Analytical similarity assessment of MYL-1402O to reference Bevacizumab.

BACKGROUND: Bevacizumab (BEV) is a recombinant humanized monoclonal immunoglobulin G1 antibody that binds to vascular endothelial growth factor (VEGF)-A and acts as an antiangiogenic agent. It is approved for treatment of many cancer indications, including metastatic colorectal cancer and nonsquamous non-small cell lung cancer. RESEARCH DESIGN AND METHODS: The analytical similarity of the BEV biosimilar MYL-1402O to reference BEV sourced from the European Union and United States was assessed using physicochemical and functional tests to support the clinical development of MYL-1402O. Assessment of physicochemical and analytical similarity showed that MYL-1402O has the same amino acid sequence and similar posttranslational modification profile as the reference BEV products. RESULTS: The functional and biologic activity of MYL-1402O assessed using inhibition of VEGF-induced cell proliferation in human umbilical vein endothelial cells, inhibition of VEGF-induced VEGF receptor 2 phosphorylation, and fragment antigen and fragment crystallizable receptor binding, was comparable to reference BEV products. CONCLUSIONS: The totality of the data assessment confirms the high degree of similarity of MYL-1402O to reference BEV with respect to physicochemical and in vitro functional properties. The product quality data presented here, along with data from phase 1 clinical studies, demonstrate the similarity of MYL-1402O to reference BEV products, supporting further clinical development of this BEV biosimilar.

Physicochemical and functional characterization of trastuzumab-dkst, a trastuzumab biosimilar.

Aims: Preclinical comparative similarity studies of trastuzumab-dkst, a Herceptin® biosimilar, are reported. Materials & methods: Primary sequence and higher order structure and pharmacological mechanisms of action were compared using multiple techniques. Pharmacokinetics and repeat-dose toxicity were assessed in cynomolgus monkeys. Results: Primary structures were identical; secondary and tertiary structures were highly similar. Non-significant differences were observed for charge heterogeneity. Twelve of 13 glycan species were highly similar, with slightly higher total mannose levels in trastuzumab-dkst. FcγR and FcRn binding activity was highly similar. Each drug equally inhibited HER2+ cell proliferation, demonstrating equivalent relative potency in mediating HER2+ cell cytolysis by antibody-dependent cellular cytotoxicity. Pharmacokinetic and toxicological profiles in cynomolgus monkeys were similar. Conclusion: Trastuzumab-dkst, US-licensed trastuzumab and EU-approved trastuzumab demonstrate high structural and functional similarity.

Identification, characterization and control of a sequence variant in monoclonal antibody drug product: a case study.

Sequence variants (SV) in protein bio therapeutics can be categorized as unwanted impurities and may raise serious concerns in efficacy and safety of the product. Early detection of specific sequence modifications, that can result in altered physicochemical and or biological properties, is therefore desirable in product manufacturing. Because of their low abundance, and finite resolving power of conventional analytical techniques, they are often overlooked in early drug development. Here, we present a case study where trace amount of a sequence variant is identified in a monoclonal antibody (mAb) based therapeutic protein by LC-MS/MS and the structural and functional features of the SV containing mAb is assessed using appropriate analytical techniques. Further, a very sensitive selected reaction monitoring (SRM) technique is developed to quantify the SV which revealed both prominent and inconspicuous nature of the variant in process chromatography. We present the extensive characterization of a sequence variant in protein biopharmaceutical and first report on control of sequence variants to < 0.05% in final drug product by utilizing SRM based mass spectrometry method during the purification steps.

Physicochemical and functional characterization of MYL-1501D, a proposed biosimilar to insulin glargine.

