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.     

Comparison of spatial variation in otolith chemistry of two fish species and relationships with water chemistry and otolith growth

Spatial variation in the chemistry (Mg, Mn, Sr and Ba) of recently deposited otolith material (last 20–30 days of life) was compared between two demersal fish species; snapper Pagrus auratus (Sparidae) and sand flathead Platycephalus bassensis (Platycephalidae), that were collected simultaneously at 12 sites across three bays in Victoria, south-eastern Australia. Otolith chemistry was also compared with ambient water chemistry and among three sampling positions adjacent to the proximal otolith margin. For both species, variation in otolith chemistry among bays was significant for Ba, Mn and Sr; however, differences among bays were only similar between species for Ba and Mn. Only Ba showed significant variation at the site level. Across the 12 sites, mean otolith Ba levels were significantly positively correlated between species. Further, although incorporation rates differed, mean ambient Ba levels for both species were positively correlated with ambient Ba levels. Spatial variation in multi-element otolith chemistry was also broadly similar between species and with multi-element water chemistry. Partition coefficients clearly indicated species-specific incorporation of elements into otoliths. Mg and Mn were consistently higher in snapper than sand flathead otoliths (mean ±s .d ., Mg snapper 22·1 ± 3·8 and sand flathead 9·9 ± 1·5 μg g⁻¹, Mn snapper 4·4 ± 2·6 and sand flathead 0·5 ± 0·3 μg g⁻¹), Sr was generally higher in sand flathead otoliths (sand flathead 1570 ± 235 and snapper 1346 ± 104 μg g⁻¹) and Ba was generally higher in snapper otoliths (snapper 12·1 ± 12·8 and sand flathead 1·8 ± 1·4 μg g⁻¹). For both species, Mg and Mn were higher in the faster accreting regions of the otolith margin, Sr was lower in the slower accreting region and Ba showed negligible variation among the three sampling regions. This pattern was consistent with the higher Mg and Mn, and generally lower Sr observed in the faster accreting snapper otoliths. It is hypothesized that the differences between species in the incorporation of these elements may be at least partly related to differences in metabolic and otolith accretion rate. Although rates of elemental incorporation into otoliths appear species specific, for elements such as Ba where incorporation appears consistently related to ambient concentrations, spatial variation in otolith chemistry should show similarity among co-occurring species.     

Selective interactions between vertebrate polycomb homologs and the SUV39H1 histone lysine methyltransferase suggest that histone H3-K9 methylation contributes to chromosomal targeting of Polycomb group proteins

Polycomb group (PcG) proteins form multimeric chromatin-associated protein complexes that are involved in heritable repression of gene activity. Two distinct human PcG complexes have been characterized. The EED/EZH2 PcG complex utilizes histone deacetylation to repress gene activity. The HPC/HPH PcG complex contains the HPH, RING1, BMI1, and HPC proteins. Here we show that vertebrate Polycomb homologs HPC2 and XPc2, but not M33/MPc1, interact with the histone lysine methyltransferase (HMTase) SUV39H1 both in vitro and in vivo. We further find that overexpression of SUV39H1 induces selective nuclear relocalization of HPC/HPH PcG proteins but not of the EED/EZH2 PcG proteins. This SUV39H1-dependent relocalization concentrates the HPC/HPH PcG proteins to the large pericentromeric heterochromatin domains (1q12) on human chromosome 1. Within these PcG domains we observe increased H3-K9 methylation. Finally, we show that H3-K9 HMTase activity is associated with endogenous HPC2. Our findings suggest a role for the SUV39H1 HMTase and histone H3-K9 methylation in the targeting of human HPC/HPH PcG proteins to modified chromatin structures.     

Sex-specific foraging behaviour in a monomorphic seabird

Sexual differences in the foraging behaviour of parents have been observed in a number of sexually sizedimorphic birds, particularly seabirds, and the usual inference has been that these sex-specific differences are mediated primarily by differences in body size. To test this explanation, we compared the foraging behaviour of parents in a monomorphic seabird species, the northern gannet Morus bassanus. Using specially designed instruments and radio telemetry we found that individuals of both sexes were consistent in the directions and durations of their foraging trips. However, there were significant differences in the foraging behaviour of males and females. Female gannets were not only more selective than males in the areas where they foraged, but they also made longer, deeper dives and spent more time on the sea surface than males. As the sexes are morphologically similar in this species, then these differences are unlikely to have been mediated by body size. Our work highlights the need to investigate sexual differences in the foraging behaviour of seabirds and other species more closely, in order to test alternative theories that do not rely on differences in body size.     

Sequence variation in the androgen receptor gene is not a common determinant of male sexual orientation.

To test the hypothesis that DNA sequence variation in the androgen receptor gene plays a causal role in the development of male sexual orientation, we have (1) measured the degree of concordance of androgen receptor alleles in 36 pairs of homosexual brothers, (2) compared the lengths of polyglutamine and polyglycine tracts in the amino-terminal domain of the androgen receptor in a sample of 197 homosexual males and 213 unselected subjects, and (3) screened the the entire androgen receptor coding region for sequence variation by PCR and denaturing gradient-gel electrophoresis (DGGE) and/or single-strand conformation polymorphism analysis in 20 homosexual males with homosexual or bisexual brothers and one homosexual male with no homosexual brothers, and screened the amino-terminal domain of the receptor for sequence variation in an additional 44 homosexual males, 37 of whom had one or more first- or second-degree male relatives who were either homosexual or bisexual. These analyses show that (1) homosexual brothers are as likely to be discordant as concordant for androgen receptor alleles; (2) there are no large-scale differences between the distributions of polyglycine or polyglutamine tract lengths in the homosexual and control groups; and (3) coding region sequence variation is not commonly found within the androgen receptor gene of homosexual men. The DGGE screen identified two rare amino acid substitutions, ser205-to-arg and glu793-to-asp, the biological significance of which is unknown.     

The removal of volatile ketone mixtures from air in biofilters

The work reported concerns the removal of mixtures of two ketones, methyl ethyl ketone (MEK) and methyl isobutyl ketone (MIBK), which find wide application as industrial solvents, from effluent air streams in downward flow biofilters operating at relative humidities in excess of 95 percent. The inlet concentrations of the two pollutants were 300 mg m^–3 MEK and 330 mg m^–3 MIBK. Maximum elimination capacities achieved were 50 g m^–3h^–1 for MEK and 20 g m^–3h^–1 for MIBK. Marked interaction between the elimination of the two ketones was observed and established biophysical models for the kinetic analysis of biofilter operation proved inadequate as far as the complex processes involved in multi-component biodegradable vapour elimination were concerned. The complexity of such systems requires further definition and the development of appropriate models for process evaluation and design.     

Differential cDNA cloning by enzymatic degrading subtraction (EDS).

We describe a new method, called enzymatic degrading subtraction (EDS), for the construction of subtractive libraries from PCR amplified cDNA. The novel features of this method are that i) the tester DNA is blocked by thionucleotide incorporation; ii) the rate of hybridization is accelerated by phenol-emulsion reassociation; and iii) the driver cDNA and hybrid molecules are enzymatically removed by digestion with exonucleases III and VII rather than by physical partitioning. We demonstrate the utility of EDS by constructing a subtractive library enriched for cDNAs expressed in adult but not in embryonic rat brains.