The work of the team headed by Dr. Jocelyn Dupuis has led to the development of the first safe, sensitive and non-invasive molecular imaging agent designed for early diagnosis and monitoring of pulmonary hypertension. Dr. Dupuis was able to demonstrate that PulmoBind could replace the tests currently used in clinical trials to establish an early diagnosis and monitor treatment efficacy.

“This breakthrough brings great hope to patients with pulmonary hypertension because of the life-changing impact of benefiting from earlier treatment,” said Dr. Dupuis.

Pulmonary hypertension (PH) is a common, potentially life- threatening disease characterized by a progressive narrowing of the blood vessels in the lungs. It causes a gradual increase in shortness of breath, which leads to significant disability. There is currently no cure for pulmonary hypertension, and the development of effective medication is complicated by the lack of non-invasive tests that can screen for the disease at an early stage and monitor its progression. That is why this discovery gives so much hope to people with this disease.

PulmoBind is a new radiopharmaceutical product developed jointly by Dr. François Harel from the MHI’s Nuclear Medicine Department and Alain Fournier, PhD, a peptide chemist at INRS–Institut Armand-Frappier.

“There is currently no equivalent to PulmoBind capable of performing a functional imaging of pulmonary circulation. We take pride in this product for which we carried out all stages of development, from the basic research to its human application. We were able to achieve this using the state-of-the-art equipment at the MHI’s Nuclear Medicine Department,” explains Dr. François Harel, nuclear cardiology specialist.

According to Dr. Jean-Claude Tardif, director of the Montreal Heart Institute Research Centre and professor of medicine at Université de Montréal, “this discovery and the development of PulmoBind by Dr. Jocelyn Dupuis and Dr. Francois Harel could truly change the lives of patients with pulmonary hypertension by improving early diagnosis and personalized monitoring. Once again, the Montreal Heart Institute has demonstrated its leadership position in cardiovascular precision medicine with this remarkable advancement.”

The project itself was backed by the CQDM and that organization says it  is pleased with the results yielded by the project to date. “We are extremely proud to have supported this project from the very beginning. We also salute the contribution of mentor Michael Klimas from Merck in the development of a positron emission tomography (PET) imaging version of PulmoBind. Results are very impressive and point to a bright future,” said Diane Gosselin, CEO of CQDM.

“I wish to express my gratitude to CQDM for believing in our project’s potential from the outset. Its clinical progress would not have been possible without their substantial financing of $2.8 M,” added Dr. Dupuis.

The results of this project will lead to a Phase 3 study extending three years and involving 350 patients across 10 centres in North America.

Esperas says it will develop ESP-01 in the province of Québec for the next three and a half years, in patients with advanced or metastatic cancer including metastatic triple-negative breast, colorectal or ovarian cancer.

The project will begin with a first-in-human clinical trial, to determine the safety and efficacy of ESP-01, followed by Phase 2 clinical trials, to be conducted at major Montreal hospitals, the Centre Hospitalier de l’Université de Montréal (CHUM), the Segal Cancer Center at the Jewish General Hospital (JGH) and the McGill University Hospital Center (MUHC) to which other Quebec centers may be added.

“We are pleased to collaborate with Esperas on this important clinical study,” said Dr. Gerald Batist, scientific director and co-founder of Q-CROC. “Through this partnership, we will realize our mission, which is to significantly improve the anti-cancer drug development ecosystem, to multiply patient treatment options, and to reduce the financial burden on the health care system through, in part, improvement of the clinical research infrastructure in Quebec.”

Esperas Pharma Inc. was founded in April 2015 by a US$16.5 million financing led by TVM Life Science Ventures VII, a venture capital fund domiciled in Montréal, and co-investor Fonds de solidarité FTQ. Esperas acquired the worldwide rights to develop and commercialize ESP-01 from Eli Lilly and Company (Lilly). ESP-01 is an oral investigational medicine which specifically inhibits cancer cell division or unchecked proliferation.

“The management team of Esperas Pharma Inc. is delighted to initiate the development of this anticancer medicine in Québec and to provide Quebec cancer patients with first access to this promising development candidate,” said Caroline Fortier, CEO of Esperas Pharma Inc. “Furthermore, the company is proud to collaborate with the Q-CROC network and renowned centers such as the CHUM the JGH and the MUHC.”

