A robust robotic high-throughput antibody purification platform.

Monoclonal antibodies (mAbs) have become the fastest growing segment in the drug market with annual sales of more than 40 billion US$ in 2013. The selection of lead candidate molecules involves the generation of large repertoires of antibodies from which to choose a final therapeutic candidate. Improvements in the ability to rapidly produce and purify many antibodies in sufficient quantities reduces the lead time for selection which ultimately impacts on the speed with which an antibody may transition through the research stage and into product development. Miniaturization and automation of chromatography using micro columns (RoboColumns(®) from Atoll GmbH) coupled to an automated liquid handling instrument (ALH; Freedom EVO(®) from Tecan) has been a successful approach to establish high throughput process development platforms. Recent advances in transient gene expression (TGE) using the high-titre Expi293F™ system have enabled recombinant mAb titres of greater than 500mg/L. These relatively high protein titres reduce the volume required to generate several milligrams of individual antibodies for initial biochemical and biological downstream assays, making TGE in the Expi293F™ system ideally suited to high throughput chromatography on an ALH. The present publication describes a novel platform for purifying Expi293F™-expressed recombinant mAbs directly from cell-free culture supernatant on a Perkin Elmer JANUS-VariSpan ALH equipped with a plate shuttle device. The purification platform allows automated 2-step purification (Protein A-desalting/size exclusion chromatography) of several hundred mAbs per week. The new robotic method can purify mAbs with high recovery (>90%) at sub-milligram level with yields of up to 2mg from 4mL of cell-free culture supernatant.

Outcomes of resection of extra-adrenal pheochromocytomas/paragangliomas in the laparoscopic era: a comparison with adrenal pheochromocytoma.

INTRODUCTION: Laparoscopic adrenalectomy (LA) is the standard for removal of adrenal pheochromocytomas (pheos), but laparoscopic (LAP) resection of paragangliomas (PGs) is controversial. This study analyzes our results of resection of PGs in the LAP era. METHODS: A retrospective record review of all patients who underwent resection of intra-abdominal PGs from 1998 to 2011 was performed. Pre- and postoperative clinical, radiologic, biochemical, and pathologic data for LAP resection of PGs were compared with patients who underwent LA for adrenal pheo (LA pheo; n = 62). Statistical analysis was performed and data are reported as mean ± SD. RESULTS: Fifteen patients had resection of PGs (6 OPEN, 9 LAP) and 62 had LA pheo. Most common PG locations were perirenal or renal hilum (n = 6) and para-aortic (n = 4). One LAP PG was converted to OPEN due to inflammation from a prior biopsy. Mean age of LAP PGs was 45.3 ± 13.2 years, and mean tumor size was 3.3 ± 2.1 cm. OPEN PGs were larger (5.1 vs. 3.3 cm), had shorter operative times (173 vs. 254 min), and longer hospitalization (5.7 vs. 2.6 days) and ICU stays (1.33 vs. 0.22 days) compared with LAP PGs (p ≤ 0.05). Compared with LA pheo, operative times for LAP PG were significantly longer (254 vs. 175 min, p = 0.001) but other outcomes were similar. Complications occurred in 5.9 % of LA pheos, 22 % of LAP PGs and 67 % of OPEN PGs. CONCLUSIONS: Patients with paragangliomas can safely benefit from LAP resection with outcomes similar to adrenal pheos. In the absence of a need for contiguous organ resection, LAP resection of paragangliomas seems to be the preferred surgical approach.

Physicomechanical evaluation of absorbable and nonabsorbable barrier composite meshes for laparoscopic ventral hernia repair.

BACKGROUND: This study aimed to compare the physicomechanical properties of composite prostheses for laparoscopic ventral hernia repair (LVHR) through standard testing and a proposed classification system. METHODS: Seven prostheses (four with absorbable barriers and 3 with nonabsorbable barriers) were evaluated. The barrier layer was removed, after which the area of the interstices and the diameter of the filaments were determined. The barrier layer was left intact during thickness, density, suture retention strength, tear resistance, uniaxial tensile, and ball-burst testing. Specimens were oriented parallel or perpendicular to their longest dimension during testing. One-way analysis of variance (ANOVA) with Tukey's posttest or an unpaired, two-tailed t-test was performed to determine whether differences existed due to mesh or orientation, and a p value<0.05 was considered significant. RESULTS: Significant differences were observed between mesh types and due to the orientation of the mesh during testing. Of the absorbable barrier meshes, Bard Sepramesh IP Composite demonstrated the greatest suture retention and tear strengths, followed by C-QUR mesh. Of the permanent barrier meshes, DUALMESH demonstrated the greatest suture retention strength in the perpendicular direction, followed by Bard Composix E/X. DUALMESH and Bard Composix E/X demonstrated equivalent suture retention strength in the parallel direction and equivalent tear resistance in both testing directions. All meshes demonstrated tensile strengths greater than the physiologically relevant range of 16-32 N/cm. CONCLUSIONS: This study provided a basic understanding of how the structural aspects of each mesh design influence functionality. Differences between composite barrier prostheses commonly used for LVHR were observed due to barrier type, mesh type, and orientation. A set of standard testing techniques and a classification system also were presented to define fully the properties of these materials.

