Enrichment and recovery of oligonucleotide impurities by N-Rich twin-column continuous chromatography.

N-Rich is a twin-column continuous chromatography technology well suited for small-scale isolation and the enrichment of product related impurities. For the first time, N-Rich was used for impurity isolation from a double-stranded RNA (dsRNA) therapeutic synthetic oligonucleotide (ON), produced by solid-phase synthesis. By employing the N-Rich process, where the desired impurities are recycled and selectively enriched, and interfering substances are depleted, it was possible to obtain substantial amounts of high purity marginal impurities with a reproducible, automatized, and productive method. The productivity-purity tradeoff inherent to traditional impurity isolation methods, i.e., analytical chromatography, was effectively alleviated. Using N-Rich, satisfactory purity values and mass recoveries of several low-concentrated impurities could be obtained simultaneously. A performance comparison demonstrated an up to 15-fold increase for purity values and up to 20-fold mass impurity isolation and concentration with the N-Rich technology in comparison to conventional isolation procedures, drastically reducing processing times, manual handling, and waste production.

Continuous countercurrent chromatographic twin-column purification of oligonucleotides: The role of the displacement effect.

Oligonucleotides (ONs) are breaking through in the biopharmaceutical industry as a promising class of biotherapeutics. The main success of these molecules is due to their peculiar way of acting in the cellular process, regulating the gene expression and hence influencing the protein synthesis at a pretranslational level. Although the Food and Drug Administration (FDA) already approved a few ON-based therapeutics, their production cost strongly limits large-scale manufacturing: a situation that can be alleviated through process intensification. In this study, we address this problem by developing an efficient and continuous chromatographic purification process for ONs. In particular, we considered the chromatographic purification of an ON crude prepared by chemical synthesis using anion exchange resins. We demonstrate that in this system the competitive adsorption of the various species on the same sites of the resin leads to the displacement of the more weakly adsorbing species by the more strongly adsorbing ones. This phenomenon affects the behavior of the chromatographic units and it has been investigated in detail. Then, we developed a continuous countercurrent solvent gradient purification (MCSGP) process, which can significantly improve the productivity and buffer consumption compared to a classical single-column, batch chromatographic process.

Enrichment and purification of peptide impurities using twin-column continuous chromatography.

N-Rich is a twin-column chromatography process that enriches target compounds relative to other components in a mixture, thereby facilitating their isolation and characterization. This study demonstrates the performance of N-Rich for isolation of Angiotensin II peptide impurities compared with standard analytical and preparative chromatography approaches. Peptides have diverse chemical properties and are produced using a wide range of methods, resulting in products with complex impurity profiles. The characterization of impurities for clinical development is essential but obtaining high purity samples in sufficient quantities is often a difficult task when using standard chromatographic techniques. In contrast, by using cyclic continuous chromatography with UV-based process control, N-Rich enables automatic on-column accumulation of target impurities while other compounds in the mixture are depleted. This has multiple advantages compared to standard techniques. Firstly, at the end of the cyclic accumulation phase the highly enriched target is eluted in one step with high purity and concentration. This means fewer fractions for analysis are generated and up-concentration steps are reduced. Secondly, the purification of target impurities using semi-preparative scale chromatography becomes viable, even if initial resolution is poor compared to analytical HPLC. This allows for very significant increases in productivity for purification of difficult to isolate impurities. This study demonstrates two N-Rich strategies: Example 1: Purification of µg quantities of multiple Angiotensin II impurities with a >9-fold increase in productivity compared to analytical HPLC. Example 2: Specific isolation of 1 mg of a critical impurity at 88% purity. 79-fold increase in productivity and a 69-fold reduction in solvent consumption compared to analytical HPLC.

Purification of a GalNAc-cluster-conjugated oligonucleotide by reversed-phase twin-column continuous chromatography.

Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) is a continuous chromatography technique used to maximize purification yields compared to traditional batch purification methods. Here we apply MCSGP for the reversed phase purification of a N-acetylgalactosamine (GalNAc)-cluster-conjugated DNA-LNA gapmer oligonucleotide therapeutic using a twin-column chromatography system. Based on a batch process as a starting point, MCSGP was designed, optimized and compared with the batch process regarding process performance and scale-up requirements. Product yields increased from 52.7% using batch chromatography to 91.5% using MCSGP, with purity, productivity, and buffer consumption otherwise comparable. In a manufacturing scenario, use of MCSGP would allow the downscaling of oligonucleotide synthesis by 42.5%, which would result in a significant cost reduction and increased throughput. Moreover, the equipment, chemicals and methodology used in MCSGP are analogous to a standard reversed phase purification allowing for a "like for like" transition to the upgraded MCSGP process.