Overall, the budget did not disappoint. Much needed investments in basic research have been significantly improved over the previous government’s commitments. A new investment of $2 billion was announced beginning this year for a Post-Secondary Institutions Strategic Investment Fund. The fund covers 50% of eligible infrastructure projects and
presumably will be administer by, or will replace the Canada Foundation for Innovation. The tri-council funding was also increased by an additional $95 million/year beginning this year, in addition to the previously committed $46 million/year announced by the previous government, for a total $141 million/year.

Additional investments in basic research include:

• Mitacs- $14 million over 2 years
• Genome Canada- $237.2 million over 4 years
• CDRD- $32M over 2 years (beginning next year)
• Stem Cell Network-$12 million over 2 years
• Perimeter Institute- $50 million over 5 years (beginning next year)
• Brain Canada Foundation- $20 million over 3 years

For innovation derived entrepreneurs and businesses on the other hand, the budget does not provide a clear understanding on how life science companies may benefit. The budget commits up to $800 million over four years (starting in 2017) for innovative networks and clusters, but it is unclear how this money will be disbursed. The government states that it is committed to helping “high-impact firms scale-up”, but there is no specific funding allocated to this commitment. The NRC-IRAP program was however provided with an additional $50 million investment for the 2016-17 year.

In summary, the budget is good news if you’re in academia, but whether Canadian businesses will also grow and prosper from these investments is yet to be determined.

Highlights from the budget are included below.

Excerpts on Innovation from the Canadian Federal Budget 2016

Compiled directly from budget (Source: Department of Finance, Budget 2016)
Highlights and section titles by R. Merson

Academic Infrastructure
Recognizing the value to Canada of strong post-secondary institutions, Budget 2016 proposes to provide up to $2 billion over three years, starting in 2016–17, for a new Post-Secondary Institutions Strategic Investment Fund, a time-limited initiative that will support up to 50 per cent of the eligible costs of infrastructure projects at post-secondary institutions and affiliated research and commercialization organizations, in collaboration with provinces and territories. This initiative is aimed at enhancing and modernizing research and commercialization facilities on Canadian campuses, as well as industry-relevant training facilities at college and polytechnic institutions, and projects that reduce greenhouse gas emissions and improve the environmental sustainability of these types of facilities.

The new Fund will support investments of the following types:

• A university could convert under-utilized space into new research labs that advance its excellence in a specialized field of strength;
• A college could modernize or create sector-specific training facilities, including capacity for advanced areas such as Red Seal trades;
• On-campus incubators and accelerators could be expanded to increase and improve support for entrepreneurs and start-ups as they develop strategies to grow their business;
• College and university facilities that support prototype development or proof-of-principle assessment could receive investments in order to better serve the needs of industry partners; and
• Post-secondary institutions could retrofit existing buildings for research and development or advanced training activities with more energy efficient heating systems and pursue Leadership in Energy and Environmental Design standards.

Discovery Research

Recognizing the fundamental role of investigator-led discovery research in an innovative society, Budget 2016 proposes to provide an additional $95 million per year, starting in 2016–17, on an ongoing basis to the granting councils—the highest amount of new annual funding for discovery research in more than a decade. These funds will be allocated as follows:

• $30 million for the Canadian Institutes of Health Research;
• $30 million for the Natural Sciences and Engineering Research Council;
• $16 million for the Social Sciences and Humanities Research Council; and
• $19 million for the Research Support Fund to support the indirect costs borne by post-secondary institutions in undertaking federally sponsored research.

Together with the funding provided to the granting councils in Budget 2015 of $46 million in 2016–17 and ongoing, a total of $141 million in new annual resources will be made available to the granting councils going forward.

Additional Investments in World Class Research

Mitacs, a national not-for-profit organization, builds partnerships between academia, industry and the world to create a more innovative Canada. Budget 2016 proposes to provide $14 million over two years, starting in 2016–17, to the Mitacs Globalink program. This funding will support 825 internships and fellowships annually, helping Canadian universities to attract top students from around the world and enabling Canadian students to take advantage of training opportunities abroad.

Genome Canada, a not-for-profit organization established in 2000, plays a central role in helping to identify possibilities and seize opportunities for Canada in the accelerating field of genomics. To continue to support leading genomics researchers and promising scientific breakthroughs, Budget 2016 proposes to provide $237.2 million in 2016–17 to support the pan-Canadian activities of Genome Canada to the end of 2019–20.