Insulin glargine is a long-acting analogue of human insulin that has been used to manage hyperglycemia in patients with diabetes mellitus (DM) for nearly 20 years. Insulin glargine has a relatively constant concentration-time profile that mimics basal levels of insulin and allows for once-daily administration. MYL-1501D is a biosimilar insulin glargine designed to offer greater access of insulin glargine to patients, with comparable efficacy and safety to the marketed reference product. We conducted a comprehensive panel of studies based on a formal analysis of critical quality attributes to characterize the structural and functional properties of MYL-1501D and reference insulin glargine products available in the United States and European Union. MYL-1501D was comprehensively shown to have high similarity to the reference products in terms of protein structure, metabolic activity (both in vitro cell-based assays and in vivo rabbit bioassays), and in vitro cell-based assays for mitogenic activity. The structural analyses demonstrated that the primary protein sequence was identical, and secondary and tertiary structures are similar between the proposed biosimilar and the reference products. Insulin receptor binding affinity and phosphorylation studies also established analytical similarity. MYL-1501D demonstrated high similarity in different metabolic assays of glucose uptake, adipogenesis activity, and inhibition of stimulated lipolysis. Rabbit bioassay studies showed MYL-1501D and EU-approved insulin glargine are highly similar to US-licensed insulin glargine. These product quality studies show high similarity between MYL-1501D and licensed or approved insulin glargine products and suggest the potential of MYL-1501D as an alternative cost-effective treatment option for patients and clinicians.

Identification and quantification of product-related quality attributes in bio-therapeutic monoclonal antibody via a simple, and robust cation-exchange HPLC method compatible with direct online detection of UV and native ESI-QTOF-MS analysis.

Modern analytical ion-exchange chromatography is one of the conventional tools used for assessment of product-related quality attributes in bio-therapeutic monoclonal antibodies (mAbs). Here, we present an approach to resolve, identify, and quantify product-related substances of therapeutic mAb at its intact molecular level by cation exchange (CIEX) HPLC coupled directly to electrospray ionization - quadrupole time of flight mass spectrometry (ESI-QTOF-MS). This method utilizes pH gradient elution mode comprised of ammonium formate buffer components, and a weak cation exchange column as stationary phase. Furthermore, ion-mobility mass spectrometry (IM-MS) provided additional insights on its higher order structure. Also, orthogonal assays such as conventional CIEX-HPLC, high resolution capillary isoelectric focusing, peptide mapping, spectroscopic, and fluorescence methods were used considerably to support the findings. Additionally, an in vitro assay was included to assess the associated impact on Fc mediated function. Overall, the developed method with simultaneous detection of UV peak area percentage at 280 nm and native ESI-MS is found to be a rapid and robust analytical tool for direct assessment of structural and purity attributes, process optimization, product development, and to decipher the relevant role of micro-variants on quality, stability, and clinical outcomes.

Correction: T cell activation and differentiation is modulated by a CD6 domain 1 antibody Itolizumab.

[This corrects the article DOI: 10.1371/journal.pone.0180088.].

T cell activation and differentiation is modulated by a CD6 domain 1 antibody Itolizumab.

CD6 is associated with T-cell modulation and is implicated in several autoimmune diseases. We previously demonstrated that Itolizumab, a CD6 domain 1 (CD6D1) specific humanized monoclonal antibody, inhibited the proliferation and cytokine production by T lymphocytes stimulated with anti-CD3 antibody or when co-stimulated with ALCAM. Aberrant IL-17 producing CD4+ helper T-cells (Th17) have been identified as pivotal for the pathogenesis of certain inflammatory autoimmune disorders, including psoriasis. Itolizumab has demonstrated efficacy in human diseases known to have an IL-17 driven pathogenesis. Here, in in vitro experiments we show that by day 3 of human PBMC activation using anti-CD3 and anti-CD28 co-stimulation in a Th17 polarizing milieu, 15-35% of CD4+ T-cells overexpress CD6 and there is an establishment of differentiated Th17 cells. Addition of Itolizumab reduces the activation and differentiation of T cells to Th17 cells and decreases production of IL-17. These effects are associated with the reduction of key transcription factors pSTAT3 and RORγT. Further, transcription analysis studies in these conditions indicate that Itolizumab suppressed T cell activation by primarily reducing cell cycle, DNA transcription and translation associated genes. To understand the mechanism of this inhibition, we evaluated the effect of this anti-human CD6D1 mAb on ALCAM-CD6 as well as TCR-mediated T cell activation. We show that Itolizumab but not its F(ab')2 fragment directly inhibits CD6 receptor hyper-phosphorylation and leads to subsequent decrease in associated ZAP70 kinase and docking protein SLP76. Since Itolizumab binds to CD6 expressed only on human and chimpanzee, we developed an antibody binding specifically to mouse CD6D1. This antibody successfully ameliorated the incidence of experimental autoimmune encephalitis in the mice model. These results position CD6 as a key molecule in sustaining the activation and differentiation of T cells and an important target for modulating autoimmune diseases.

