M. Reza Nejadnik

Formulation, Delivery and Stability of Bone Morphogenetic Proteins for Effective Bone Regeneration

Bone morphogenetic proteins (BMPs) are responsible for bone formation during embryogenesis and bone regeneration and remodeling. The osteoinductive action of BMPs, especially BMP-2 and BMP-7, has led to their use in a range of insurmountable treatments where intervention is required for effective bone regeneration. Introduction of BMP products to the market, however, was not without reports of multiple complications and side effects. Aiming for optimization of the therapeutic efficacy and safety, efforts have been focused on improving the delivery of BMPs to lower the administered dose, localize the protein, and prolong its retention time at the site of action. A major challenge with these efforts is that the protein stability should be maintained. With this review we attempt to shed light on how the stability of BMPs can be affected in the formulation and delivery processes. We first provide a short overview of the current standing of the complications experienced with BMP products. We then discuss the different delivery parameters studied in association with BMPs, and their influence on the efficacy and safety of BMP treatments. In particular, the literature addressing the stability of BMPs and their possible interactions with components of the delivery system as well as their sensitivity to conditions of the formulation process is reviewed. In summary, recent developments in the fields of bioengineering and biopharmaceuticals suggest that a good understanding of the relationship between the formulation/delivery conditions and the stability of growth factors such as BMPs is a prerequisite for a safe and effective treatment.

IgG-loaded hyaluronan-based dissolving microneedles for intradermal protein delivery.

Dissolving microneedles are an attractive approach for non-invasive delivery of drugs via the skin, particularly when the doses are in the microgram or low-milligram range. The aim of the study was to develop hyaluronan-based, monoclonal IgG-loaded microneedles for intradermal delivery enabling efficient penetration and rapid dissolution in the skin while preserving protein stability. Microscopic analysis showed successful preparation of sharp microneedles with the tip length of ~280 μm and with up to 10% (w/w) of IgG content. The water content of the microneedles was ~12% and was not affected by the protein content. The protein distribution was uniform within microneedle tips and individual arrays but some array-to-array variation of IgG level within a single preparation batch was detected. After dissolution of microneedle arrays in PBS, N80% of protein was recovered and no conformational changes were detected by fluorescence spectroscopy. At submicron level, only weak and reversible interaction between HA and IgG was found by asymmetric flow field flow fractionation analysis after the dissolution of prepared microneedles. Although, the formation of insoluble micron-size particles was detected by flow imaging microscopy the IgG amount incorporated into these particles was negligible (b5%). Finally, microneedles were able to penetrate into the epidermis of ex vivo human skin followed by the rapid dissolution of the microneedle tips in the skin. After 10 min of application, the majority of the original tip length was dissolved and IgG and hyaluronan were co-deposited until a depth of 150-200 μm in the skin. In conclusion, developed hyaluronan-based dissolving microneedles allow rapid noninvasive intradermal protein delivery.

Intradermal vaccination with hollow microneedles : a comparative study of various protein antigen and adjuvant encapsulated nanoparticles

ABSTRACT In this study, we investigated the potential of intradermal delivery of nanoparticulate vaccines to modulate the immune response of protein antigen using hollow microneedles. Four types of nanoparticles covering a broad range of physiochemical parameters, namely poly (lactic‐co‐glycolic) (PLGA) nanoparticles, liposomes, mesoporous silica nanoparticles (MSNs) and gelatin nanoparticles (GNPs) were compared. The developed nanoparticles were loaded with a model antigen (ovalbumin (OVA)) with and without an adjuvant (poly(I:C)), followed by the characterization of size, zeta potential, morphology, and loading and release of antigen and adjuvant. An in‐house developed hollow‐microneedle applicator was used to inject nanoparticle suspensions precisely into murine skin at a depth of about 120 &mgr;m. OVA/poly(I:C)‐loaded nanoparticles and OVA/poly(I:C) solution elicited similarly strong total IgG and IgG1 responses. However, the co‐encapsulation of OVA and poly(I:C) in nanoparticles significantly increased the IgG2a response compared to OVA/poly(I:C) solution. PLGA nanoparticles and liposomes induced stronger IgG2a responses than MSNs and GNPs, correlating with sustained release of the antigen and adjuvant and a smaller nanoparticle size. When examining cellular responses, the highest CD8+ and CD4+ T cell responses were induced by OVA/poly(I:C)‐loaded liposomes. In conclusion, the applicator controlled hollow microneedle delivery is an excellent method for intradermal injection of nanoparticle vaccines, allowing selection of optimal nanoparticle formulations for humoral and cellular immune responses. Graphical abstract Figure. No caption available.