Oligonucleotides: Current Trends and Innovative Applications in the Synthesis, Characterization, and Purification.

Oligonucleotides (ONs) are gaining increasing importance as a promising novel class of biopharmaceuticals. Thanks to their fundamental role in gene regulation, they can be used to develop custom-made drugs (also called N-to-1) able to act on the gene expression at pre-translational level. With recent approvals of ON-based therapeutics by the Food and Drug Administration (FDA), a growing demand for high-quality chemically modified ONs is emerging and their market is expected to impressively prosper in the near future. To satisfy this growing market demand, a scalable and economically sustainable ON production is needed. In this paper, the state of the art of the whole ON production process is illustrated with the aim of highlighting the most promising routes toward the auspicated market-size production. In particular, the most recent advancements in both the upstream stage, mainly based on solid-phase synthesis and recombinant technology, and the downstream one, focusing on chromatographic techniques, are reviewed. Since ON production is projected to expand to the large scale, automatized multicolumn countercurrent technologies will reasonably be required soon to replace the current ones based on batch single-column operations. This consideration is supported by a recent cutting-edge application of continuous chromatography for the ON purification.

Novel multispecific heterodimeric antibody format allowing modular assembly of variable domain fragments.

Multispecific antibody formats provide a promising platform for the development of novel therapeutic concepts that could facilitate the generation of safer, more effective pharmaceuticals. However, the production and use of such antibody-based multispecifics is often made complicated by: 1) the instability of the antibody fragments of which they consist, 2) undesired inter-subunit associations, and 3) the need to include recombinant heterodimerization domains that confer distribution-impairing bulk or enhance immunogenicity. In this paper, we describe a broadly-applicable method for the stabilization of human or humanized antibody Fv fragments that entails replacing framework region IV of a Vκ1/VH3-consensus Fv framework with the corresponding germ-line sequence of a λ-type VL chain. We then used this stable Fv framework to generate a novel heterodimeric multispecific antibody format that assembles by cognate VL/VH associations between 2 split variable domains in the core of the complex. This format, termed multispecific antibody-based therapeutics by cognate heterodimerization (MATCH), can be applied to produce homogeneous and highly stable antibody-derived molecules that simultaneously bind 4 distinct antigens. The heterodimeric design of the MATCH format allows efficient in-format screening of binding domain combinations that result in maximal cooperative activity.

Characterisation of a Novel Anti-CD52 Antibody with Improved Efficacy and Reduced Immunogenicity.

Anti-CD52 therapy has been shown to be effective in the treatment of a number of B cell malignancies, hematopoietic disorders and autoimmune diseases (including rheumatoid arthritis and multiple sclerosis); however the current standard of treatment, the humanized monoclonal antibody alemtuzumab, is associated with the development of anti-drug antibodies in a high proportion of patients. In order to address this problem, we have identified a novel murine anti-CD52 antibody which has been humanized using a process that avoids the inclusion within the variable domains of non-human germline MHC class II binding peptides and known CD4+ T cell epitopes, thus reducing its potential for immunogenicity in the clinic. The resultant humanized antibody, ANT1034, was shown to have superior binding to CD52 expressing cells than alemtuzumab and was more effective at directing both antibody dependent and complement dependent cell cytotoxicity. Furthermore, when in the presence of a cross-linking antibody, ANT1034 was more effective at directly inducing apoptosis than alemtuzumab. ANT1034 also showed superior activity in a SCID mouse/human CD52 tumour xenograft model where a single 1 mg/Kg dose of ANT1034 led to increased mouse survival compared to a 10 mg/Kg dose of alemtuzumab. Finally, ANT1034 was compared to alemtuzumab in in vitro T cell assays in order to evaluate its potential to stimulate proliferation of T cells in peripheral blood mononuclear cells derived from a panel of human donors: whereas alemtuzumab stimulated proliferation in a high proportion of the donor cohort, ANT1034 did not stimulate proliferation in any of the donors. Therefore we have developed a candidate therapeutic humanized antibody, ANT1034, that may have the potential to be more efficacious and less immunogenic than the current standard anti-CD52 therapy.