Fracture neck of femur in Factor XIII deficiency: Was better outcome possible?

The fracture in a Factor XIII deficiency patient is being reported for the first time in the literature. We report a displaced fracture neck of femur in a 17-year-old boy with Factor XIII deficiency. Open reduction and internal fixation was done 8 days after the following the initial injury. Two units each of fresh frozen plasma and cryoprecipitate were given perioperatively to prevent excessive bleeding. No perioperative bleeding complications were encountered. At 18-months follow-up, the fracture had united with evidence of avascular necrosis. The fracture neck of femur in a child or young adult needs to be reduced and stabilized at the earliest to prevent devastating complications. Its occurrence in a patient with Factor XIII deficiency is to be managed like in a normal patient, but with extra perioperative care. Undue delay in fixation as happened in this case should be avoided for a better outcome.

The sphericity deviation score: a quantitative radiologic outcome measure of Legg-Calvé Perthes disease applicable at the stage of healing and at skeletal maturity.

AIM: This study aimed to determine if a new measure of sphericity of the femoral head, the Sphericity Deviation Score (SDS), could be estimated reliably at the stage of healing of Legg-Calvé-Perthes disease (LCPD) and to determine if the SDS at the healing stage reflects the outcome at skeletal maturity. METHODS: The SDS was measured with image analysis software on radiographs of 120 children with unilateral LCPD at healing of the disease and at skeletal maturity. The reproducibility of measurement was assessed. SDS values for hips in different Stulberg classes were calculated. On the basis of SDS values at healing of 82 children, a logistic regression model was developed to determine the probability of a good outcome at skeletal maturity. The validity of the model was tested on another 38 children. RESULTS: The SDS values at the stage of healing and at skeletal maturity were comparable. The SDS values were lowest for Stulberg Class I hips and highest for Class IV and V hips. The validated regression model showed a very high probability of a good outcome (Stulberg Class I or II) at skeletal maturity if the SDS at the stage of healing was below 10. CONCLUSIONS: A reliable estimate of the outcome of LCPD at skeletal maturity can be made by computing the SDS as soon as the disease heals.

Quantitative measures for evaluating the radiographic outcome of Legg-Calvé-Perthes disease.

BACKGROUND: Large sample sizes are needed if discrete outcome measures are used to perform outcome studies, whereas more practicable sample sizes are sufficient if continuous outcome measures are used. Recognizing the need to devise reliable continuous outcome measures for assessing the outcomes of treatment of Legg-Calvé-Perthes disease, we evaluated a method of quantifying the shape and size of the femoral head and the femoral-acetabular relationship from computer images of anteroposterior and lateral radiographs with the help of image-analysis software. METHODS: Radiographs of 121 skeletally mature individuals with healed Legg-Calvé-Perthes disease were analyzed, and the sphericity deviation score, femoral head enlargement, femoral neck growth inhibition, and composite femoral congruency arc were calculated. RESULTS: Each of these measurements was reproducible, with distinctly different values for hips in each of the Stulberg classes. Measures of sphericity and congruence (the sphericity deviation score and the composite femoral congruency arc) could very clearly discriminate between spherical and aspherical hips, and there was a strong correlation between these two measurements. CONCLUSIONS: The study shows that it is possible to have reliable quantitative measures of the outcome of Legg-Calvé-Perthes disease at skeletal maturity.

Aging-dependent changes in rat heart mitochondrial glutaredoxins--Implications for redox regulation.