Launched in 2007, the Centre for Drug Research and Development is a not-for-profit corporation located in Vancouver on the campus of the University of British Columbia. Its mandate is to identify and translate promising health research discoveries from universities across Canada into new medicines and therapies, a process that is both challenging and costly. To date, the Centre has advanced the commercialization of promising new therapies, secured commitments from leading pharmaceutical firms, attracted foreign investments to Canada, and affirmed its leadership on the global stage by championing the creation of the Global Alliance of Leading Drug Discovery and Development Centres. Budget 2016 proposes to provide up to $32 million over two years, starting in 2017–18, to fuel the growth of the Centre’s promising pipeline and contribute to fully reaping the benefits of Canada’s significant investments in health research.

The Stem Cell Network was created in 2001 to act as a catalyst for enabling the translation of stem cell research into clinical applications, commercial products and public policy. To further support Canadian strengths in this highly promising field, Budget 2016 proposes to provide up to $12 million over two years, starting in 2016–17, to support the Network’s research, training and outreach activities.

The Perimeter Institute for Theoretical Physics in Waterloo, Ontario, is an independent centre devoted to foundational research in theoretical physics. Since its creation in 1999, the Institute has built a global reputation for its exceptional research environment and has demonstrated outstanding scientific merit, which has helped to attract top-tier researchers to Canada. The Institute also hosts hundreds of international researchers each year, trains promising new researchers, and undertakes outreach activities with students, teachers and members of the general public. Along with the University of Waterloo’s Institute for Quantum Computing, the Perimeter Institute is a key institution in the region’s Quantum Valley innovation ecosystem, which fuels Canada’s leadership in new quantum technologies expected to transform and create new industries. Budget 2016 proposes to provide $50 million over five years, beginning in 2017–18, to the Perimeter Institute to strengthen its position as a world-leading research centre for theoretical physics. Each federal dollar will be matched by two dollars from the Institute’s other partners.

The Brain Canada Foundation is a national, charitable organization that raises funds to foster advances in neuroscience discovery research, with the aim to enhance understanding and improve health care for those affected by neurological injury and disease. To help increase understanding of the brain and brain health, Budget 2016 proposes to provide up to $20 million over three years, starting in 2016–17, for the Brain Canada Foundation’s Canada Brain Research Fund, which supports competitively awarded, collaborative, multidisciplinary brain health and brain disorder research projects. Federal funding for this initiative will be matched by resources raised from other non-government partners of the Brain Canada Foundation.

Strengthening Innovative Networks and Clusters

Translating Canada’s science and technology strengths into successful, globally competitive companies requires the private sector, post-secondary institutions, governments and other stakeholders to work together more strategically to achieve greater impact. Connections between knowledge producers and users— including researchers and firms—and collaboration within supply chains driven by market opportunities create value through innovation and support economic growth. Information gaps and coordination challenges may prevent these linkages from being developed to their full potential, impacting the strength of innovation ecosystems. To help address these challenges, Budget 2016 proposes to make available up to $800 million over four years, starting in 2017–18, to support innovation networks and clusters as part of the Government’s upcoming Innovation Agenda.

Helping High-Impact Firms Scale Up

The Government recognizes the vital role that high-impact firms play in creating jobs and generating economic growth. Having more firms realize their untapped growth potential supports a growing and innovative economy. However, these fast-growing firms tend to face common challenges at predictable points along their growth paths. By coordinating federal support such as financing solutions, advisory services and export and innovation support from key federal delivery organizations, these firms are better placed to invest in innovation and secure the talent and capital that will enable their success in the global marketplace.

Consistent with the Innovation Agenda’s goals to better coordinate and align support for Canadian innovators, Budget 2016 proposes to launch a new initiative in 2016–17 to help high-impact firms scale up and further their global competitiveness. Under this client-centric approach, firms will be able to access coordinated services tailored to their needs at crucial transition points, from key organizations starting with Innovation, Science and Economic Development Canada, the Business Development Bank of Canada, Export Development Canada, the National Research Council’s Industrial Research Assistance Program, Global Affairs Canada’s Trade Commissioner Service and the Regional Development Agencies. The initiative aims to target 1,000 firms in the first few years, and expand to more firms thereafter.

