Do Plant Cells Secrete Exosomes Derived from Multivesicular Bodies?

Multivesicular bodies (MVBs) are spherical endosomal organelles containing small vesicles formed by inward budding of the limiting membrane into the endosomal lumen. In mammalian red cells and cells of immune system, MVBs fuse with the plasma membrane in an exocytic manner, leading to release their contents including internal vesicles into the extracellular space. These released vesicles are termed exosomes. Transmission electron microscopy studies have shown that paramural vesicles situated between the plasma membrane and the cell wall occur in various cell wall-associated processes and are similar to exosomes both in location and in morphology. Our recent studies have revealed that MVBs and paramural vesicles proliferate when cell wall appositions are rapidly deposited beneath fungal penetration attempts or during plugging of plasmodesmata between hypersensitive cells and their intact neighboring cells. This indicates a potential secretion of exosome-like vesicles into the extracellular space by fusion of MVBs with the plasma membrane. This MVB-mediated secretion pathway was proposed on the basis of pioneer studies of MVBs and paramural vesicles in plants some forty years ago. Here, we recall the attention to the occurrence of MVB-mediated secretion of exosomes in plants.Key Words: cell wall, endocytosis, endosome, exocytosis, exosome, multivesicular body, paramural bodyMultivesicular bodies (MVBs) are spherical endosomal organelles containing a number of small vesicles formed by inward budding of the limiting membrane into the endosomal lumen.¹ MVBs contain endocytosed cargoes and deliver them into lysosomal/vacuolar compartments for degradation. They also incorporate newly synthesized proteins destined for lysosomal/vacuolar compartments.² In mammalian cells of hematopoietic origin, endosomal MVBs function in removal of endocytosed surface proteins in an exocytic manner. They are redirected to the plasma membrane, where they release their contents including internal vesicles into the extracellular space by membrane fusion. The released vesicles are termed exosomes.³ During reticulocyte maturation to erythrocyte, a group of surface proteins, such as the transferrin receptor, become obsolete and are discarded via MVB-mediated secretion.³ Time-course transmission electron microscopy (TEM) first revealed that colloidal gold-transferrin was internalized into MVBs via receptor-mediated endocytosis and then transferrin together with its receptor were delivered into the extracellular space via the fusion of MVBs with the plasma membrane of reticulocytes.⁴ Some other cell types of hematopoietic origin, such as activated platelets, cytotoxic T cells and antigen-presenting cells, also secrete exosomes. Exosomes thus may play a role in various physiological processes other than discarding obsolete proteins.³Our recent TEM studies provided ultrastructural evidence on the enhanced vesicle trafficking in barley leaf cells attacked by the biotrophic powdery mildew fungus. Multivesicular compartments including MVBs, intravacuolar MVBs, and paramural bodies turned out to proliferate in intact host cells during formation of cell wall appositions (papilla response), in the hypersensitive response, and during accommodation of haustoria.^5,6 MVBs proliferated in the cytoplasm of haustorium-containing epidermal cells during compatible interactions and near sites of cell wall-associated oxidative microburst either during the papilla response or during the hypersensitive response. Because MVBs in plant cells have been demonstrated to be endosomal compartments,^7–9 they may participate in internalization of nutrients from the apoplast of intact haustorium-containing epidermal cells and sequestration of damaged membranes and deleterious materials originating from the oxidative microburst.^5,6 The presence of intravacuolar MVBs with double limiting membranes (Fig. 1A) indicates an engulfment of MVBs by the tonoplast and a vacuole-mediated autophagy of MVBs.^5,6 MVBs, as prevacuolar compartments in plant cells,⁹ thus probably deliver their contents into the central vacuole via both the fusion with the tonoplast and the engulfment by the tonoplast (Fig. 2A and B). On the other hand, paramural bodies, in which small vesicles are situated between the cell wall and the plasma membrane, were associated with cell wall appositions deposited beneath fungal penetration attempts (Fig. 1B) or around hypersensitive cells including sites of plugged plasmodesmata (Fig. 1C and D).^5,6 Because paramural vesicles are similar to exosomes both in location and in morphology, we speculated that MVBs fuse with the plasma membrane in an exocytic manner to form paramural bodies.^5,6 Endocytosed cell surface materials in endosomal MVBs may be reused and delivered together with newly synthesized materials in Golgi apparatus-derived vesicles to cell wall appositions, which are deposited rapidly to prevent fungal penetration (Fig. 2A) or to contain hypersensitive cell death (Fig. 2B). MVBs thus may be driven along two distinct pathways to deliver their contents into either central vacuole or extracellular space.Open in a separate windowFigure 1Multivesicular compartments in intact cells in barley leaves attacked by the barley powdery mildew fungus. (A) An intravacuolar multivesicular body (MVB) with double limiting membranes in an intact epidermal cell (EC) adjacent to a hypersensitive epidermal cell (EC*). The arrows point to the outer limiting membrane, which is seemingly derived from the tonoplast. Note that neighboring intravacuolar vesicles (in between two arrowheads) may result from degradation of double limiting membranes of intravacuolar MVBs or may be delivered into the vacuole by MVB-fusion with the tonoplast. (B) Paramural vesicles (arrowheads) in a paramural body associated with cell wall appositions (asterisk) deposited by an intact epidermal cell. (C) A multivesicular body (MVB) in contact with a paramural body (PMB) (a nonmedian section) associated with cell wall appositions (asterisk) deposited by an intact mesophyll cell adjacent to a hypersensitive mesophyll cell. Note that cell wall appositions