Physicochemical and biological similarity assessment of LBAL, a biosimilar to adalimumab reference product (Humira®).

LBAL was developed as an adalimumab (Humira®) biosimilar using Chinese hamster ovary cell lines. Comparable quality, safety, and efficacy between a biosimilar and its reference product should be ensured for regulatory approval. Here, we present the results of a comprehensive physicochemical and biological characterization between LBAL and Humira®. As physicochemical attributes, primary and higher-order structure, N-glycan profile, and disulfide linkage were investigated. Biological attributes were evaluated by target/receptor binding analysis and in vitro/ex vivo cell-based assays, which are linked to mechanisms of action. As a result, LBAL had the identical amino acid sequence, similar post-translational modifications and N-/C-terminal variants, and comparable primary, secondary, and tertiary structures and disulfide linkage profile. However, some differences in N-glycan profiles were observed. Biological activities, including tumor necrosis factor (TNF) binding, TNF-neutralization, apoptosis, Fc receptor binding, and complement-dependent cytotoxicity, were largely consistent. Despite a slightly lower antibody-dependent cellular cytotoxicity activity in LBAL, this difference was not significant under physiological conditions. As indicated, this extensive analytical characterization and functional comparison assessment showed that LBAL was similar to Humira®, with minor differences of no clinical relevance. Taken together, our comparative assessment of physicochemical and biological attributes demonstrated that LBAL is structurally and functionally very similar to Humira®, supporting the biosimilarity of clinical efficacy and safety.

Comprehensive Physicochemical and Biological Characterization of the Proposed Biosimilar Darbepoetin Alfa, LBDE, and Its Originator Darbepoetin Alfa, NESP®.

BACKGROUND: For regulatory approval, the comparability of a biosimilar product to an originator product should be ensured through thorough physicochemical and biological characterization. OBJECTIVE: To evaluate the biosimilarity between LBDE, the proposed biosimilar darbepoetin alfa, and NESP®, its originator, we performed a comprehensive physicochemical and biological characterization study. METHODS: Primary and higher-order protein structures were analyzed using Lys-C peptide mapping with liquid chromatography-mass spectrometry (LC-MS), disulfide bond identification, circular dichroism, and fluorescence spectroscopy. Glycosylation and isoform distribution were analyzed using MS, LC, and capillary zone electrophoresis. Size variants were evaluated with size-exclusion chromatography-high-performance liquid chromatography (SEC-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Biological characterization included binding affinity for human erythropoietin receptor, in vitro cell proliferation, and in vivo potency. Pharmacokinetics (PK) were evaluated using rats through two injection routes. RESULTS: Non-reducing and reducing Lys-C peptide mapping showed a highly similar peak profile, confirming that LBDE and NESP® have the same primary structure and disulfide bonds. Glycosylation and isoform analyses showed that the attached N-glycan and O-glycan structures were the same and their relative contents were similar. Spectroscopic analysis of LBDE showed indistinguishable spectra with NESP®. For both LBDE and NESP®, a very small amount of size variants was found in SEC-HPLC, and no minor bands were detected in SDS-PAGE. Furthermore, LBDE did not show any difference with NESP® in the in vitro and in vivo functional analyses. PK parameters of LBDE were in good agreement with those of NESP®. CONCLUSION: LBDE shows high similarity to NESP® with regard to structure and function.

Cell type-specific activation of intracellular transglutaminase 2 by oxidative stress or ultraviolet irradiation: implications of transglutaminase 2 in age-related cataractogenesis.

