Sandeep Nema

Protein aggregation--pathways and influencing factors.

Proteins generally will tend to aggregate under a variety of environmental conditions in comparison with small drug molecules. The extent of aggregation is dependent on many factors that can be broadly classified as intrinsic (primary, secondary, tertiary or quaternary structure) or extrinsic (environment in which protein is present, processing conditions, etc). These protein aggregates may exhibit less desirable characteristics like reduced or no biological activity, potential for immunogenicity or other side effects. Protein aggregation remains one of the major challenges in the development and commercialization of biotechnology products. This article is intended to review and discuss the latest understandings in protein aggregation pathways and the possible extrinsic factors that affect or control the protein aggregation process.

Immunogenicity of protein aggregates--concerns and realities.

Protein aggregation is one of the key challenges in the development of protein biotherapeutics. It is a critical product quality issue as well as a potential safety concern due to the increased immunogenicity potential of these aggregates. The overwhelming safety concern has led to an increased development effort and regulatory scrutiny in recent years. The main purposes of this review are to examine the literature data on the relationship between protein aggregates and immunogenicity, to highlight the linkage and existing inconsistencies/uncertainties, and to propose directions for future investigations/development.

In vitro transdermal delivery of therapeutic antibodies using maltose microneedles.

This paper investigates the microneedle-mediated in vitro transdermal delivery of human IgG as a model protein and demonstrates its applicability to deliver a monoclonal antibody. Microchannels created by the treatment of maltose microneedles in full thickness hairless rat skin were visualized using methylene blue staining. Cryostat sections were prepared and stained using hematoxylin and eosin to locate the depth of penetration. In vitro penetration studies were conducted using freshly excised full thickness hairless rat skin and various parameters like needle length, number of needles and effect of donor concentration were examined. Pathway of IgG transport across skin was confirmed by immunohistochemical (IHC) studies. A monoclonal antibody was delivered under optimized conditions. Methylene blue was taken up by microchannels indicating disruption of the stratum corneum and cryosections showed that microneedles just reached the dermis. Human IgG delivery increased with increase in arrays of microneedles, concentration and length of microneedles. IHC studies demonstrated that IgG follows microchannels for transport across the skin. Transdermal delivery was also demonstrated for the monoclonal antibody. In conclusion, maltose microneedles provide a means for the transdermal delivery of macromolecules.

Molecular charge mediated transport of a 13 kD protein across microporated skin

Transport of proteins across the skin is highly limited owing to their hydrophilic nature and large molecular size. This study was conducted to assess the skin transport abilities of a model protein across hairless rat skin during iontophoresis alone and in combination with microneedles as a function of molecular charge. The effect of microneedle pretreatment on electroosmotic flow was also investigated. Skin permeation experiments were carried out in vitro using daniplestim (DP) (MW, 12.76 kD; isoelectric point, 6.2) as a model protein molecule. The effect of molecular charge on protein transport was evaluated by performing studies in two different buffers--TRIS (pH 7.5) and acetate (pH 4.0). Iontophoretic transport mechanisms of DP varied with respect to molecular charge on the protein. The combination approach (iontophoresis and microneedles) gave much higher flux values compared to iontophoresis alone at both pH 4.0 and pH 7.5, however, the delivery in this case was also found to be charge dependent. The findings of this study indicate that electroosmosis persisted upon microporation, thus retaining skins permselective properties. This enables us to explore the combination of microneedles and iontophoresis as a potential approach for delivery of proteins.

Critical considerations for developing nucleic acid macromolecule based drug products.

Protein expression therapy using nucleic acid macromolecules (NAMs) as a new paradigm in medicine has recently gained immense therapeutic potential. With the advancement of nonviral delivery it has been possible to target NAMs against cancer, immunodeficiency and infectious diseases. Owing to the complex and fragile structure of NAMs, however, development of a suitable, stable formulation for a reasonable product shelf-life and efficacious delivery is indeed challenging to achieve. This review provides a synopsis of challenges in the formulation and stability of DNA/m-RNA based medicines and probable mitigation strategies including a brief summary of delivery options to the target cells. Nucleic acid based drugs at various stages of ongoing clinical trials are compiled.

Case Study: Parecoxib: A Prodrug of Valdecoxib

Parecoxib, a weakly acidic drug (pKa 4.9), was developed as a COX-2 inhibitor for the management and treatment of acute pain. It was designed to be a water-soluble (> 50 mg/mL in normal saline), parenterally safe prodrug form of valdecoxib, or 4-(5-methyl-3-phenyl-4-isoxazolyl) benzenesulfonamide, a diaryl-substituted isoxazole. Valdecoxib is a sparingly water soluble (10 µ/mL), weakly acidic drug with a pKa of 9.8. Valdecoxib is commercially available as Bextra, an oral formulation for the management of acute pain, chronic pain, osteoarthritis (OA), rheumatoid arthritis (RA), and primary dysmenorrhea.

Effects of Drying Process on an IgG1 Monoclonal Antibody Using Solid-State Hydrogen Deuterium Exchange with Mass Spectrometric Analysis (ssHDX-MS)

PurposeLyophilization and spray drying are widely used to manufacture solid forms of therapeutic proteins. Lyophilization is used to stabilize proteins vulnerable to degradation in solution, whereas spray drying is mainly used to prepare inhalation powders or as an alternative to freezing for storing bulk drug substance. Both processes impose stresses that may adversely affect protein structure, stability and bioactivity. Here, we compared lyophilization with and without controlled ice nucleation, and spray drying for their effects on the solid-state conformation and matrix interactions of a model IgG1 monoclonal antibody (mAb).MethodsSolid-state conformation and matrix interactions of the mAb were probed using solid-state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS), and solid-state Fourier transform infrared (ssFTIR) and solid-state fluorescence spectroscopies.ResultsmAb conformation and/or matrix interactions were most perturbed in mannitol-containing samples and the distribution of states was more heterogeneous in sucrose and trehalose samples that were spray dried.ConclusionsThe findings demonstrate the sensitivity of ssHDX-MS to changes weakly indicated by spectroscopic methods, and support the broader use of ssHDX-MS to probe formulation and process effects on proteins in solid samples.

Probing the Conformation of an IgG1 Monoclonal Antibody in Lyophilized Solids Using Solid-State Hydrogen–Deuterium Exchange with Mass Spectrometric Analysis (ssHDX-MS)

Therapeutic proteins are often formulated as lyophilized products to improve their stability and prolong shelf life. The stability of proteins in the solid-state has been correlated with preservation of native higher order structure and/or molecular mobility in the solid matrix, with varying success. In the studies reported here, we used solid-state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS) to study the conformation of an IgG1 monoclonal antibody (mAb) in lyophilized solids and related the extent of ssHDX to aggregation during storage in the solid phase. The results demonstrate that the extent of ssHDX correlated better with aggregation rate during storage than did solid-state Fourier-transform infrared (ssFTIR) spectroscopic measurements. Interestingly, adding histidine to sucrose at different formulation pH conditions decreased aggregation of the mAb, an effect that did not correlate with structural or conformational changes as measured by ssFTIR or ssHDX-MS. Moreover, peptide-level ssHDX-MS analysis in four selected formulations demonstrated global changes across the structure of the mAb when lyophilized with sucrose, trehalose, or mannitol, whereas site-specific changes were observed when lyophilized with histidine as the sole excipient.