Consistent manufacturing and quality control of a highly complex recombinant polyclonal antibody product for human therapeutic use

The beneficial effect of antibody therapy in human disease has become well established mainly for the treatment of cancer and immunological disorders. The inherent monospecificity of mAbs present limitations to mAb therapy which have become apparent notably in addressing complex entities like infectious agents or heterogenic endogenous targets. For such indications mixtures of antibodies comprising a combination of specificities would convey more potent biological effect which could translate into therapeutic efficacy. Recombinant polyclonal antibodies (rpAb) consisting of a defined number of well-characterized mAbs constitute a new class of target specific antibody therapy. We have developed a cost-efficient cell banking and single-batch manufacturing concept for the production of such products and demonstrate that a complex pAb composition, rozrolimupab, comprising 25 individual antibodies can be manufactured in a highly consistent manner in a scaled-up manufacturing process. We present a strategy for the release and characterization of antibody mixtures which constitute a complete series of chemistry, manufacturing, and control (CMC) analytical methods to address identity, purity, quantity, potency, and general characteristics. Finally we document selected quality attributes of rozrolimupab based on a battery of assays at the genetic-, protein-, and functional level and demonstrate that the manufactured rozrolimupab batches are highly pure and very uniform in their composition.     

Removal of virus from boar semen spiked with porcine circovirus type 2

The virus porcine circovirus type 2 (PCV2) is associated with different disease entities, including reproductive failure. The objective of this study was to investigate the use of a semen processing technique for the elimination of infectious PCV2 in semen. PCV2 was chosen as a model virus because of its small size, high resistance to inactivation and as a known risk factor for boar semen contamination. Aliquots of ejaculates were spiked with PCV2 and processed by a double processing technique, consisting of Single Layer Centrifugation on Androcoll?-P followed by a "swim-up" procedure. Samples were collected from the resulting fractions during the selection process and analyzed for the presence of infectious PCV2. Virus titres were determined by performing a 50% tissue culture infective dose assay (TCID(50)) by end point dilution and with the use of an indirect peroxidise monolayer assay technique. With an initial infectious virus titre of 3.25-3.82 (TCID(50))/50μL the two-step sperm selection method eliminated 2.92±0.23 logs of infectious PCV2, corresponding to more than 99% reduction. Sperm quality was not affected by the selection procedure.     

Agriculturalists and pastoralists: Bronze Age economy of the Murghab alluvial fan,southern Central Asia

Archaeological investigations of pastoral economies often emphasize exchange relations with agricultural populations, though for Bronze Age Eurasia the notion of a ubiquitous ‘pastoral realm’ has masked various forms of mixed subsistence economies. In Central Asia, there are few attempts to specifically identify the domestic crops utilized by mobile pastoralists or what they may suggest about the role of agriculture in mobile pastoral production or subsistence strategies. This study reports the macrobotanical remains from two Late/Final Bronze Age (ca. 1950–1300 bc) mobile pastoralist habitation sites in the Murghab alluvial fan region of southern Turkmenistan. We compare our results with published macrobotanical data from contemporary agricultural settlements in the Murghab region, as well as with other sites in broader prehistoric Eurasia. We find that mobile pastoralists in the Murghab utilized some of the same domestic crops as their sedentary neighbors. While the data presented here do not preclude the possibility that mobile pastoralists may have practiced some low-investment cultivation (particularly of millet), we hypothesize an economic model that places mobile pastoralists in direct contact with nearby sedentary farming communities through exchange for pre-processed grains. These results highlight one of the possible strategies of mobile pastoral subsistence in Central Asia, and are a further step toward identifying the various degrees of agricultural involvement in the conceptually outdated pastoral realm of Eurasia.     

Reduced Expression of the Polymeric Immunoglobulin Receptor in Pancreatic and Periampullary Adenocarcinoma Signifies Tumour Progression and Poor Prognosis

The polymeric immunoglobulin receptor (pIgR) is a key component of the mucosal immune system that mediates epithelial transcytosis of immunoglobulins. High pIgR expression has been reported to correlate with a less aggressive tumour phenotype and an improved prognosis in several human cancer types. Here, we examined the expression and prognostic significance of pIgR in pancreatic and periampullary adenocarcinoma. The study cohort encompasses a consecutive series of 175 patients surgically treated with pancreaticoduodenectomy for pancreatic and periampullary adenocarcinoma in Malmö and Lund University Hospitals, Sweden, between 2001–2011. Tissue microarrays were constructed from primary tumours (n = 175) and paired lymph node metastases (n = 105). A multiplied score was calculated from the fraction and intensity of pIgR staining. Classification and regression tree analysis was used to select the prognostic cut-off. Unadjusted and adjusted hazard ratios (HR) for death and recurrence within 5 years were calculated. pIgR expression could be evaluated in 172/175 (98.3%) primary tumours and in 96/105 (91.4%) lymph node metastases. pIgR expression was significantly down-regulated in lymph node metastases as compared with primary tumours (p = 0.018). Low pIgR expression was significantly associated with poor differentiation grade (p<0.001), perineural growth (p = 0.027), lymphatic invasion (p = 0.016), vascular invasion (p = 0.033) and infiltration of the peripancreatic fat (p = 0.039). In the entire cohort, low pIgR expression was significantly associated with an impaired 5-year survival (HR = 2.99, 95% confidence interval (CI) 1.71–5.25) and early recurrence (HR = 2.89, 95% CI 1.67–4.98). This association remained significant for survival after adjustment for conventional clinicopathological factors, tumour origin and adjuvant treatment (HR = 1.98, 95% CI 1.10–3.57). These results demonstrate, for the first time, that high tumour-specific pIgR expression signifies a more favourable tumour phenotype and that low expression independently predicts a shorter survival in patients with pancreatic and periampullary cancer. The mechanistic basis for the putative tumour suppressing properties of pIgR in these cancers merits further study.     

