal characterization of biomolecules, are getting intense attention in drug development. This paper presents a novel MEMS-based differential scanning calorimeter (DSC) for direct thermal characterization of protein samples. The DSC consisted of a pair of temperature sensors made by vanadium oxide (VOx) film with a temperature coefficient of resistivity of -0.025/K at 300 K, a microfluidic device with high thermal insulation (2.8 K/mW), and a Peltier heater for linear temperature scanning. The DSC exhibited high sensitivity (6.1 µV/µW), low noise (0.4 µW), high scanning rate (45 K/min), and low sample consumption volume (0.63 µL). The MEMS DSC was verified by measuring the temperature-induced denaturation of lysozyme at different pH, and then used to study the thermal stability of a monoclonal antibody (mAb), an antigen-binding fragment (Fab), and a dual variable domain immunoglobulin (DVD-Ig) at pH = 6. The results showed that lysozyme is a stable protein in the pH range of 4.0-8.0. The protein stability study revealed that the transition temperatures of the intact Fab fragment, mAb, and DVD proteins were comparable with conformational stability results obtained using conventional commercial DSC. These studies demonstrated that the MEMS DSC is an effective tool for directly understanding the thermal stability of antibodies in a high-throughput and low-cost manner compared to conventional calorimeters.

Micro-differential scanning calorimeter for liquid biological samples.

We developed an ultrasensitive micro-DSC (differential scanning calorimeter) for liquid protein sample characterization. This design integrated vanadium oxide thermistors and flexible polymer substrates with microfluidics chambers to achieve a high sensitivity (6 V/W), low thermal conductivity (0.7 mW/K), high power resolutions (40 nW), and well-defined liquid volume (1 µl) calorimeter sensor in a compact and cost-effective way. We further demonstrated the performance of the sensor with lysozyme unfolding. The measured transition temperature and enthalpy change were in accordance with the previous literature data. This micro-DSC could potentially raise the prospect of high-throughput biochemical measurement by parallel operation with miniaturized sample consumption.

Dose levels in particulate-containing formulations impact anti-drug antibody responses to murine monoclonal antibody in mice.

Dosage levels and particulate contents of therapeutic protein formulations are potential factors that impact immunogenicity of protein therapeutics. Here, we evaluated the effect of dose levels on the immunogenicity of protein particulates formed by adsorbing a murine monoclonal IgG2c/κ antibody (mAb1) onto silicone oil microdroplets, glass, or aluminum hydroxide (Alhydrogel) microparticles. Immune responses to these particulate-containing preparations were compared against responses to solutions of mAb1 that had been ultracentrifuged to minimize particle levels. Formulations containing 5 or 500 µg of adsorbed mAb1 were administered subcutaneously to C57BL/6J or BALB/c mice. Antidrug antibodies (ADAs) were detected using an isotype-specific enzyme-linked immunosorbent assay (ELISA) method or a chemiluminescence method. Sera from BALB/c mice showed greater ADA responses to administration of particles at the 5-µg dose level than at the 500-µg dose level. In sera from C57BL/6J mice, ADA levels detected by ELISA were independent of the particle dose levels tested. ADAs were not detected in sera from C57BL/6J mice performing the chemiluminescence technique. In conclusion, mice administered formulations of a murine antibody adsorbed onto silicone oil microdroplets, glass microparticles, or Alhydrogel(®) showed greater ADA responses that those that received particle-free mAb1 preparations, and responses were greater for formulations containing lower doses of antibody. .

Investigation of the immunogenicity of different types of aggregates of a murine monoclonal antibody in mice.

PURPOSE: The potential contribution of protein aggregates to the unwanted immunogenicity of protein pharmaceuticals is a major concern. In the present study a murine monoclonal antibody was utilized to study the immunogenicity of different types of aggregates in mice. Samples containing defined types of aggregates were prepared by processes such as stirring, agitation, exposure to ultraviolet (UV) light and exposure to elevated temperatures. METHODS: Aggregates were analyzed by size-exclusion chromatography, light obscuration, turbidimetry, infrared (IR) spectroscopy and UV spectroscopy. Samples were separated into fractions based on aggregate size by asymmetrical flow field-flow fractionation or by centrifugation. Samples containing different types and sizes of aggregates were subsequently administered to C57BL/6 J and BALB/c mice, and serum was analyzed for the presence of anti-IgG1, anti-IgG2a, anti-IgG2b and anti-IgG3 antibodies. In addition, the pharmacokinetic profile of the murine antibody was investigated. RESULTS: In this study, samples containing high numbers of different types of aggregates were administered in order to challenge the in vivo system. The magnitude of immune response depends on the nature of the aggregates. The most immunogenic aggregates were of relatively large and insoluble nature, with perturbed, non-native structures. CONCLUSION: This study shows that not all protein drug aggregates are equally immunogenic.