Tyr(682) in the intracellular domain of APP regulates amyloidogenic APP processing in vivo.

BACKGROUND: The pathogenesis of Alzheimer's disease is attributed to misfolding of Amyloid-ß (Aß) peptides. Aß is generated during amyloidogenic processing of Aß-precursor protein (APP). Another characteristic of the AD brain is increased phosphorylation of APP amino acid Tyr(682). Tyr(682) is part of the Y(682)ENPTY(687) motif, a docking site for interaction with cytosolic proteins that regulate APP metabolism and signaling. For example, normal Aß generation and secretion are dependent upon Tyr(682) in vitro. However, physiological functions of Tyr(682) are unknown. METHODOLOGY/PRINCIPAL FINDINGS: To this end, we have generated an APP Y682G knock-in (KI) mouse to help dissect the role of APP Tyr(682) in vivo. We have analyzed proteolytic products from both the amyloidogenic and non-amyloidogenic processing of APP and measure a profound shift towards non-amyloidogenic processing in APP KI mice. In addition, we demonstrate the essential nature of amino acid Tyr(682) for the APP/Fe65 interaction in vivo. CONCLUSIONS/SIGNIFICANCE: Together, these observations point to an essential role of APP intracellular domain for normal APP processing and function in vivo, and provide rationale for further studies into physiological functions associated with this important phosphorylation site.

Evidence that the Amyloid beta Precursor Protein-intracellular domain lowers the stress threshold of neurons and has a "regulated" transcriptional role.

BACKGROUND: Regulated intramembrane proteolysis of the beta-amyloid precursor protein by the gamma-secretase yields two peptides. One, amyloid-beta, is the major component of the amyloid plaques found in Alzheimer's disease patients. The other, APP IntraCellular Domain, has been involved in regulation of apoptosis, calcium flux and gene transcription. To date, a few potential target genes transcriptionally controlled by AID, alone or complexed with Fe65/Tip60, have been described. Although the reports are controversial: these include KAI1, Neprilysin, p53, EGFR, LRP and APP itself. Furthermore, p53 has been implicated in AID mediated susceptibility to apoptosis. To extend these findings, and assess their in vivo relevance, we have analyzed the expression of the putative target genes and of the total brain basal transriptoma in transgenic mice expressing AID in the forebrain. Also, we have studied the susceptibility of primary neurons from such mice to stress and pro-apoptotic agents. RESULTS: We found that AID-target genes and the mouse brain basal transcriptoma are not influenced by transgenic expression of AID alone, in the absence of Fe65 over-expression. Also, experiments conducted on primary neurons from AID transgenic mice, suggest a role for AID in sensitizing these cells to toxic stimuli. Overall, these findings hint that a role for AID, in regulating gene transcription, could be induced by yet undefined, and possibly stressful, stimuli in vivo. CONCLUSION: Overall, these data suggest that the release of the APP intracellular domain may modulate the sensitivity of neuronal cells to toxic stimuli, and that a transcriptional role of AID could be inscribed in signaling pathways thatare not activated in basal conditions.

GLUT1 and GLUT8 in endometrium and endometrial adenocarcinoma.

Glucose is provided to cells by a family of glucose transport facilitators known as GLUTs. These transporters are expressed in a tissue specific manner and are overexpressed in many primary tumors of these tissues. Regulation of glucose transport facilitator expression has been demonstrated in endometrial tissue and endometrial adenocarcinoma. The following experiments were conducted to quantify and localize the expression of GLUT1 and GLUT8 in benign endometrium and compare this expression to endometrial cancer. Endometrial tissue samples were obtained from random hysterectomy specimens of patients with benign indications for surgery and endometrial cancer. Immunoblot and immunolocatization studies were performed using GLUT1 and GLUT8 specific antisera. Endometrial samples from 65 women who had undergone hysterectomy were examined (n=38 benign, n=27 malignant). A 44 and a 35.4 kDa immunoreacive species was demonstrated in endometrium and endometrial cancer for GLUT1 and GLUT8, respectively. Upregulation of GLUT1 expression was demonstrated with increasing grade of tumors (P<0.002). GLUT8 expression was increased in all tumor subtypes compared to atrophic endometrium (P<0.001). Apical localization by GLUT1 and GLUT8 was demonstrated in endometrial glands. GLUT1 and GLUT8 demonstrated diffuse intracellular localization in the cancer subtypes. GLUT1 and GLUT8 are expressed in both human endometrium and endometrial cancer. There appears to be a step-wise progression in GLUT1 and GLUT8 expression as tumor histopathology worsens. GLUT1 and GLUT8 may be important markers in tumor differentiation, as well as providing energy to rapidly dividing tumor cells.