Helping Small and Medium-Sized Companies to Innovate and Grow

The National Research Council’s Industrial Research Assistance Program supports innovative and growth-oriented small and medium-sized companies through advisory services, research and development project funding and networking. While further work to develop the Innovation Agenda takes place, Budget 2016 proposes to provide the Program with an additional $50 million in 2016–17 to increase the number of companies served by the Program’s highly qualified Industrial Technology Advisors nationwide. This funding complements the additional investments being proposed to support work experience for recent graduates through the Youth Employment Strategy.

This article was republished with the permission of Merson Consulting Inc. and the author, Robert Merson.

For additional information about funding for innovation in Canada contact:

Robert Merson, Management Consultant

T: 416:669-0159
E: [email protected]
W: www.mersoncorp.com/mci

The Thermo Scientific™ KingFisher™ Duo Prime automated purification system offers a fast way to extract high quality nucleic acids in low-to medium-throughput labs. With the Thermo Scientific™ E1-ClipTip™ Adjustable Tip Spacing Equalizer Multi-channel Electronic Pipette it is possible to enhance the extraction process by using only one tool to do all of the pipetting – adding samples, reagents and magnetic beads on the deep-well 96-plate or 12-well elution strip can be done entirely using one pipette.

The E1-ClipTip Equalizer 6-channel, 15-1250 μL pipette will save valuable lab time and reduce the risk of incurring injuries related to the repetitive motions required in the process.

Introduction

The samples for DNA extraction with the KingFisher Duo Prime purification system may vary from plant leaves to mouse ears, or any organism of interest. The sample can be extracted using a variety of sample vessels available; e.g. blood tubes of different sizes, 15 mL/50 mL conical tubes, microcentrifuge tubes or microplate formats.

The KingFisher Duo Prime purification system works in standard 24- and 96-plate formats and typically there is a need to use several pipettes of different volume settings to fill the reagents on the plate and the elution strip. However, the need to switch between pipettes has been removed.

Thermo Scientific KingFisher deepwell 96-plate and KingFisher Duo Prime elution strips can easily be filled using the E1-ClipTip Equalizer pipette. The adjustable tip spacing feature makes it possible to transfer samples from tubes to plates, and dispense magnetic particles and buffers effortlessly with multi-dispensing pipetting functionality. It is also easy to program, save and name a ready-to-use protocol to fill a plate for routine applications in the Programs mode. E1-ClipTip pipettes feature a unique Matrix function that utilizes step-based programming which allows the creation of fully customized pipetting protocols. As such, this provides an ideal option for users that require a variety of pipetting functions in a unique or specific order to complement different research protocols.

In this article, the simplicity of using the E1-ClipTip Equalizer 6-channel, 15-1250 μL pipette to dispense samples and reagents on the King-Fisher Duo Prime is described with an example of DNA extraction from human cells. One pipette is used for all sample and reagent steps with easy access to useful functions such as mixing and multi-dispensing, to increase pipetting efficiency.

Materials and Methods

The E1-ClipTip Equalizer 6-channel, 15-1250 μL pipette was programmed for pipetting all samples and reagents for DNA purification from human cells using the KingFisher Cell and Tissue DNA Kit on the KingFisher Duo Prime purification system. 12 parallel samples of 5 x 105 HeLa cells were used for testing. Cell lysis was performed according to kit instructions and the lysed samples were pipetted
from microcentrifuge tubes to the KingFisher deep-well 96-plate using a pre-programmed E1-ClipTip Equalizer 6-channel, 15-1250 μL pipette.

Two programs were generated using the Matrix function. One program was created to guide users through the sample row pipetting
“KF CT Sample row Duo” and another one for pipetting washing and elution buffers “KF CT Buffers Duo.” Shortcuts can be created for the most commonly used programs on the E1- ClipTip main menu for straightforward and quick access in the laboratory.

KingFisher Cell and Tissue DNA Kit instructions were followed to adjust steps and volumes on the E1-ClipTip program according to Table 1. Thermo Scientific ClipTip 1250 pipette tips were used in all pipetting steps. The samples and reagents were pipetted on the KingFisher deep-well 96 plate except for the Elution Buffer, which was pipetted on the KingFisher Duo elution strip.

In order to achieve uniform results, it is extremely important that a homogenous solution of magnetic beads is reached. To ensure this evenness of bead distribution, the magnetic beads are mixed using the pipette, before aspiration of the solution for multiple dispense using a single channel of the E1-ClipTip pipette.