Transglutaminase (TGase) 2 is a ubiquitously expressed enzyme that modifies proteins by cross-linking or polyamination. An aberrant activity of TGase 2 has implicated its possible roles in a variety of diseases including age-related cataracts. However, the molecular mechanism by which TGase 2 is activated has not been elucidated. In this report, we showed that oxidative stress or UV irradiation elevates in situ TGase 2 activity. Neither the expression level nor the in vitro activity of TGase 2 appeared to correlate with the observed elevation of in situ TGase 2 activity. Screening a number of cell lines revealed that the level of TGase 2 activation depends on the cell type and also the environmental stress, suggesting that unrecognized cellular factor(s) may specifically regulate in situ TGase 2 activity. Concomitantly, we observed that human lens epithelial cells (HLE-B3) exhibited about 3-fold increase in in situ TGase 2 activity in response to the stresses. The activated TGase 2 catalyzed the formation of water-insoluble dimers or polymers of alphaB-crystallin, betaB(2)-crystallin, and vimentin in HLE-B3 cells, providing evidence that TGase 2 may play a role in cataractogenesis. Thus, our findings indicate that in situ TGase 2 activity must be evaluated instead of in vitro activity to study the regulation mechanism and function of TGase 2 in biological and pathological processes.

Transglutaminase 2 inhibits Rb binding of human papillomavirus E7 by incorporating polyamine.

Transglutaminase 2 (TGase 2) is one of a family of enzymes that catalyze protein modification through the incorporation of polyamines into substrates or the formation of protein crosslinks. However, the physiological roles of TGase 2 are largely unknown. To elucidate the functions of TGase 2, we have searched for its interacting proteins. Here we show that TGase 2 interacts with E7 oncoprotein of human papillomavirus type 18 (HPV18) in vitro and in vivo. TGase 2 incorporates polyamines into a conserved glutamine residue in the zinc-binding domain of HPV18 E7 protein. This modification mediates the inhibition of E7's Rb binding ability. In contrast, TGase 2 does not affect HPV16 E7, due to absence of a glutamine residue at this polyamination site. Using E7 mutants, we demonstrate that TGase 2-dependent inhibition of HPV E7 function correlates with the presence of the polyamination site. Our results indicate that TGase 2 is an important cellular interfering factor and define a novel host-virus interaction, suggesting that the inability of TGase 2 to inactivate HPV16 E7 could explain the high prevalence of HPV16 in cervical cancer.

Differential incorporation of biotinylated polyamines by transglutaminase 2.

Polyamine incorporation or cross-linking of proteins, post-translational modifications mediated by transglutaminase 2 (TGase 2), have been implicated in a variety of physiological functions including cell adhesion, extracellular matrix formation and apoptosis. To better understand the intracellular regulation mechanism of TGase 2, the properties of biotinylated polyamines as substrates for determining in situ TGase activity were analyzed. We synthesized biotinylated spermine (BS), and compared the in vitro and in situ incorporation of BS with that of biotinylated pentylamine (BP), which is an artificial polyamine derivative. When measured in vitro, BP showed a significantly higher incorporation rate than BS. In contrast, in situ incorporation of both BS and BP was not detected even in TGase 2-overexpressed 293 cells. Cells exposed to high calcium showed a marked increase of BP incorporation but not of BS. These data indicate that the in situ activity of TGase 2 gives different results with different substrates, and suggest the possibility of overrepresentation of in situ TGase 2 activity when assayed with BP. Therefore, careful interpretation or evaluation of in situ TGase 2 activity may be required.

Improved immunodetection of human papillomavirus E7.

Human papillomavirus E7 (HPV E7) is a viral oncoprotein that plays an important role in cervical carcinogenesis through binding with retinoblastoma protein (Rb). Inactivation of Rb by E7 is necessary but not sufficient for cellular transformation, suggesting other protein-protein interactions are required for E7-mediated cellular transformation aside from the interaction with Rb. However, studies on the oncogenic function of HPV E7 have been limited by its poor immunoreactivity. In this report, we show that the fixation of purified recombinant HPV E7 on blotted nitrocellulose membrane with glutaldehyde markedly enhanced the immunoreactivity of HPV E7 protein. Using HeLa and Caski cell lines which are infected with HPV 18 and HPV 16, respectively, we demonstrated that native HPV E7 proteins also could be detected by this method. These results therefore can provide the experimental conditions for detection of HPV E7 proteins with greater sensitivity and may help to analyze E7 functions.