Management of a semi‐natural grassland; results from a 15‐year‐old experiment in southern Sweden

Abstract. Vegetation changes in a semi‐natural grassland of wooded meadow type that had been grazed for centuries are described following the introduction of various management regimes: mowing each year, mowing every third year, burning, mechanical removal of woody plants, chemical treatment of woody plants, continuous grazing and abandonment. The experiment was established in southern Sweden in 1972 and has been in progress for 15 years. In 1972, 1980 and 1986 the botanical composition in these plots was investigated in permanent subplots. The study clearly demonstrates that mowing or grazing is necessary to preserve community structure and that mowing is to be preferred in cases where maintaining species richness is of primary concern. Mowing every third year delayed vegetation change and prevented woody species from spreading. Therefore, periodic mowing might be an alternative way to preserve the flora. In contrast, yearly burning does not seem to be a viable management in this type of semi‐natural grassland. To preserve the open landscape regeneration of woody plants has to be prevented. However, in plots where woody plants were removed the typical grassland flora declined. Abandonment resulted in closed forest.     

Cell surface orifices of caveolae and localization of caveolin to the necks of caveolae in adipocytes

Caveolae are noncoated invaginations of the plasma membrane that form in the presence of the protein caveolin. Caveolae are found in most cells, but are especially abundant in adipocytes. By high-resolution electron microscopy of plasma membrane sheets the detailed structure of individual caveolae of primary rat adipocytes was examined. Caveolin-1 and -2 binding was restricted to the membrane proximal region, such as the ducts or necks attaching the caveolar bulb to the membrane. This was confirmed by transfection with myc-tagged caveolin-1 and -2. Essentially the same results were obtained with human fibroblasts. Hence caveolin does not form the caveolar bulb in these cells, but rather the neck and may thus act to retain the caveolar constituents, indicating how caveolin participates in the formation of caveolae. Caveolae, randomly distributed over the plasma membrane, were very heterogeneous, varying in size between 25 and 150 nm. There was about one million caveolae in an adipocyte, which increased the surface area of the plasma membrane by 50%. Half of the caveolae, those larger than 50 nm, had access to the outside of the cell via ducts and 20-nm orifices at the cell surface. The rest of the caveolae, those smaller than 50 nm, were not open to the cell exterior. Cholesterol depletion destroyed both caveolae and the cell surface orifices.     

Structural properties of recombinant nonrepetitive and repetitive parts of major ampullate spidroin 1 from Euprosthenops australis: implications for fiber formation

Spider dragline silk proteins, spidroins, have a tripartite composition; a nonrepetitive N-terminal domain, a central repetitive region built up from many iterated poly-Ala and Gly rich blocks, and a C-terminal nonrepetitive domain. It is generally believed that the repetitive region forms intermolecular contacts in the silk fibers, while precise functions of the terminal domains have not been established. Herein, thermal, pH, and salt effects on the structure and aggregation and/or polymerization of recombinant N- and C-terminal domains, a repetitive segment containing four poly-Ala and Gly rich coblocks, and combinations thereof were studied. The N- and C-terminal domains have mainly alpha-helical structure, and interestingly, both form homodimers. Dimerization of the end domains allows spidroin multimerization independent of the repetitive part. Reduction of an intersubunit disulfide in the C-terminal domain lowers the thermal stability but does not affect dimerization. The repetitive region shows helical secondary structure but appears to lack stable folded structure. A protein composed of this repetitive region linked to the C-terminal domain has a mainly alpha-helical folded structure but shows an abrupt transition to beta-sheet structures upon heating. At room temperature, this protein self-assembles into macroscopic fibers within minutes. The secondary structures of none of the domains are altered by pH or salt. However, high concentrations of phosphate affect the tertiary structure and accelerate the aggregation propensity of the repetitive region. Implications of these results for dragline spidroin behavior in solution and silk fiber formation are discussed.     

A sequence-specific function for the N-terminal signal-like sequence of the TonB protein

TonB is a proline-rich protein which provides a functional link between the inner and outer membranes of Gram-negative bacteria. TonB is anchored to the inner membrane via an N-terminal signal-like sequence and spans the periplasm, interacting with transport receptors in the outer membrane. We have investigated the role of the N-terminal signal-like peptide in TonB function. Replacement of the N-terminal sequence with heterologous sequences indicates that it has at least three distinct rotes in TonB function: (i) to facilitate translocation of TonB across the cytoplasmic membrane; (ii) to anchor TonB to the cytoplasmic membrane; (iii) a sequence-specific functional interaction with the ExbBD proteins.     

Kinetics of the H-ATPase in chromatophores from Rhodospirillum rubrum: Effect of the electrical transmembrane potential on the rate constants

The effect of the electrical potential on the H⁺-ATPase of Rhodospirillum rubrum is examined. It is shown that the forward reaction rate (ATP synthesis) is increased by a factor of 10 during illumination while the reversed rate is only slightly decreased. This indicates that the electrical potential across the membrane affects the rate constants mainly by increasing the forward rate constants rather than decreasing the reversed rate constants in order to go from net hydrolysis to net synthesis.