Clinical and animal studies have documented that hearts of the elderly are more susceptible to ischemia/reperfusion damage compared to young adults. Recently we found that aging-dependent increase in susceptibility of cardiomyocytes to apoptosis was attributable to decrease in cytosolic glutaredoxin 1 (Grx1) and concomitant decrease in NF-κB-mediated expression of anti-apoptotic proteins. Besides primary localization in the cytosol, Grx1 also exists in the mitochondrial intermembrane space (IMS). In contrast, Grx2 is confined to the mitochondrial matrix. Here we report that Grx1 is decreased by 50-60% in the IMS, but Grx2 is increased by 1.4-2.6 fold in the matrix of heart mitochondria from elderly rats. Determination of in situ activities of the Grx isozymes from both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria revealed that Grx1 was fully active in the IMS. However, Grx2 was mostly in an inactive form in the matrix, consistent with reversible sequestration of the active-site cysteines of two Grx2 molecules in complex with an iron-sulfur cluster. Our quantitative evaluations of the active/inactive ratio for Grx2 suggest that levels of dimeric Grx2 complex with iron-sulfur clusters are increased in SSM and IFM in the hearts of elderly rats. We found that the inactive Grx2 can be fully reactivated by sodium dithionite or exogenous superoxide production mediated by xanthine oxidase. However, treatment with rotenone, which generates intramitochondrial superoxide through inhibition of mitochondrial respiratory chain Complex I, did not lead to Grx2 activation. These findings suggest that insufficient ROS accumulates in the vicinity of dimeric Grx2 to activate it in situ.

Glutaredoxin regulates apoptosis in cardiomyocytes via NFkappaB targets Bcl-2 and Bcl-xL: implications for cardiac aging.

Cardiomyocyte apoptosis is a well-established contributor to irreversible injury following myocardial infarction (MI). Increased cardiomyocyte apoptosis is associated also with aging in animal models, exacerbated by MI; however, mechanisms for this increased sensitivity to oxidative stress are unknown. Protein mixed-disulfide formation with glutathione (protein glutathionylation) is known to change the function of intermediates that regulate apoptosis. Since glutaredoxin (Grx) specifically catalyzes protein deglutathionylation, we examined its status with aging and its influence on regulation of apoptosis. Grx1 content and activity are decreased by approximately 40% in elderly (24-mo) Fischer 344 rat hearts compared to adult (6-mo) controls. A similar extent of Grx1 knockdown in H9c2 cardiomyocytes led to increased apoptosis, decreased NFkappaB-dependent transcriptional activity, and decreased production (mRNA and protein) of anti-apoptotic NFkappaB target genes, Bcl-2 and Bcl-xL. Knockdown of Bcl-2 and/or Bcl-xL in wild-type H9c2 cells to the same extent ( approximately 50%) as observed in Grx1-knockdown cells increased baseline apoptosis; and knockdown of Bcl-xL, but not Bcl-2, also increased oxidant-induced apoptosis analogous to Grx1-knockdown cells. Natural Grx1-deficient cardiomyocytes isolated from elderly rats also displayed diminished NFkappaB activity and Bcl-xL content. Taken together, these data indicate diminution of Grx1 in elderly animals contributes to increased apoptotic susceptibility via regulation of NFkappaB function.

Targeted disruption of the glutaredoxin 1 gene does not sensitize adult mice to tissue injury induced by ischemia/reperfusion and hyperoxia.

To understand the physiological function of glutaredoxin, a thiotransferase catalyzing the reduction of mixed disulfides of protein and glutathione, we generated a line of knockout mice deficient in the cytosolic glutaredoxin 1 (Grx1). To our surprise, mice deficient in Grx1 were not more susceptible to acute oxidative insults in models of heart and lung injury induced by ischemia/reperfusion and hyperoxia, respectively, suggesting that either changes in S-glutathionylation status of cytosolic proteins are not the major cause of such tissue injury or developmental adaptation in the Glrx1-knockout animals alters the response to oxidative insult. In contrast, mouse embryonic fibroblasts (MEFs) isolated from Grx1-deficient mice displayed an increased vulnerability to diquat and paraquat, but they were not more susceptible to cell death induced by hydrogen peroxide (H(2)O(2)) and diamide. A deficiency in Grx1 also sensitized MEFs to protein S-glutathionylation in response to H(2)O(2) treatment and retarded deglutathionylation of the S-glutathionylated proteins, especially for a single prominent protein band. Additional experiments showed that MEFs lacking Grx1 were more tolerant to apoptosis induced by tumor necrosis factor alphaplus actinomycin D. These findings suggest that various oxidants may damage the cells via distinct mechanisms in which the action of Grx1 may or may not be protective and Grx1 may exert its function on specific target proteins.

What is the functional significance of the unique location of glutaredoxin 1 (GRx1) in the intermembrane space of mitochondria?