Positherapy: targeted nuclear therapy of breast cancer with 18F-2-deoxy-2-fluoro-D-glucose.

Breast cancer remains a major cause of cancer death in women in the United States. Novel therapies are needed for patients when standard treatments are ineffective. We have recently shown on a cellular level the therapeutic potential of positrons in malignancy. Here, we report for the first time positron therapy with (18)F-2-deoxy-2-fluoro-D-glucose ((18)F-FDG) in a breast cancer animal model to affect tumor growth rate and survival (positherapy). We used xenografted mammary tumors in nude mice using Notch mammary cancer cells which also express ras oncogene. Notch xenografted tumors actively took up (18)F-FDG with a tumor to normal tissue ratio of 3.24. Tumor-bearing mice were treated with 2.5 mCi (18)F-FDG, which is equivalent to the physiological human maximum tolerated dose. Positherapy resulted in both significant prolongation of survival and decrease in tumor growth rate in comparison with nontreated controls. Immunoblot of Notch tumors showed the presence of glucose transporters (GLUT) 1, 4, and 8. Substantial differences between GLUT1, GLUT4, and GLUT8 were observed in their distribution within the tumor mass. Whereas GLUT4 and GLUT8 were distributed relatively homogeneously throughout the tumor, GLUT1 was confined to necrotic areas. Immunofluorescence double labeling was used to determine cellular localization of GLUTs. GLUT1 was expressed mostly at the cell membrane. GLUT4 and GLUT8 were mostly localized to cytoplasmic compartments with some GLUT4 expressed at or near the cell membrane in close proximity to GLUT1. Thus, GLUT1 was likely responsible for the (18)F-FDG uptake by tumor cells with some possible contribution from GLUT4. These results are important for the development of positherapy with (18)F-FDG for refractory metastatic breast and other cancers.

Implications of glucose transporter protein type 1 (GLUT1)-haplodeficiency in embryonic stem cells for their survival in response to hypoxic stress.

Glucose transporter protein type 1 (GLUT1) is a major glucose transporter of the fertilized egg and preimplantation embryo. Haploinsufficiency for GLUT1 causes the GLUT1 deficiency syndrome in humans, however the embryo appears unaffected. Therefore, here we produced heterozygous GLUT1 knockout murine embryonic stem cells (GT1+/-) to study the role of GLUT1 deficiency in their growth, glucose metabolism, and survival in response to hypoxic stress. GT1(-/-) cells were determined to be nonviable. Both the GLUT1 and GLUT3 high-affinity, facilitative glucose transporters were expressed in GT1(+/+) and GT1(+/-) embryonic stem cells. GT1(+/-) demonstrated 49 +/- 4% reduction of GLUT1 mRNA. This induced a posttranscriptional, GLUT1 compensatory response resulting in 24 +/- 4% reduction of GLUT1 protein. GLUT3 was unchanged. GLUT8 and GLUT12 were also expressed and unchanged in GT1(+/-). Stimulation of glycolysis by azide inhibition of oxidative phosphorylation was impaired by 44% in GT1(+/-), with impaired up-regulation of GLUT1 protein. Hypoxia for up to 4 hours led to 201% more apoptosis in GT1(+/-) than in GT1(+/+) controls. Caspase-3 activity was 76% higher in GT1(+/-) versus GT1(+/+) at 2 hours. Heterozygous knockout of GLUT1 led to a partial GLUT1 compensatory response protecting nonstressed cells. However, inhibition of oxidative phosphorylation and hypoxia both exposed their increased susceptibility to these stresses.