Antibody responses in mice to particles formed from adsorption of a murine monoclonal antibody onto glass microparticles.

Immunogenicity of therapeutic monoclonal antibodies (mAbs) is a concern because of the effects of anti-drug antibodies (ADAs) on therapeutic efficacy. Particulate matter has been suggested as a potential contributing factor to immunogenicity. In this study, we investigated ADA levels in mice in response to administration of a murine immunoglobulin G (IgG)2c/κ mAb (mAb1) that was generated in C57BL/6J mice. Particles of mAb1 were formed by adsorbing the protein to glass microparticles. Formulations containing microparticles were administered subcutaneously to mice of either the syngeneic strain, C57Bl/6J, or the allogeneic strain, BALB/c. ADA levels were measured using an isotype-specific enzyme-linked immunosorbent assay method. Whereas BALB/c mice showed strong IgG1 and IgG2b responses against both the particulate and native mAb1 samples, adsorption of mAb1 to particles rendered it slightly more immunogenic than its native, soluble form. In BALB/c mice, immunoglobulin M (IgM) was produced after the first week of injections and then faded gradually. In contrast, C57BL/6J mice showed moderate IgM, IgG1, IgG2b, and IgG3 responses to injections of glass particle-adsorbed mAb1. ADA responses were higher in the allogeneic BALB/c mice, which do not produce mAbs of the IgG2c/κ isotype. Thus, the presence of both foreign epitopes and particles may be important in inducing ADA responses.

Periodontal wound healing/regeneration following implantation of recombinant human growth/differentiation factor-5 in a beta-tricalcium phosphate carrier into one-wall intrabony defects in dogs.

OBJECTIVE: Recombinant human growth/differentiation factor-5 (rhGDF-5) is being evaluated as a candidate therapy in support of periodontal regeneration. The objective of this study was to evaluate periodontal wound healing/regeneration following the application of rhGDF-5 on a particulate beta-tricalcium phosphate (beta-TCP) carrier using an established defect model. MATERIALS AND METHODS: Bilateral 4 x 5 mm (width x depth), one-wall, critical-size, intrabony periodontal defects were surgically created at the mandibular second and fourth pre-molar teeth in 15 Beagle dogs. Unilateral defects in five animals received rhGDF-5/beta-TCP (Scil Technology GmbH); five animals received beta-TCP solo; and five animals served as sham-surgery controls. Contralateral sites received treatments reported elsewhere. The animals were sacrificed following an 8-week healing interval for histological examination. RESULTS: Clinical healing was generally uneventful. Sites implanted with rhGDF-5/beta-TCP exhibited greater enhanced cementum and bone formation compared with beta-TCP and sham-surgery controls; cementum regeneration averaged (+/- SD) 3.83 +/- 0.73 versus 1.65 +/- 0.82 and 2.48 +/- 1.28 mm for the controls (p<0.05). Corresponding values for bone regeneration height averaged 3.26 +/- 0.30 versus 1.70 +/- 0.66 and 1.68 +/- 0.49 mm (p<0.05), and bone area 10.45 +/- 2.26 versus 6.31 +/- 2.41 and 3.00 +/- 1.97 mm(2) (p<0.05). Cementum regeneration included cellular/acellular cementum with or without a functionally oriented periodontal ligament. A non-specific connective tissue attachment was evident in the sham-surgery control. Controls exhibited mostly woven bone with primary osteons, whereas rhGDF-5/beta-TCP sites showed a noticeable extent of lamellar bone. Sites receiving rhGDF-5/beta-TCP or beta-TCP showed some residual beta-TCP granules apparently undergoing biodegradation without obvious differences between the sites. Sites receiving beta-TCP alone commonly showed residual beta-TCP granules sequestered in the connective tissue or fibrovascular marrow. CONCLUSION: rhGDF-5/beta-TCP has a greater potential to support the regeneration of the periodontal attachment. Long-term studies are necessary to confirm the uneventful maturation of the regenerated tissues.

Periodontal wound healing/regeneration following implantation of recombinant human growth/differentiation factor-5 (rhGDF-5) in an absorbable collagen sponge carrier into one-wall intrabony defects in dogs: a dose-range study.