The “KF_TissueDNA_Duo” protocol was started on the KingFisher Duo Prime purification system to perform the DNA purification. Once filled, the deep-well 96-plate and 12-well elution strip were inserted into the instrument following the instructions on the user interface. The Thermo Scientific™ Multiskan™ GO microplate spectrophotometer was then used to determine DNA quantity and quality by common absorbance measurements.

Results

Genomic DNA (gDNA) purification from HeLa cells resulted in even yields of high quality gDNA. The resulting agarose gel shows clear bands indicating intact gDNA, and the absorbance spectra shows extracted gDNA to be pure without remains of proteins or other impurities (Figures 1a and 1b).

Conclusions

The E1-ClipTip Equalizer 6-channel, 15-1250 μL pipette with adjustable tip spacing is an ideal solution for dispensing samples and reagents for nucleic acid purification using the KingFisher Duo Prime purification system. By using the E1-ClipTip Equalizer 6-channel pipette, it is possible to use just one pipette and one size of pipette tip to easily dispense all reagents across all protocol steps, when dispensing volumes from 25 to 800 µL. The adjustable tip spacing helps to reduce repetitive motions, thus increasing personnel safety and lab ergonomics, while reducing pipetting time when working between different labware formats. As a result, laboratory efficiency is increased.

About the Author
Sini Suomalainen is an Applications Manager with Thermo Fisher Scientific.

While surface plasmon resonance and bio-layer interferometry are widely accepted and highly valued tools for screening studies of drug candidate molecules, the data they provide are only as good as the solutions loaded onto the instruments. Preliminary assessment of the quality of the sample proteins and solutions is imperative for reliable binding results.

Dynamic light scattering is commonly used to evaluate protein aggregation, degradation and solution quality. However, in the context of high-throughput screening, conventional dynamic light scattering detection is just not feasible, since these instruments work with single-sample microcuvettes and the amount of labour required would be quite extensive. The DynaPro Plate Reader II overcomes this obstacle as it measures dynamic light scattering in situ in industry-standard microwell plates, performing automated, nonperturbative quality assessments with minimal time and effort.

After analysis on the DynaPro, a ‘heat map’ created by the software offers a quick visual scan of the aggregation state in each well. Detailed particle size distributions may be examined more closely. The operator can readily determine which solutions are suitable for binding assays in order to ensure confidence in the results.

The plates can then simply be transferred to the SPR or BLI instrument with no intermediate fluid handling or perturbation. This seamless work flow greatly enhances productivity in drug discovery.

Introduction

Surface Plasmon Resonance (SPR) and Bio Layer Interferometry (BLI) are powerful and widely-used techniques for high-throughput screening and discovery of candidate biotherapeutics. In high-throughput SPR, molecules identified as key targets for treatment are immobilized on a chip, and solutions of potential binding partners injected at one or more concentrations in order to assess affinity
and kinetics of interaction. In BLI, the targets or candidates are immobilized on fiber optic probes and dipped into microwells containing solutions of the opposing molecules. The outcome of the screen is the selection of one or more candidate therapeutic molecules with advantageous properties, such as high affinity and rapid binding kinetics.

In a typical screen, dozens to hundreds of candidates may be tested, so many high-throughput SPR instruments are designed to draw sample from standard microwell plates and inject into microfluidic channels. The analyte flows over the immobilized target molecule, where good candidates settle rapidly onto the chip surface via specific association with the target epitope. A similar process occurs in BLI, except no microfluidics are involved. An optical probe then provides a signal proportional to the increase in surface-bound mass, and the analysis of these signals over multiple analyte concentrations yields affinity and kinetics.

An oft-overlooked obstacle to effective candidate selection is sample quality. The purity and solution properties of molecules employed in SPR and BLI screening may impact the measurements adversely in two ways: 1) data quality and 2) microfluidic integrity. Fortunately, a valuable tool is readily available to address sample quality analysis.

High-throughput dynamic light scattering (HT-DLS) with the Wyatt DynaPro ® Plate Reader II illuminates protein quality without perturbing the solution, performing measurements in the same microwell plates used by high-throughput discovery screening platforms. HT-DLS enables rapid evaluation of sample solutions, prior to their loading onto the SPR or BLI instrument, catching material of poor quality before it has the chance to plug the microfluidics or lead to wasting of valuable time and resources on meaningless measurements. As an added benefit, DLS can determine the analyte’s diffusion coefficient, an important property in SPR experiments for identifying mass transfer limitations. More information regarding dynamic light scattering may be found at www.wyatt.com/DLS.