deposit beside an intercellular space (IS). The arrows point to the tonoplast. (D) A paramural body (PMB) associated with cell wall appositions (asterisks) blocking plasmodesmata (in between two arrowheads) at the side of an intact mesophyll cell (MC) underlying a hypersensitive epidermal cell (EC*). The arrows point to the tonoplast. CV, central vacuole; CW, cell wall; MB, microbody. Bars, 1µm.Open in a separate windowFigure 2Hypothetical diagram of delivery of endocytosed cell surface materials via MVBs into the central vacuole or the extracellular space where intact barley cells deposit cell wall appositions. (A) Deposition of cell wall appositions (asterisk) beneath powdery mildew penetration attempts. AGT, appressorial germ tube; PP, penetration peg. (B) Deposition of cell wall appositions (asterisks) against constricted plasmodesmata (PD) between a hypersensitive epidermal cell (EC) penetrated by the powdery mildew fungus and an underlying mesophyll cell (MC). H, haustorium. Arrows and numbers show pathways of vesicle trafficking. 1, Secretion of Golgi-derived vesicles containing newly synthesized materials; G, Golgi body; TGN, trans-Golgi network; 2, Endocytosis of cell surface materials from coated pits (coated open circles) via coated vesicles (coated circles) to multivesicular bodies (MVB); 3, Delivery of endocytosed materials for degradation inside the central vacuole (CV) via membrane fusion between MVBs and the tonoplast (T); small broken circles, vesicles in degradation; 4, Delivery of endocytosed materials for degradation inside the central vacuole via engulfment of MVBs by the tonoplast; large broken circles; MVB limiting membranes in degradation; 5, delivery of endocytosed materials into the extracellular space for deposition of cell wall appositions (asterisks) via membrane fusion between MVBs and the plasma membrane (PM). CW, cell wall; PMB, paramural body. PD0, 1, 2, 3 and 4 represent stages of plugging plasmodesmata. PD0, open plasmodesmata between two intact mesophyll cells (MC) subjacent to the hypersensitive epidermal cell (EC); PD1, constriction of plasmodesmata by callose (grey dots) deposition at plasmodesmal neck region; PD2, constricted plasmodesmata associated with plasmodesma-targeted secretion; PD3, further blocking of plasmodesmata by deposition of cell wall appositions; PD4, completely blocked plasmodesmata.Earlier than the discovery in animal cell systems,⁴ it was proposed in two independent papers in 1967 that the fusion of MVBs with the plasma membrane might result in the release of small vesicles into the extracellular space in fungi and in higher plants.^10,11 Several lines of evidence support the occurrence of MVB-mediated secretion of exosome-like vesicles in plants. First, vesicles of the same morphology as MVB internal vesicles have been observed in extracellular spaces or paramural spaces in various types of plant cells in various plant species by TEM.¹² An early study on endocytosis by soybean protoplasts also showed small extracellular vesicles attaching on the plasma membrane.⁸ Second, cooccurrence of MVBs and paramural vesicles has been observed in processes of cell proliferation, cell differentiation, and cell response to abiotic and biotic stress. Examples are cell plate formation,^13,14 secondary wall thickening,^15,16 cold hardness,^17,18 and deposition of cell wall appositions upon pathogen attack.^5,6,19–21 Third, identical molecular components, such as arabinogalactan proteins^22,23 and peroxidases,⁶ have been immunolocalized in both MVBs and paramural bodies. Despite these pieces of evidence, a conclusive demonstration of MVB-mediated secretion of exosomes in plants requires further exploration.The presently available experimental systems, approaches, and membrane markers may allow future demonstration of MVB-mediated secretion of exosomes in plants. Recent in vivo real-time observation and colocalization of cell surface and endosomal markers have already revealed that endosomes filled with endocytosed preexisting cell wall and plasma membrane materials are rapidly delivered to cytokinetic spaces to form cell plates in dividing tobacco, Arabidopsis, and maize cells.²⁴ Because TEM observed paramural bodies attaching to cell plates¹³ and MVBs in the vicinity of cell plates during all stages of cell plate formation,^14,25,26 MVBs and paramural bodies may participate in delivery of endocytosed building blocks to cell plates. Jiang''s and Robinson''s labs together developed a transgenic tobacco BY-2 cell line stably expressing a YFP-labeled vacuolar sorting receptor protein and antibodies against the vacuolar sorting receptor protein localized to the limiting membrane of MVBs.⁹ These tools together with live cell imaging and immunoelectron microscopy may allow visualization of MVB-fusion to the new plasma membrane, of vacuolar sorting receptors in both the limiting membrane of MVBs and the new plasma membrane, and of identical cell plate components in both internal vesicles of MVBs and paramural vesicles.In spite of obvious differences in plant and animal cytokinesis, the generation of cell plates by cell-plate-directed fusion of endosomes resembles the plugging of midbody canals by midbody-directed endosomes to separate daughter cells at the terminal phase of animal cytokinesis.²⁷ Likely, functional similarities of the fusion between endosomal MVBs and the plasma membrane to eliminate unwanted cell contents may also exist in maturation of mammalian red blood cells and plant sieve elements in the sense that the fusion of MVBs with the plasma membrane may occur during maturation of the latter.²⁸ On the other hand, although plant cells may secrete MVB-derived exosomes in defense response upon pathogen attack,^5,6 plant cell walls rule out the direct intercellular communication during the immune response mediated by exosomes in the circulation of mammals.³ In contrast, plasmodesma-directed secretion of exosomes would block the cell-to-cell communication between hypersensitive cells and their neighboring cells during hypersensitive response.⁵ Further exploration will lead us to a better understanding of similarities and differences of exosome secretion between plants and animals.     