Glutaredoxins (GRx) catalyze reversible protein glutathionylation. They are implicated in sulfhydryl homeostasis and regulation of redox signal transduction, controlling various cellular processes like DNA synthesis, defense against oxidative stress, apoptosis signaling, and DNA-binding of transcription factors. Two isoforms of GRx are well characterized in mammals: GRx1, the "cytosolic" form, and GRx2, the "mitochondrial" form. Here we report documentation of GRx1 in mitochondria, localized exclusively in the intermembrane space and segregated from GRx2, localized exclusively in the mitochondrial matrix. We hypothesize that GRx1 and GRx2 in their unique locations regulate different functions of the mitochondria via reversible S-glutathionylation.

Glutathione supplementation potentiates hypoxic apoptosis by S-glutathionylation of p65-NFkappaB.

In murine embryonic fibroblasts, N-acetyl-L-cysteine (NAC), a GSH generating agent, enhances hypoxic apoptosis by blocking the NFkappaB survival pathway (Qanungo, S., Wang, M., and Nieminen, A. L. (2004) J. Biol. Chem. 279, 50455-50464). Here, we examined sulfhydryl modifications of the p65 subunit of NFkappaB that are responsible for NFkappaB inactivation. In MIA PaCa-2 pancreatic cancer cells, hypoxia increased p65-NFkappaB DNA binding and NFkappaB transactivation by 2.6- and 2.8-fold, respectively. NAC blocked these events without having an effect on p65-NFkappaB protein levels and p65-NFkappaB nuclear translocation during hypoxia. Pharmacological inhibition of the NFkappaB pathway also induced hypoxic apoptosis, indicating that the NFkappaB signaling pathway is a major protective mechanism against hypoxic apoptosis. In cell lysates after hypoxia and treatment with N-ethylmaleimide (thiol alkylating agent), dithiothreitol (disulfide reducing agent) was not able to increase binding of p65-NFkappaB to DNA, suggesting that most sulfhydryls in p65-NFkappaB protein were in reduced and activated forms after hypoxia, thereby being blocked by N-ethylmaleimide. In contrast, with hypoxic cells that were also treated with NAC, dithiothreitol increased p65-NFkappaB DNA binding. Glutaredoxin (GRx), which specifically catalyzes reduction of protein-SSG mixed disulfides, reversed inhibition of p65-NFkappaB DNA binding in extracts from cells treated with hypoxia plus NAC and restored NFkappaB activity. This finding indicated that p65-NFkappaB-SSG was formed in situ under hypoxia plus NAC conditions. In cells, knock-down of endogenous GRx1, which also promotes protein glutathionylation under hypoxic radical generating conditions, prevented NAC-induced NFkappaB inactivation and hypoxic apoptosis. The results indicate that GRx-dependent S-glutathionylation of p65-NFkappaB is most likely responsible for NAC-mediated NFkappaB inactivation and enhanced hypoxic apoptosis.

Activation of pregnane X receptor disrupts glucocorticoid and mineralocorticoid homeostasis.

The pregnane X receptor (PXR) was isolated as a xenobiotic receptor that regulates responses to various xenobiotic agents. In this study, we show that PXR plays an important endobiotic role in adrenal steroid homeostasis. Activation of PXR by genetic (transgene) or pharmacological (ligand, such as rifampicin) markedly increased plasma concentrations of corticosterone and aldosterone, the respective primary glucocorticoid and mineralocorticoid in rodents. The increased levels of corticosterone and aldosterone were associated with activation of adrenal steroidogenic enzymes, including cytochrome P450 (CYP)11a1, CYP11b1, CYP11b2, and 3beta-hydroxysteroid dehydrogenase. The PXR-activating transgenic mice also exhibited hypertrophy of the adrenal cortex, loss of glucocorticoid circadian rhythm, and lack of glucocorticoid responses to psychogenic stress. Interestingly, the transgenic mice had normal pituitary secretion of ACTH and the corticosterone-suppressing effect of dexamethasone was intact, suggesting a functional hypothalamus-pituitary-adrenal axis despite a severe disruption of adrenal steroid homeostasis. The ACTH-independent hypercortisolism in the PXR-activating transgenic mice is reminiscent of the pseudo-Cushing's syndrome in patients. The glucocorticoid effect appears to be PXR specific, as the activation of constitutive androstane receptor in transgenic mice had little effect. We propose that PXR is a potential endocrine disrupting factor that may have broad implications in steroid homeostasis and drug-hormone interactions.