The Importance of Analyte Quality I: Impurities
Like most techniques, SPR and BLI are subject to the unavoidable, fundamental law of experimental science succinctly put as ‘garbage in, garbage out.’ Analyte quality, so often ignored, is actually quite critical to obtaining accurate and meaningful measurements that ultimately lead to selection of the best therapeutic candidates – and hence the best clinical results. Impurities of low molecular weight, such as extractables and leachables, usually have relatively low impact on SPR and BLI measurements. However, large impurities, such as aggregates and foreign particles (both described by the recently coined – and very apt – term of ‘nanocrud’, see www.chi-peptalk.com/biologics-formulation) can wreak havoc on measurements by both techniques. Large impurities contribute to four basic types of experimental uncertainty in SPR and BLI measurements: noisy signals, spurious signals, inaccurate concentrations, and skewed kinetics.

The evanescent optical fields that probe binding do not extend very far into the solution, typically a few hundreds of nanometers. And yet, any nanoparticle or aggregate passing within that distance from the surface of the chip or fiber probe will result in a signal spike approximately proportional to its mass. Consider a 100 x 100 μm² surface immobilized with bound ligand and illuminated by the SPR beam. Exposing this surface to a concentration of analyte >> K_d results in full coverage and a maximum binding signal. Now, consider a single contaminating nanoparticle of ~5μm diameter in that analyte solution. Since the contaminant contains the same volume as the bound analyte, upon passing very close to the chip or probe surface, this particle can generate a noise spike in the binding signal equivalent to all of the bound analyte. Smaller nanoparticles and aggregates, that may be present in larger numbers in a sample of low quality, will produce a steady stream of small signal fluctuations leading to a degraded optical response. Both effects are represented in the simulated sensorgrams shown in Figure 2.

Analyte aggregates may or may not be active. If active and present in appreciable quantities (e.g., > 5% total analyte protein mass), the aggregates will bind to the immobilized ligand and generate an SPR or BLI signal larger than that of the expected monomeric interaction, skewing the binding response towards a higher estimated affinity and on rate. Aggregates presenting multiple binding sites may exhibit ‘avidity’ effects – interacting simultaneously with multiple immobilized molecules or exhibiting a reduced dissociation rate by hopscotching along the surface of the chip, spuriously leading to an extreme overestimate of affinity. If analyte aggregates are inactive, the effective concentration will be lower than the measured total concentration, leading to decreased binding and an apparent decrease in affinity. Either way, aggregates lead to incorrect quantification of candidate binding properties (Figure 3).

The addition of active non-monomeric species with different diffusion properties and binding kinetics than the monomer may also adversely impact the time-dependent sensorgrams. Since the analysis assumes a single binding species with unique on and off rates, the presence of multiple binding species will create binding curves that can-not be fit correctly under the standard assumptions.

The Importance of Analyte Quality II: Self-Associating Analyte
Standard SPR and BLI analyses require that the analyte be monomeric in solution at the concentrations employed in the experiment. The previous section addressed the adverse impact of irreversibly aggregated material on the analysis. Poorly formulated or otherwise ‘sticky’ analytes may self-associate reversibly as well as irreversibly. When this is true, the analyte monomer is in dynamic equilibrium with small oligomers, such as dimers or tetramers, and the actual concentration of monomer varies with protein concentration. Once again the effect on the final measurement depends on the activity and presentation of binding sites, where active oligomers lead to overestimates of affinity and inactive aggregates lead to underestimates (Figure 4). Moreover, presentation of multiple binding sites may lead to avidity effects and gross overestimates of affinity.

The Importance of Substrate Protein Quality
Much as aggregated analytes can lead to experimental errors, so too can aggregated immobilized proteins. Inparticular, the presence of protein aggregates on the chip or fiber probe surface will most likely lead to a decrease in active material or in the average number of exposed epitopes per immobilized mass. Consequentially aggregated substrate proteins decrease the apparent affinity.