Frasnian vertebrate taphonomy and sedimentology of macrofossil concentrations from the Langsēde Cliff,Latvia

Luk?evi?s, E., Ahlberg, P.E., Stinkulis, ?., Vasi?kova, J. & Zupi??, I. 2011: Frasnian vertebrate taphonomy and sedimentology of macrofossil concentrations from the Langsēde Cliff, Latvia. Lethaia, Vol. 45, pp. 356–370. The siliciclastic sequence of the Upper Devonian of Kurzeme, Western Latvia, is renowned for abundant vertebrate fossils, including the stem tetrapods Obruchevichthys gracilis and Ventastega curonica. During the first detailed taphonomic study of the vertebrate assemblage from the Ogre Formation cropping out at the Langsēde Cliff, Imula River, abundant vertebrate remains have been examined and identified as belonging to one psammosteid, two acanthodian and three sarcopterygian genera; the placoderm Bothriolepis maxima dominates the assemblage. Besides fully disarticulated placoderm and psammosteid plates, separate sarcopterygian scales and teeth, and acanthodian spines, partly articulated specimens including complete distal segments of Bothriolepis pectoral fins, Bothriolepis head shields and sarcopterygian lower jaws have been found. The size distribution of the placoderm bones demonstrates that the individuals within the assemblage are of approximately uniform age. Distinct zones have been traced within the horizontal distribution of the bones. These linear zones are almost perpendicular to the dominant dip azimuth of the cross‐beds and ripple‐laminae and most probably correspond to the depressions between subaqueous dunes. Concavity ratio varies significantly within the excavation area. The degree of fragmentation of the bones and disarticulation of the skeletons suggest that the carcasses were reworked and slightly transported before burial. Sedimentological data suggest deposition in a shallow marine environment under the influence of rapid currents. The fossiliferous bed consists of a basal bone conglomerate covered by a cross‐stratified sandstone with mud drapes, which is in turn overlain by ripple laminated sandstone, indicating the bones were buried by the gradual infilling of a tidal channel. All the Middle–Upper Devonian vertebrate bone‐beds from Latvia are associated with sandy to clayey deposits and have been formed in a sea‐coastal zone during rapid sedimentation episodes, but differ in fossil abundance and degree of preservation. □Agnathans, Devonian, facies analysis, fish, fossil assemblage, palaeoenvironment.     

Nutridynamics--studying the dynamics of food components in products and in the consumer

The concentrations and biological effects of nutrients, antinutrients and bioactive compounds, including microbes and their constituents, are affected by production and processing steps, the food matrix in which they reside, the way they are digested and metabolized in the human body, and whether or not and in what form they subsequently reach their target site. A new scientific concept, denoted here as 'nutridynamics', aims to unravel the dynamics of these processes by using a systematic approach to study how a food component is affected by the food matrix itself and what it does in the body. This holistic concept has potential synergy with the areas of food technology and nutrigenomics, and provides a link between food production and the mechanistic effects of bioactive ingredients.     

Spatial and temporal population genetic structure of four northeastern Pacific littorinid gastropods: the effect of mode of larval development on variation at one mitochondrial and two nuclear DNA markers

We investigated the effect of development mode on the spatial and temporal population genetic structure of four littorinid gastropod species. Snails were collected from the same three sites on the west coast of Vancouver Island, Canada in 1997 and again in 2007. DNA sequences were obtained for one mitochondrial gene, cytochrome b ( Cyt b ), and for up to two nuclear genes, heat shock cognate 70 ( HSC70 ) and aminopeptidase N intron ( APN54 ). We found that the mean level of genetic diversity and long-term effective population sizes ( N _e) were significantly greater for two species, Littorina scutulata and L. plena , that had a planktotrophic larval stage than for two species, Littorina sitkana and L. subrotundata , that laid benthic egg masses which hatched directly into crawl-away juveniles. Predictably, two poorly dispersing species, L. sitkana and L. subrotundata , showed significant spatial genetic structure at an 11- to 65-km geographical scale that was not observed in the two planktotrophic species. Conversely, the two planktotrophic species had more temporal genetic structure over a 10-year interval than did the two direct-developing species and showed highly significant temporal structure for spatially pooled samples. The greater temporal genetic variation of the two planktotrophic species may have been caused by their high fecundity, high larval dispersal, and low but spatially correlated early survivorship. The sweepstakes-like reproductive success of the planktotrophic species could allow a few related females to populate hundreds of kilometres of coastline and may explain their substantially larger temporal genetic variance but lower spatial genetic variance relative to the direct-developing species.     

Can the failure to punish promote cheating in mutualism?