Physicomechanical evaluation of polypropylene, polyester, and polytetrafluoroethylene meshes for inguinal hernia repair.

BACKGROUND: For meshes to be used effectively for hernia repair, it is imperative that engineers and surgeons standardize the terminology and techniques related to physicomechanical evaluation of these materials. The objectives of this study were to propose standard techniques, perform physicomechanical testing, and classify materials commonly used for inguinal hernia repair. STUDY DESIGN: Nine meshes were evaluated: 4 polypropylene, 1 polyester, 1 polytetrafluoroethylene, and 3 partially absorbable. Physical properties were determined through image analysis, laser micrometry, and density measurements. Biomechanical properties were determined through suture retention, tear resistance, uniaxial, and ball burst testing with specimens tested in 2 different orientations. A 1-way ANOVA with Tukey's post-test or a t-test were performed, with p < 0.05. RESULTS: Significant differences were observed due to both mesh type and orientation. Areas of interstices ranged from 0.33 ± 0.01 mm² for ProLite (Atrium Medical Corp) and C-QUR Lite (Atrium Medical Corp) Large to 4.10 ± 0.06 mm² for ULTRAPRO (Ethicon), and filament diameters ranged from 99.00 ±8.1 µm for ProLite Ultra (Atrium Medical Corp) and C-QUR Lite Small to 338.8 ± 3.7 µm for Parietex Flat Sheet TEC (Covidien). These structural characteristics influenced biomechanical properties such as tear resistance and tensile strength. ProLite Ultra, C-QUR Lite Small, ULTRAPRO and INFINIT (WL Gore & Associates) did not resist tearing as effectively as the others. All meshes exhibited supraphysiologic burst strengths except INFINIT and ULTRAPRO. CONCLUSIONS: Significant differences exist between the physicomechanical properties of polypropylene, polyester, polytetrafluoroethylene, and partially absorbable mesh prostheses commonly used for inguinal hernia repair. Orientation of the mesh was also shown to be critical for the success of meshes, particularly those demonstrating anisotropy.

Population-based detection of Lynch syndrome in young colorectal cancer patients using microsatellite instability as the initial test.

Approximately 1-2% of colorectal cancers (CRC) arise because of germline mutations in DNA mismatch repair genes, referred to as Lynch syndrome. These tumours show microsatellite instability (MSI) and loss of expression of mismatch repair proteins. Pre-symptomatic identification of mutation carriers has been demonstrated to improve survival; however, there is concern that many are not being identified using current practices. We evaluated population-based MSI screening of CRC in young patients as a means of ascertaining mutation carriers. CRC diagnosed in patients aged <60 years were identified from pathology records. No prior information was available on family history of cancer. PCR techniques were used to determine MSI in the BAT-26 mononucleotide repeat and mutation in the BRAF oncogene. Loss of MLH1, MSH2, MSH6 and PMS2 protein expression was evaluated in MSI+ tumours by immunohistochemistry. MSI+ tumours were found in 105/1,344 (7.8%) patients, of which 7 were excluded as possible Lynch syndrome because of BRAF mutation. Of the 98 "red flag" cases that were followed up, 25 were already known as mutation carriers or members of mutation carrier families. Germline test results were obtained for 35 patients and revealed that 22 showed no apparent mutation, 11 showed likely pathogenic mutations and 2 had unclassified variants. The proportion of MSI+ cases in different age groups that were estimated to be mutation carriers was 89% (<30 years), 83% (30-39), 68% (40-49) and 17% (50-59). We recommend MSI as the initial test for population-based screening of Lynch syndrome in younger CRC patients, regardless of family history.

The expression of tumor necrosis factor-alpha, its receptors and steroidogenic acute regulatory protein during corpus luteum regression.

BACKGROUND: Corpus luteum (CL) regression is known to occur as two parts; functional regression when steroidogenesis declines and structural regression when apoptosis is induced. Previous studies suggest this process occurs by the production of luteolytic factors, such as tumour necrosis factor-alpha (TNF-alpha). METHODS: We examined TNF-alpha, TNF-alpha receptors (TNFR1 and 2) and steroidogenic acute regulatory (StAR) protein expression during CL regression in albino Wistar rats. CL from Days 16 and 22 of pregnancy and Day 3 post-partum were examined, in addition CL from Day 16 of pregnancy were cultured in vitro to induce apoptosis. mRNA was quantitated by kinetic RT-PCR and protein expression examined by immunohistochemistry and Western blot analyses. RESULTS: TNF-alpha mRNA increased on Day 3 post-partum. TNFR were immunolocalized to luteal cells, and an increase in TNFR2 mRNA observed on Day 3 post-partum whilst no change was detected in TNFR1 mRNA relative to Day 16. StAR protein decreased on Day 3 post-partum and following trophic withdrawal but no change was observed following exogenous TNF-alpha treatment. StAR mRNA decreased on Day 3 post-partum; however, it increased following trophic withdrawal and TNF-alpha treatment in vitro. CONCLUSION: These results demonstrate the existence of TNFR1 and TNFR2 in rat CL and suggest the involvement of TNF-alpha in rat CL regression following parturition. Furthermore, decreased StAR expression over the same time points was consistent with the functional regression of the CL.