Presence of splice variant forms of cytochrome P4502D1 in rat brain but not in liver.

Cytochromes P450 (P450), a family of heme-containing proteins, is involved in the oxidative metabolism of both foreign and endogenous compounds. Although liver is quantitatively the major organ involved in the metabolism of most xenobiotics, there is increasing evidence that these enzymes are present in extrahepatic tissues, such as lung, kidney, brain, etc and they may contribute to the in situ metabolism of xenobiotics in these organs. The possible relationship between genetic polymorphism seen in P4502D6 and incidence of neurodegenerative diseases, such as Parkinson's disease, has prompted the characterization of P4502D enzymes in rat brain. In the present study, we demonstrate that P4502D1 (the rat homologue of human P4502D6) is constitutively expressed in rat brain and the mRNA and protein are localized predominantly in neuronal cell population in the olfactory bulb, cortex, cerebellum, and hippocampus. An alternate spliced transcript of CYP2D1 having exon 3 deletion was detected in rat brain but not in liver. Deletion of exon 3 causes frame shift and generates a stop codon at 391 bp relative to the start codon ATG leading to premature termination of translation. Thus, Northern blotting and in situ hybridization represent contributions from functional transcripts and alternate spliced variants that do not translate into functional protein. Further, the splice variant having partial inclusion of intron 6 detected in human brain was not detected in rat brain indicating that alternate spliced gene products of P450 enzymes are generated in species-specific and tissue-specific manner.

A frameshift mutation and alternate splicing in human brain generate a functional form of the pseudogene cytochrome P4502D7 that demethylates codeine to morphine.

A frameshift mutation 138delT generates an open reading frame in the pseudogene, cytochrome P4502D7 (CYP2D7), and an alternate spliced functional transcript of CYP2D7 containing partial inclusion of intron 6 was identified in human brain but not in liver or kidney from the same individual. mRNA and protein of the brain variant CYP2D7 were detected in 6 of 12 human autopsy brains. Genotyping revealed the presence of the frameshift mutation 138delT only in those human subjects who expressed the brain variant CYP2D7. Genomic DNA analysis in normal volunteers revealed the presence of functional CYP2D7 in 4 of 8 individuals. In liver, the major organ involved in drug metabolism, a minor metabolic pathway mediated by CYP2D6 metabolizes codeine (pro-drug) to morphine (active drug), whereas norcodeine is the major metabolite. In contrast, when expressed in Neuro2a cells, brain variant CYP2D7 metabolized codeine to morphine with greater efficiency compared with the corresponding activity in cells expressing CYP2D6. Morphine binds to micro-opioid receptors in certain regions of the central nervous system, such as periaqueductal gray, and produces pain relief. The brain variant CYP2D7 and micro-opioid receptor colocalize in neurons of the periaqueductal gray area in human brain, indicating that metabolism of codeine to morphine could occur at the site of opioid action. Histio-specific isoforms of P450 generated by alternate splicing, which mediate selective metabolism of pro-drugs within tissues, particularly the brain, to generate active drugs may play an important role in drug action and provide newer insights into the genetics of metabolism.

Expression, purification, and biochemical characterization of Mycobacterium tuberculosis aspartate decarboxylase, PanD.

Like all bacteria, Mycobacterium tuberculosis (Mtb) possesses the genes necessary for coenzyme A biosynthesis and metabolism. In the present work, the Mtb panD gene was PCR amplified, overexpressed, and purified by metal affinity chromatography. The recombinant Mtb panD was found to exist as a tetramer in solution. Incubation of Mtb panD at 37 degrees C for several hours resulted in a complete cleavage of the inactive (pi) form into the two subunits (alpha and beta). The cleavage was confirmed by Western blot analysis as well as by N-terminal sequencing. Cleaved Mtb panD was assayed for decarboxylase activity with L-aspartate as substrate. The kinetic parameters K(m) and k(cat) were found to be 219 microM and 0.65s(-1), respectively. These results provide the means for further studies based on the identification of the Mtb panD as well as other components of pantothenate metabolism as potential drug targets.