A key challenge in the study of mutualism is to understand the mechanisms that prevent cheating. In some systems, host retaliation against cheaters prevents the breakdown of cooperation. Here, we focus on the converse of this demonstration, and ask whether hosts that fail to retaliate are commonly inhabited by cheaters. We do so using the classic ant–plant interaction, in which plants provide ant-housing (domatia) in return for protection from herbivores. Our model system is the rattan ant-palm Korthalsia furtadoana , which grows swollen leaf sheaths as domatia and associates with two species of obligate host-ants, Camponotus sp90 and C. sp93 , and with facultative Crematogaster and 'tramp' ant species. One ant-tree species is known to retaliate by tying the growth of domatia to the successful protection of new leaves, and non-protecting cheaters are rare. In contrast, K. furtadoana grows the domatium before the new leaf develops, suggesting that sanctioning may not be possible. We experimentally simulated herbivory by cutting leaves from shoots and found no difference in the mortality and growth of domatia on such 'cheated' shoots than on controls, confirming that K. furtadoana cannot sanction non-protectors. We then investigated the intensity of protection that Camponotus and Crematogaster ant-symbionts provide K. furtadoana . We demonstrate that C. sp90 , which only inhabit half of colonised plants, vigorously protects leaves, that C. sp93 rarely protects, and that Crematogaster never protects. We then show that plants inhabited by C. sp90 have a higher growth rate than those inhabited by C. sp93 . We conclude that C. sp90 is a protection mutualist, while C. sp93 and Crematogaster are parasites, the first such demonstrations for an ant–palm interaction. The presence of commonly occurring parasites, as well as rare tramp ants, provides the first clear correlative evidence that an inability to punish results in abundant cheating.     

One-year clinical follow-up of a registry evaluating a percutaneous revascularisation strategy combining a pre-specified simple selection process with the use of a new thin-strut bare cobalt-chromium stent

Objectives. To evaluate clinical events in a specifically selected cohort of patients with obstructive coronary artery disease (CAD), using a new generation thin-strut bare cobalt-chromium coronary stent. Methods. Patients with single- or multi-vessel, stable or unstable CAD eligible for percutaneous implantation of at least one bare cobalt-chromium stent were evaluated in a single-centre registry. Prospective pre-specified criteria for bare cobalt-chromium stent implantation in our centre were: any acute ST-elevation myocardial infarction (MI), otherwise 1) de novo coronary lesion, and 2) lesion length <20 mm, and 3) reference vessel diameter >2.6 mm, and 4) no diabetes, unless reference vessel diameter >3.5 mm. Endpoints, retrospectively collected, were death, MI and clinically driven target-lesion revascularisation (TLR) and target-vessel revascularisation (TVR) after 12 months. Results. Between September 2005 and June 2007, 712 patients (48.7% one-vessel, 29.9% two-vessel, 20% three-vessel and 1.4% left main disease; 7.9% diabetics) were treated with 800 bare cobalt-chromium stents, for stable angina (40.9%), unstable angina (20.9%) or acute ST-elevation MI (38.2%). The procedural success rate was 99.3%. Peri-procedural MI rate was 2.2% in the semi-elective group. At 12 months there were 17 deaths (2.4%), of which nine non-cardiac, 20 (2.8%) MI, 19 (2.7%) TLR and 29 (4.1%) TVR. Early and late definite stent thrombosis occurred in four (0.6%) and three (0.4%) patients, respectively. Conclusion. A strategy aimed at minimising drug-eluting stent use and combining a pre-specified simple selection process with the use of a new thin-strut bare cobalt-chromium stent is safe and effective at one-year clinical follow-up. (Neth Heart J 2010;18:486-92.)     

Biotech Outsourcing Strategies cmc—Biologics stream: June 17, 2010, Copenhagen,Denmark