A Comprehensive Evaluation of Nanoparticle Tracking Analysis (NanoSight) for Characterization of Proteinaceous Submicron Particles

Nanoparticle tracking analysis (NTA) has attracted great interest for application in the field of submicron particle characterization for biopharmaceuticals. It has the virtue of direct sample visualization and particle-by-particle tracking, but the complexity of method development has limited its routine applicability. We systematically evaluated data collection and processing parameters as well as sample handling methods using shake-stressed protein samples. The camera shutter and gain were identified as the key factors influencing NTA results. We also demonstrated that sample filtration was necessary for NTA analysis if there were high numbers of micron particles, whereas the choice of filter membrane was critical for data quality. Sample dilution into corresponding formulation buffer did not affect particle size distributions in our study. Finally, NTA analysis exhibited excellent repeatability in intraday comparison of multiple measurements on the same sample and interday comparison on different batches of samples. Shaking-induced protein aggregation could also be sensitively monitored by NTA. In conclusion, NTA analysis can be used as a robust stability-indicating method for the characterization of proteinaceous submicron particles and thereby complement other analytical methods, provided that consistent sample handling and parametric settings are established for the specific case study.

No Touching! Abrasion of Adsorbed Protein Is the Root Cause of Subvisible Particle Formation During Stirring.

This study addressed the effect of contact sliding during stirring of a monoclonal antibody solution on protein aggregation, in particular, in the nanometer and micrometer size range. An overhead stirring set-up was designed in which the presence and magnitude of the contact between the stir bar and the container could be manipulated. A solution of 0.1 mg/mL of a monoclonal antibody (IgG) in phosphate buffered saline was stirred at 300 rpm at room temperature. At different time points, samples were taken and analyzed by nanoparticle tracking analysis, flow imaging microscopy, and size-exclusion chromatography. In contrast to non-contact-stirred and unstirred samples, the contact-stirred sample contained several-fold more particles and showed a significant loss of monomer. No increase in oligomer content was detected. The number of particles formed was proportional to the contact area and the magnitude of the normal pressure between the stir bar and the glass container. Extrinsic 9-(2,2-dicyanovinyl) julolidine fluorescence indicated a conformational change for contact-stirred protein samples. Presence of polysorbate 20 inhibited the formation of micron-sized aggregates. We suggest a model in which abrasion of the potentially destabilized, adsorbed protein leads to aggregation and renewal of the surface for adsorption of a fresh protein layer.

Protein–polyelectrolyte interactions: Monitoring particle formation and growth by nanoparticle tracking analysis and flow imaging microscopy

The purpose of this study was to investigate the formation and growth kinetics of complexes of proteins and oppositely charged polyelectrolytes. Equal volumes of IgG and dextran sulfate (DS) solutions, 0.01 mg/ml each in 10mM phosphate, pH 6.2, were mixed. At different time points, samples were taken and analyzed by nanoparticle tracking analysis (NTA), Micro-Flow Imaging (MFI) and size-exclusion chromatography (SEC). SEC showed a huge drop in monomer content (approximately 85%) already 2 min after mixing, while a very high nanoparticle (size up to 500 nm) concentration (ca. 9 × 10(8)/ml) was detected by NTA. The nanoparticle concentration gradually decreased over time, while the average particle size increased. After a lag time of about 1.5h, a steady increase in microparticles was measured by MFI. The microparticle concentration kept increasing up to about 1.5 × 10(6)/ml until it started to slightly decrease after 10h. The average size of the microparticles remained in the low-μm range (1-2 μm) with a slight increase and broadening of the size distribution in time. The experimental data could be fitted with Smoluchowskis perikinetic coagulation model, which was validated by studying particle growth kinetics in IgG:DS mixtures of different concentrations. In conclusion, the combination of NTA and MFI provided novel insight into the kinetics and mechanism of protein-polyelectrolyte complex formation.