AIM: Recombinant human growth/differentiation factor-5 (rhGDF-5) is being evaluated as a candidate therapy in support of periodontal regeneration. The objective of this study was to evaluate cementum and alveolar bone formation, and aberrant healing events following surgical implantation of rhGDF-5 in an absorbable collagen sponge (ACS) carrier using an established periodontal defect model. MATERIALS AND METHODS: Bilateral 4 x 5 mm (width x depth), one-wall, critical-size, intrabony periodontal defects were surgically created at the mandibular second and fourth pre-molar teeth in 15 Beagle dogs. Five animals received 1 microg/defect and five animals 20 microg/defect rhGDF-5 in unilateral defect sites. Contralateral sites received treatments reported elsewhere. Five animals received rhGDF-5/ACS with 0 (buffer control) and 100 microg/defect rhGDF-5 in contralateral defect sites. The animals were euthanized at 8 weeks post-surgery for histologic and histometric evaluation. RESULTS: Surgical implantation of rhGDF-5 stimulated significant periodontal regeneration. Cementum formation was significantly enhanced in sites implanted with rhGDF-5 (1 and 100 microg) compared with control (p<0.05). Similarly, bone formation height was significantly greater in sites receiving rhGDF-5 (1 and 100 microg) compared with control (p<0.05). There were no significant or remarkable differences in bone and cementum formation within the selected dose interval (1, 20 and 100 microg rhGDF-5). None of the control or the rhGDF-5 sites exhibited root resorption, ankylosis, or other aberrant tissue reactions. CONCLUSION: Surgical implantation of rhGDF-5/ACS may be used safely to support periodontal wound healing/regeneration in intrabony periodontal defects without complications.

Development of an injectable composite as a carrier for growth factor-enhanced periodontal regeneration.

AIM: Biomaterials are often applied in periodontal therapy; however, not always well adapted for tissue regeneration. The objective of this study was to evaluate the physico-chemical properties and biocompatibility of an injectable, in situ setting composite for growth factor-enhanced periodontal regeneration. MATERIAL AND METHODS: The composite constitutes bioresorbable poly(lactic-co-glycolic acid) (PLGA) and additives forming in situ a matrix designed as a carrier for recombinant human growth/differentiation factor-5 (rhGDF-5). In vitro characterization included the porosity, biointeraction, biodegradation, injectability, and biological activity of released rhGDF-5. Biocompatibility was compared with granular beta-tricalcium phosphate and an absorbable collagen sponge using a canine periodontal defect model. RESULTS: The PLGA composite showed a highly porous (500-1000 mum) space-providing structure. It effectively induced coagulation exhibiting an intimate interaction with the fibrin clot. The biphasic biodegradation was complete within 4 weeks. The composite was conveniently injectable (90.4+/-3.6 N) for ease of use. It exhibited a sustained rhGDF-5 release over 4 weeks (40.8%) after initial burst (3.4%) detected by ALP activity. Sites receiving the composite showed limited, if any, residuals and had no appreciable negative effect on periodontal wound healing. There were no noteworthy inflammatory lesions in sites receiving the PLGA composite. CONCLUSION: Characteristics of the PLGA composite makes it an attractive matrix to support native wound healing and rhGDF-5-enhanced periodontal regeneration.

Superior effect of MD05, beta-tricalcium phosphate coated with recombinant human growth/differentiation factor-5, compared to conventional bone substitutes in the rat calvarial defect model.

BACKGROUND: MD05 consists of beta-tricalcium phosphate (beta-TCP) coated with recombinant human growth/differentiation factor-5 (rhGDF-5) and is under evaluation as an osteoinductive and osteoconductive bone graft material for use in dental and maxillofacial applications. The objective of this study was to compare the bone regenerative properties of MD05 with those of conventional commercially available bone substitutes. METHODS: Full-thickness, 6-mm diameter, calvarial critical-size defects (two per animal) were created in adult Sprague-Dawley rats. Groups of rats were implanted with the following: 1) MD05; 2) bovine bone mineral; 3) bovine bone mineral with collagen; 4) bovine bone mineral with synthetic peptide, 5) beta-TCP (from two different manufacturers); or 6) no filling material (sham controls). Blinded macroscopic analysis, histopathologic analysis, and histomorphometric analysis were carried out 6 weeks after implantation. RESULTS: New bone formation assessed histomorphometrically was about five times greater with MD05 than with the other bone substitutes tested, and bone repair was well advanced in MD05-filled defects after 6 weeks. The extent of fibrous tissue and residual implant were significantly lower in the MD05 group. In contrast to the other materials, the use of MD05 was associated with the complete osseous bridging of the defect and with the presence of normal bone marrow. The osteoinductive effect of rhGDF-5 was apparent from the more pronounced bone ingrowth observed with MD05 compared to the beta-TCP carrier alone. All implants showed good biocompatibility. CONCLUSION: MD05 achieved superior bone regeneration compared to conventional materials and is a promising new bone substitute for dental and maxillofacial applications.