Now in its third year, the Biotech Outsourcing Strategies (BOS) meeting organized by Bio2Business took place at the Søhuset Conference Centre in Hørsholm, Copenhagen. The focus of this year''s event was the demanding and challenging area of chemistry, manufacturing and controls (CMC), and the meeting provided ample opportunity for lively discussion of the key issues surrounding this area. New for the 2010 conference, a biologics-focused lecture stream ran in parallel to the established small molecule stream. Both streams boasted a distinguished panel of keynote speakers who discussed all aspects of CMC from early stage scale-up through late stage clinical development. In addition to the keynote speakers, selected contract research organizations (CROs) gave short presentations on the solutions that they could provide to some of the challenges facing CMC. The meeting attracted more than 150 delegates from leading drug development companies and CRO service providers, and greatly facilitated the forging of new working relationships through pre-arranged one-to-one meetings. Moreover, exhibitions from event sponsors and considerable scheduled networking time over lunch and evening receptions further enhanced the highly productive and interactive nature of the meeting.Key words: outsourcing, chemistry manufacturing and controls, biopharmaceutical developments, contract manufacturingDr. Anne Bondgaard Tolstup (Symphogen) presented an overview of the key challenges for small companies involved in biopharmaceutical up-stream process (USP) development and good manufacturing practice (GMP). Founded in 2000, Symphogen focuses on cell line development and manufacturing of recombinant antibodies including polyclonal antibodies, a new class of therapeutics that Symphogen has brought into clinical development. Their therapeutic areas of interest are cancer, infectious diseases and immunoglobulin replacement therapy because there are many well-understood and novel targets for which new monoclonal antibody (mAb) products can be developed.Central to Dr. Tolstup''s presentation was a critical analysis of their experiences in contract manufacturing organization (CMO) outsourcing of three recombinant antibodies developed in-house. Symphogen''s first outsourcing experience came in 2004, which was fairly early in the life of the company, with the development of Sym001. Sym001 presented significant challenges for outsourcing because it was a new type of polyclonal antibody product comprising 25 recombinant antibodies. The challenge presented by the complexity of Sym001 was further complicated by the limited in-house experience in CMC development and a lack of equipment for USP development at Symphogen. For their USP development strategy, they wanted to employ a single batch manufacturing process that was novel at the time. Starting with 25 individual cell lines under GMP conditions, they expanded the cell lines, mixed them together and generated a polyclonal master cell bank. This was further expanded to give the polyclonal working cell bank that could then be used in USP manufacturing similar to those used for mAb production. To cope with the challenges faced with outsourcing such a novel production method, Symphogen formed an in-house CMC and regulatory team, sought advice from several experienced consultants and conducted extensive research into CMOs and regulatory agencies. After evaluation of several European CMOs, they formed a collaboration with Biovitrum (Stockholm, Sweden). The key criterion for this selection was a sense of strong commitment from the Biovitrum business development team to understand and accommodate customer demands during negotiation of technical work. Dr. Tolstup detailed the working infrastructure of the collaboration, stressing the importance of regular face-to-face meetings between the project teams, and the necessity for a joint steering team to mediate disagreements, thus keeping disputes out of the scientific groups. The collaboration was successful, and resulted in development of a consistent manufacturing process, and release and characterization tools to assess polyclonality. In 2007, Sym001 entered clinical trials and it is currently in Phase 2.In the case of Sym002, a smallpox drug comprising 26 antibodies that is being developed with funding from the United States (US) government, Dr. Tolstup discussed the difficulties that can be encountered when working with CMOs. She highlighted lack of face-to-face meetings, staff inexperience and inexperienced project management teams as less than ideal foundations for a mutually beneficial working relationship. Despite issues surrounding the collaboration, it resulted in two successful batch scale-ups of Sym002.The third CMO experience at Symphogen came in the manufacturing of Sym004, which comprises two chimeric IgG1 antibodies that target epidermal growth factor receptor domain III at uniquely positioned, non-overlapping epitopes. The manufacturing strategy for Sym004 included separate USP and down-stream process (DSP), preparation of drug substance for the two mAbs comprising Sym004, followed by formulation of the mAbs into one drug product by a 1:1 mix. They chose CMC biologics (Copenhagen, Denmark) to collaborate with because their technical equipment fit well with in-house technology at Symphogen. The scientific teams worked well together, leading to successful technology transfer and a robust and scalable USP, and Phase 1 clinical studies are currently underway in the US. Dr. Tolstup concluded by summarizing the general learning points from CMO outsourcing at Symphogen by stating that good communication, flexibility and open-mindedness are key for successful CMO collaborations.Dr. Andreas Castan showcased the implementation of quality by design (QbD) in process development at Biovitrum. Although not an entirely new concept, QbD aims to design and build quality into product and manufacturing processes, instead of simply testing for quality after production. Regulatory agencies require monitoring and process robustness in order to reduce variability of manufacturing, and this is achieved at Biovitrum by the integration of process analytical technology (PAT) into QbD. PAT is a system for designing, analyzing and controlling manufacturing through measurements of critical quality and performance attributes, with the goal of ensuring final product quality. Dr. Castan stressed how incorporation of PAT in QbD can result in reduced variation and increased process understanding, and greatly facilitates technology transfer and scale-up. Linking performance of the manufacturing process to safety and efficacy of the product was discussed, with an emphasis on the need to define the critical quality attributes needed for assessment of the impact that the manufacturing process can have on product safety and efficacy. Dr. Castan indicated that quality attributes are usually assessed by conducting release tests used to assess a small subset of product characteristics, followed by more extensive characterization. By adopting a QbD approach the focus is much more on the process that allows broader assessment of the quality attributes, rather than on analyzing a small representative sample post-manufacture. Defining the relevant critical quality attributes of a product, however, can prove challenging, and Dr. Castan discussed the need for continual reassessment of quality attributes based upon structure-function relationships identified from biological characterization studies. Additionally, he pointed out the requirement for not just one, but a combination of different quality attributes.In a detailed case study, the process whereby Biovitrum defined the design space for one of their projects was explored. The USP comprised a mammalian cell culture fed-batch process, and the DSP consisted of three chromatographic steps, one virus reduction step and one ultrafiltration/diafiltration step, followed by an extensive analytical package to assess the critical quality attributes. Initial risk assessment starts with the manufacturing process and evaluates the operating parameters for each step. Each step is then rated and assigned a risk priority number and further scientific discussion determines which parameters are most likely to have an impact on the quality attributes, and therefore require further characterization. From the process risk assessment, Biovitrum then selected the best design for the project and established that a large number of analytical methods were required.At this point, Dr. Castan touched on experimental design, specifically the difference between running one factor at a time versus a sum of experiments design, which offers a more comprehensive and structured approach to exploration of design space. The characterization of the USP bioreactor step and the DSP capture step using the more structured design of experiments approach was discussed. From the failure mode and effects analysis, operational parameters were identified and augmented with axial points and repeats resulting in a total number of experiments required. The experimental results were then used to assess the reproducibility of the center points of one parameter, which then generated a reliable model necessary for plotting contour charts. With the design space established, it was then possible to identify the desirable product operating ranges.Some of the unique challenges and strategies for outsourcing