Investigating copper-regulated protein expression in Menkes fibroblasts using antibody microarrays.

Neurodegenerative illnesses are characterized by aberrant metabolism of biometals such as copper (Cu), zinc (Zn) and iron (Fe). However, little is known about the metabolic effects associated with altered metal homeostasis. In this study, we used an in vitro model of altered Cu homeostasis to investigate how Cu regulates cellular protein expression. Human fibroblasts containing a natural deletion mutation of the Menkes (MNK) ATP7A Cu transporter (MNK deleted) were compared to fibroblasts overexpressing ATP7A (MNK transfected). Cultures of MNK-transfected (Low-Cu) cells exhibited 95% less intracellular Cu than MNK-deleted (High-Cu) cells. Comparative proteomic analysis of the two cell-lines was performed using antibody microarrays, and significant differential protein expression was observed between Low-Cu and High-Cu cell-lines. Western blot analysis confirmed the altered protein expression of Ku80, nexilin, L-caldesmon, MAP4, Inhibitor 2 and DNA topoisomerase I. The top 50 altered proteins were analysed using the software program Pathway Studio (Ariadne Genomics) and revealed a significant over-representation of proteins involved in DNA repair and maintenance. Further analysis confirmed that expression of the DNA repair protein Ku80 was dependent on cellular Cu homeostasis and that Low-Cu levels in fibroblasts resulted in elevated susceptibility to DNA oxidation.

Development of a cluster of differentiation antibody-based protein microarray.

Protein microarrays combine aspects of DNA microarrays and ELISA for the parallel interrogation of a biological sample using a multiplex of protein biomarkers. Here we report the development of a protein microarray consisting of a subset of CD antibodies and CRP. Several preparations (culture supernatant, ascites fluid and purified Ig) of each antibody were used in a forward phase protein microarray. Microarrays were fabricated using a non-contact printer delivering 300 pL (+/-30 pL) to specific locations on polyacrylamide gel-based substrates. Following production, microarrays were blocked for non-specific binding and incubated with sera conjugated directly with Cy3. Using CRP as a control biomarker, 12 clinical samples (inflammatory conditions and controls) were interrogated using the protein microarray format and results compared to CRP measured by conventional immunoassay. The data obtained from the microarray correlated with CRP assessed by immunoassay. Subsequently CRP 'positive' samples were interrogated for CD antigen expression; which revealed CD25 and CD45RO expression in all samples. Whilst this study focussed on a subset of CD antibodies, it is anticipated that this array could be expanded to include a larger number of CD antibodies and allow screening of sera from multiple conditions in order to identify disease markers.

Role of tumor necrosis factor-alpha and the modulating effect of the caspases in rat corpus luteum apoptosis.

Tumor necrosis factor-alpha (TNFalpha) is a pleiotropic cytokine that has been implicated in apoptosis of many cell systems. However, the signal transduction of TNFalpha during the structural and functional regression of the corpus luteum (CL) is largely unknown. In this study, we investigate the role of TNFalpha in rat CL apoptosis and the involvement of monocyte chemoattractant protein-1 (MCP-1) and the modulating effect of the caspases in this process. An in vivo study of CL during pregnancy and postpartum using immunohistochemistry and Western blot analysis indicated that increases in TNFalpha correspond with luteal apoptosis approaching term (Day 22) and at postpartum (Day 3). CL apoptosis was further investigated using a whole-CL culture model of tropic withdrawal. An increase was observed in both low molecular weight (MW) DNA fragmentation and TUNEL staining from 0 h to 8 h in culture. CL apoptosis in vitro was associated with increased protein expression of both TNFalpha and MCP-1 as measured by immunohistochemistry and Western blot analysis. Using a whole-CL culture model, apoptosis was induced in vitro by TNFalpha as demonstrated by a dose-dependent increase in DNA fragmentation. Treatment of luteal cells with TNFalpha and both specific caspase inhibitors (Z-DEVD-FMK, Z-VEID-FMK, Z-IETD-FMK) or a general caspase inhibitor (Boc-D-FMK) prevented the effect of TNFalpha. CL regression involves the apoptotic deletion of luteal cells; the results of this study suggest that TNFalpha is possibly involved in this process. The observed increases in MCP-1 expression suggest the coordination of TNFalpha expression with the infiltration and activation of macrophages. Furthermore, the results demonstrate the importance of the caspases in the TNFalpha signal transduction pathway and suggest a hierarchy within the caspase family.