Measurement of the Average Mass of Proteins Adsorbed to a Nanoparticle by Using a Suspended Microchannel Resonator

We assessed the potential of a suspended microchannel resonator (SMR) to measure the adsorption of proteins to nanoparticles. Standard polystyrene beads suspended in buffer were weighed by a SMR system. Particle suspensions were mixed with solutions of bovine serum albumin (BSA) or monoclonal human antibody (IgG), incubated at room temperature for 3 h and weighed again with SMR. The difference in buoyant mass of the bare and protein-coated polystyrene beads was calculated into real mass of adsorbed proteins. The average surface area occupied per protein molecule was calculated, assuming a monolayer of adsorbed protein. In parallel, dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and zeta potential measurements were performed. SMR revealed a statistically significant increase in the mass of beads because of adsorption of proteins (for BSA and IgG), whereas DLS and NTA did not show a difference between the size of bare and protein-coated beads. The change in the zeta potential of the beads was also measurable. The surface area occupied per protein molecule was in line with their known size. Presented results show that SMR can be used to measure the mass of adsorbed protein to nanoparticles with a high precision in the presence of free protein.

Post-production handling and administration of protein pharmaceuticals and potential instability issues

The safety and efficacy of protein pharmaceuticals depend not only on biological activity but also on purity levels. Impurities may be process related because of limitations in manufacturing or product related because of protein degradation occurring throughout the life history of a product. Although the pharmaceutical biotechnology industry has made great progress in improving bulk and drug product manufacturing as well as company-controlled storage and transportation conditions to minimize the level of degradation, there is less control over the many factors that may subsequently affect product quality after the protein pharmaceuticals are released and shipped by the manufacturer. Routine handling or unintentional mishandling of therapeutic protein products may cause protein degradation that remains unnoticed but can potentially compromise the clinical safety and efficacy of the product. In this commentary, we address some potential risks associated with (mis)handling of protein pharmaceuticals after release by the manufacturer. We summarize the environmental stress factors that have been shown to cause protein degradation and that may be encountered during typical handling procedures of protein pharmaceuticals in a hospital setting or during self-administration by patients. Moreover, we provide recommendations for improvements in product handling to help ensure the quality of protein pharmaceuticals during use.

Potential Issues With the Handling of Biologicals in a Hospital

A master student, who surveyed the procedures in a hospital pharmacy with regard to the handling of biologicals, identified several issues that might have jeopardized product quality. This case may be a tip of the iceberg and illustrates the urgent need for a better education of end-users about how to handle biologicals.

Development of PLGA nanoparticle loaded dissolving microneedles and comparison with hollow microneedles in intradermal vaccine delivery

Graphical abstract Figure. No caption available. &NA; Skin is an attractive but also very challenging immunisation site for particulate subunit vaccines. The aim of this study was to develop hyaluronan (HA)‐based dissolving microneedles (MNs) loaded with PLGA nanoparticles (NPs) co‐encapsulating ovalbumin (OVA) and poly(I:C) for intradermal immunisation. The NP:HA ratio used for the preparation of dissolving MNs appeared to be critical for the quality of MNs and their dissolution in ex vivo human skin. Asymmetrical flow field‐flow fractionation and dynamic light scattering were used to analyse the NPs released from the MNs in vitro. Successful release of the NPs depended on the drying conditions during MN preparation. The delivered antigen dose from dissolving MNs in mice was determined to be 1 &mgr;g OVA, in NPs or as free antigen, by using near‐infrared fluorescence imaging. Finally, the immunogenicity of the NPs after administration of dissolving MNs (NP:HA weight ratio 1:4) was compared with that of hollow MN‐delivered NPs in mice. Immunization with free antigen in dissolving MNs resulted in equally strong immune responses compared to delivery by hollow MNs. However, humoral and cellular immune responses evoked by NP‐loaded dissolving MNs were inferior to those elicited by NPs delivered through a hollow MN. In conclusion, we identified several critical formulation parameters for the further development of NP‐loaded dissolving MNs.