biologics were evaluated by Dr. Torben Lund-Hansen (Lund-Hansen Consulting ApS). The talk began with an overview of how biologics differ from small molecules specifically with respect to their size, complex and often relatively undefined mechanism of activity, and requirement for post-translational modification. In addition, the source material for biologics can potentially lead to the transmission of adventitious agents that can be hard to identify and may not be easily measurable. As a result, there is a need for substantial in-process control and validation. Dr. Lund-Hansen highlighted how the bio-friendly nature of the manufacturing process aids the susceptibility of the process to microbial contamination, but that it is simply not possible to employ aseptic processing throughout, especially in the drug substance purification.In discussing the pre-clinical attributes of biologics, the difficulties with establishing the pharmacokinetics (PK) of biologics such as antibodies were raised by Dr. Lund-Hansen. While it is possible to measure the antibody in humans or animals for three to four weeks, the biological function may last for many more months, suggesting that PK may not be the most suitable method for assessing biologics. Furthermore, all biologics have an immunogenic potential that is obviously desirable in vaccines, but is a serious unwanted effect that can occur in single and chronic dosing of therapeutics. For example, in some cases up to 20–30% of patients may have an immune response to a protein drug, and this response may in turn cause a substantial decrease in the drug''s efficacy. As a consequence, regulatory agencies have issued very strict guidelines for measuring immunogenicity, which should be of utmost consideration when developing biologics.Another challenging aspect of biologics development is that manufacturing facilities as well as the products must meet regulatory agencies'' specifications. Indeed, prior to obtaining a license the manufacturing facility must undergo inspection while being fully operational and manufacturing the complete product for which a biologics license is required. With this as context, Dr. Lund-Hansen discussed the challenges surrounding CMO selection for biologics. He advised choosing a strong and experienced CMO that can take the product all the way to market so that there is no need for technology transfers during the clinical development process. Furthermore, he stressed the need to work very closely with the CMO to understand the way the CMO works, and, crucially, not try to change their methods. The expectations from a customer point of view were discussed with respect to who is ultimately responsible for ensuring that CMC standards are met. Sometimes CMOs do not take responsibility for CMC and it can often be a sticking point in negotiations between customers and CMOs because of the large degree of risk involved.In assessing the interconnecting relationships between CMOs, sponsors and regulatory agencies, Dr. Lund-Hansen pointed out that quite often the sponsors are relatively inexperienced compared with regulatory agencies and CMOs that have worked with multiple sponsors. He called upon the CMO to educate without intimidating the customer, to be transparent in order to generate trust, and to communicate clearly and precisely the CMO strategy and cost. Additionally, the need to offer the customer a choice was raised with respect to the degree of flexibility within the working contract, and he suggested that a stretched time-line should result in a lower price to the customer.The presentation concluded with an evaluation of the huge amount of risk involved in the development of biotechnology products. Two types of risk were identified: the project risk, which should be carried by the customer and the technical risk, which should be shared between the customer and the CMO because the CMO should be in a better position to mitigate technical risk. Despite this, most CMOs prefer not to assume the technical risk, and this can lead to problems in contract negotiation.The next session featured short presentations from three CROs highlighting the technological solutions that they could provide to various CMC functions. Dr. Michael Becker summarized the technologies that Solvias offers in biopharmaceutical analysis. Solvias focuses on ensuring that the analytical technology that they develop for the customer meets deadlines and milestones to enable efficient product development. They provide analytical support from characterization of proteins through quality control methods to support stability studies, comparability studies and method transfer to CMO sites, focusing mainly on drug substance and drug development. Solvias adopted a fit for purpose approach that allows for innovation to meet customer demands, and they also seek to standardize wherever possible. The key technologies they offer are analysis by fragmented antibody capillary electrophoresis, peptide mapping and routine methods for amino acid analysis, and DNA sequencing.Eva Balslev Jørgensen described the biomanufacturing solutions offered by Novozymes BioPharma. In 2007, Novozymes BioPharma created BioBusiness as a division of Novozymes to strengthen the Novozymes brand position outside enzymes and offer value for customers interested in products with non-enzyme activities. They structure their services into three areas: manufacturing technologies, biomedical applications and manufacturing supplements. In manufacturing technologies, they offer a novel yeast expression system and an albumin infusion technology that can be used as a stabilizer for protein-based drugs. For biomedical applications, they developed the Recombumin excipient for vaccine formulation, albucult^®, which is a stabilizing agent and Hyacare^®, which is used for co-formulation in drug delivery. With respect to manufacturing supplements, they offer cell culture ingredients such as recombinant transferrin and recombinant human growth factors.The final CRO presentation was delivered by Dr. Roman Hlodan (Patheon) who discussed points to consider when developing biopharmaceuticals. Dr. Hlodan began with a look at factors affecting the development pathway by discussing business models, resources, drug delivery and expectations of investors. Regarding formulation choices, Dr. Hlodan suggested that perhaps refrigerated or ambient stored solutions offer the best option as they are cheap, easy to transport and carry the potential to be smoothly transferred to a more desirable delivery such as pre-filled syringe. If this type of formulation is not viable for the drug product, frozen solutions, which can minimize risk to product stability or lyophiles, which offer convenient transportation and storage, can be considered. To conclude his talk, Dr. Hlodan discussed some of the challenges that can arise when manufacturing, including drug substance availability, technology transfer and analytical considerations.The afternoon session began with discussion of approaches to biopharmaceutical formulation and development. Dr. Niels Johansen (ALK) noted that the company focuses on development of biopharmaceutical products for the diagnosis, treatment and prevention of allergies. Allergies are an immunological overreaction to allergens that exist in substances such as pollen, animal hair or food, and the prevalence of allergic diseases is increasing. Most treatments involve simple avoidance and symptomatic medicine, i.e., drugs that control symptoms but not the allergy. Allergy vaccinations, which involve injections of controlled doses of purified and standardized allergens extracted from natural allergen sources, are currently the only treatment that can change the course of the disease. Over time, the injections lead to desensitization of the immune system towards the allergen.Traditionally, allergy vaccinations are administered via subcutaneous injections over a period of three years and are used in the treatment of pollen, grass, dust, animal and bee/wasp venom allergies. In 2005 an alternative vaccination method, sublingual immunotherapy (SLIT), was developed. SLIT provides the potential for a more patient-friendly treatment and ALK strives to deliver an allergy vaccine that can provide fast drug release, solid dosage form for sublingual administration and good shelf life.In discussing drug product formulation, Dr. Johansen examined the requirements for compatibility between the complex mixture of proteins that make up the drug substance and the desired patient-friendly sublingual administration route. He also emphasized the importance of having a carefully designed manufacturing process because protein allergens are highly sensitive to heat and mechanical stress during processing. The question of carrying out in-house drug formulation and delivery development versus outsourcing to a CMO was then raised, and the conclusion was that the only viable option available to ALK as a small company was outsourcing to a CMO. Regarding CMO selection, Dr. Johansen emphasized examination of the reputation, available technology, and the size of the CMO with respect to it being able to take the product through development to market, when choosing a CMO. ALK formed a collaboration with Catalent, UK, the developer of Zydis^®, which is a freeze-dried oral solid dosage formulation technology that allows instant dissolution in the mouth and results in immediate release of the drug. Furthermore, the manufacturing processes of Zydis^® were ideal for formulation of proteins, which are generally stable in freeze-dried formulation. In 2006, ALK launched Grazax^®, which is a fast dissolving oral tablet developed using Zydis^® technology for the treatment of grass pollen-induced rhinitis and conjunctivitis.The strategies for creating outsourcing partnerships at Genmab were discussed by Dr Jesper Valbjørn. Genmab develops fully human antibodies for the treatment of cancers, particularly those for which there is an unmet medical need. Currently, the company has 9 Phase 3 studies of ofatumumab (Arzerra) and 2 Phase 3 studies of zalutumumab on-going, and 3 additional antibodies (daratumumab, RG4930, RG1512) in clinical development. In 2009 their first product, ofatumumab, was approved in the US for the treatment of chronic lymphocytic leukemia. Dr. Valbjørn began by examining the process for selecting a CMO. He noted that selection should be based upon the strategy and needs of the project, and he made clear distinctions between a strategic selection process, which is usually a long term, capacity-driven plan adopted by global companies and a tactical process, which is usually project-driven and includes a limited budget. Additionally, he emphasized that the need to plan for the lifetime of a project before seeking a CMO is key for the success of the project. In a more detailed breakdown of the selection process, Dr. Valbjørn described a method whereby a number of selection criteria are established, weighted and then used to screen potential CMOs.Contracting and project definition were touched upon briefly within the context of the overall process that starts with proposal requests, and includes technical and QA site visits with a review of findings and the forging of technical and quality agreements. Dr. Valbjørn stressed the importance of taking adequate time for this process because getting the agreement right from the start results in a win/win for both sides. Once the contract is in place, the next step is to effectively manage interactions with the CMO. At Genmab, this is done by setting well-defined expectations, goals and responsibilities and having alignment of teams across the company and the CMO. Dr. Valbjørn cited communication, including regular face-to-face meetings, as being important to the success of the project. Disputes are dealt with by a contract manger to keep disagreements out of the technical teams, and joint steering meetings occur every six to twelve months to evaluate performance and align expectations.With regard to technology transfer from the company to the CMO, Dr. Valbjørn stated that transparency with transfer development documentation is necessary in order to prevent problems arising at a later date. He also suggested running the process in the CMO''s development laboratory and training the CMO technicians in specialized analytical methods at your own site. The need to be data-driven and not to make assumptions about the knowledge of the CMO was also stressed. Dr. Valbjørn concluded the presentation with an analysis of the manufacturing process with respect to the engineering run and the GMP run. Engineering runs help to test the scale and performance of the process and minimize risk for the GMP run. Many CMOs are reluctant to carry out a GMP run without an initial engineering run; if the engineering run is eliminated, then there is a requirement to know the process and product well, and to communicate your priorities and risk profile/tolerance to the CMO upfront.Dr. Per Edebrink (RecipharmCobra Biologics) discussed finding the optimal characterization strategy for clinical study material. He began by giving a summary of critical quality attributes, looking at what processes can influence them and stressed the need to define them early in order to determine what characterization is required and aid design of the analytical test package. Although, it may not always be possible to know the critical quality attributes early in the pharmaceutical development, it is possible to predict what may be required from the International Conference on Harmonisation (ICH) guidelines for biopharmaceuticals. In particular, Dr. Edebrink highlighted Q6B, which outlines what structural characterization and physicochemical properties are needed. Regarding potential critical quality attributes of mAbs, Dr. Edebrink mentioned a number of different product variations, including Fc glycosylation, fragmentation in the hinge region, disulfide shuffling and lysine truncation. It is possible to identify some of these variations using intact mass protein analysis, and some example spectra were provided, including a reversed-phase high-performance liquid chromatography-ultraviolet (RP-HPLC-UV) mass spectrometry (MS) spectrum of a mAb showing the free light chain, hinge region fragments and the intact IgG.On the topic of glycoproteins, Dr. Edebrink discussed the effect that glycosylation may have on functional activity. Indeed, glycosylation can affect the biological properties of the glycoprotein, resulting in possible immunogenetic effects, and it can also reduce both the in vivo half-life and the shelf life of the glycoprotein. However, there are many ways to measure the extent of glycosylation of a glycoprotein and Dr. Edebrink showed how electrospray mass spectrometry, high performance anion exchange chromatography and matrix-assisted laser desorption/ionization MS can be used to assess glycoprotein glycosylation. It is also possible to test for sialylation, which can affect the half-life or activity of some biologics. LC-MS can be used to analyze the degree of sialylation and for peptide mapping. The final potential critical quality attribute (CQA) examined was deamidation, which can adversely affect activity. Deamidation is the degradation of asparagine and aspartate that proceeds via a succinamide intermediate. It is possible to test for deamidation using a number of different techniques and examples of using isoelectric focusing and peptide mapping coupled to LC-MS were shown. To conclude, Dr. Edebrink reiterated the need to identify potential CQAs as early as possible using the available knowledge, and assess the potential effect on the product. Furthermore, he highlighted peptide mapping by LC-MS as a powerful tool to provide detailed information on the presence and distribution of product variants.To conclude a highly informative and productive meeting, Dr. Jesper-Sonne Johansen (Novo Nordisk) presented a case study on world-class CMC development processes in mammalian, yeast and bacteria cell lines. In 2006, Novo Nordisk initiated a program to improve their CMC processes and set the ambitious goal of doubling the number of new projects in development within five years, which necessitated a doubling of capacity. They began by mapping all the CMC development processes, such as fermentation and analytical processes, and examined the number of people, time and resources allocated to each process, and assessed if there was room for improvement. They also examined how other companies carried out their CMC development and investigated opportunities for improvement and standardization of technologies going into the CMC process.The company then made two major modifications to their processes. First, they changed from continuous CMC development to a two-step development process in which the first step is to develop suitable processes for production of Phase 1 and 2 drug substance, focusing on delivering a high-quality product through a process that might not be optimized. The second step involves development of a robust manufacturing process and enhanced product knowledge. When implementing this new two-step development process, the decision was made to maintain resources present in Novo Nordisk and not to increase outsourcing.The second change that was implemented was the ‘five initiatives’ process. When examining project transfer from research to development and from development to production, they observed that a substantial amount of knowledge was lost. In order to combat this problem, they established technology transfer teams at each handover one year before the transition would occur to ensure efficient transitions. In addition, they set up technology groups that examined the technology used across research, development and production and, where possible, introduced standardized techniques.Regarding the qualitative outcome of these changes, for the two-step development process the result was a short and focused development time with clear goals and agreements on tasks. In the handover process, the transfer teams co-operated successfully and created knowledge across the chain from research to development to production. For the technology standardization, they developed standard guidelines for purifications and analytical methods. The same technology is now used in many projects, which has resulted in predictable outcomes of processes, and has greatly increased process knowledge. Remarkably, when Novo Nordisk reviewed the effect of implementing these approaches in standard development projects over two years, they found that overall resource use was half the previous level. Overall, the overhaul of CMC development at Novo Nordisk resulted in a two-fold increase in productivity between 2006 and 2008, and they were able to successfully double the number of development projects without increased use of resources.     

Step Process for Selecting and Testing Surrogates and Indicators of Afrotemperate Forest Invertebrate Diversity

Background

The diversity and complexity of invertebrate communities usually result in their exclusion from conservation activities. Here we provide a step process for assessing predominantly ground-dwelling Afrotemperate forest invertebrates'' (earthworms, centipedes, millipedes, ants, molluscs) potential as surrogates for conservation and indicators for monitoring. We also evaluated sampling methods (soil and litter samples, pitfall traps, active searching quadrats and tree beating) and temporal (seasonal) effects.

Methodology/Principal Findings

Lack of congruence of species richness across taxa indicated poor surrogacy potential for any of the focus taxa. Based on abundance and richness, seasonal stability, and ease of sampling, molluscs were the most appropriate taxon for use in monitoring of disturbance impacts. Mollusc richness was highest in March (Antipodal late summer wet season). The most effective and efficient methods were active searching quadrats and searching litter samples. We tested the effectiveness of molluscs as indicators for monitoring by contrasting species richness and community structure in burned relative to unburned forests. Both species richness and community structure changed significantly with burning. Some mollusc species (e.g. Macroptychia africana) showed marked negative responses to burning, and these species have potential for use as indicators.

Conclusions/Significance

Despite habitat type (i.e., Afrotemperate forest) being constant, species richness and community structure varied across forest patches. Therefore, in conservation planning, setting targets for coarse filter features (e.g., habitat type) requires fine filter features (e.g., localities for individual species). This is especially true for limited mobility taxa such as those studied here. Molluscs have high potential for indicators for monitoring, and this requires broader study.