Where’s the Drain Opener?
Severely aggregated or otherwise impure material bearing large particulates can lead to another highly detrimental effect: clogged microfluidics in multichannel SPR. These fluidic channels tend to be narrow but long and are prone to plugging by agglomerated proteins or other ‘nanocrud.’ The occurrence of clogging events in the middle of a screen of dozens or hundreds of candidates can ruin the efforts of weeks, if not months, of protein expression, purification and preparation – including all the work devoted to analytical method development and assessments of SPR immobilization protocols.

Recovery of plugged microfluidics might be as simple as replacing a chip or involve lengthy system cleaning and maintenance. In any case, the damages can amount to many thousands of dollars, even before accounting for lost productivity.

Dynamic Light Scattering to the Rescue!
Dynamic Light Scattering (DLS) is a non-invasive, non-perturbative optical technique that measures the size distribution of nanoparticles in solution/suspension, from less than 1 nm up to several micrometers. DLS relies on the principles of Brownian motion to determine diffusion rates of particles in solution. The information is transformed by DYNAMICS® software into a particle size distribution which can be evaluated to determine whether or not the solution may safely be injected into SPR microfluidics and whether or not the SPR or BLI measurements will produce reliable results.

DYNAMICS provides automated analysis and visualization of DLS results as a heat map indicating good, intermediate, and poor protein quality. The entire process may be completed rapidly prior to loading onto the interaction apparatus simply by transferring the microwell plate into the DynaPro HT-DLS system, running the sample screen, and then (when not contraindicated) loading the same microwell plate onto the SPR or BLI instrument. Microwells that show low-quality material can then be deselected in the interaction screening protocol.

Molecules and nanoparticles in solution or suspension ‘jitter’ due to Brownian motion, a consequence of the thermal energy of solvent molecules and the momentum imparted to the nanoparticles by collisions. In DLS, a laser beam impinges on the nanoparticles and is partially scattered in all directions. The light waves scattered by different nanoparticles reach the detectors at different phases and so interfere constructively or destructively at the detector depending on the specific phase difference between them, as shown in Figure 5.

The measured intensity of scattered light fluctuates over the time scale characteristic of diffusion. DLS captures the rates of fluctuation to determine translational diffusion coefficients D_t. As described in Figure 6, diffusion coefficients are transformed to particle sizes via the Stokes-Einstein equation: R_h=k_BT/6πηD_t, where R_h is the particle’s hydrodynamic radius, k_B is Boltzmann’s constant, _T the absolute temperature, and η the solvent viscosity.

Figure 7 shows three typical %-intensity size distribution determined by DLS. The red and blue curves arise from monomodal populations of BSA (R_h = 4 nm) and polystyrene spheres of radius 50 nm, respectively. The green curve arises from a solution containing a high proportion of monomeric protein, R_h~4 nm, as well as some large aggregates with an R_h of about 50 nm. Because the scattered intensity is proportional to molar mass, the %-intensity curve is heavily weighted toward large particulates, and in fact, the total mass of polystyrene beads is much less than that of the BSA. The actual amount of aggregate vs. monomeric protein may be estimated via the %-mass size distribution graph (not shown).

DLS does not have sufficient resolution to discriminate monomers from dimers or other small oligomers; in general, it can only resolve populations of nanoparticles that differ in size by 3 ro 5x in radius (equivalent to about 100x in mass). However, the presence of small aggregates is inferred via the width of the peak (known as polydispersity) or shifts in the average value of R_h for the population.

HT-DLS Does the Job, Quickly and Easily
Traditional DLS takes place in a microcuvette, manually, one sample at a time. It would not be feasible to test all the hundreds of candidates to be screened in this manner, though cuvette-based DLS could still be valuable for other quality assays. On the other hand, the DynaPro Plate Reader II brings the power of HT-DLS to bear on quality assessment for the target and all the candidates, thanks to its microwell-plate based format with in situ, non-perturbative measurements.

With no fluidics, the DynaPro presents no concern for potential carryover of samples between the wells. Measurements may be completely rapid, typically requiring 10 to 30 seconds per well including transition time between wells. The entire screen is set up to proceed unattended in the DYNAMICS software package.

In HTS-DLS applications, DYNAMICS is usually configured to bin the data as a heat map based on poor, intermediate and high quality size distributions according to bin definitions specified by the user. For example, a sample which shows a single, narrow peak at a size corresponding to that of the analyte may be classified as high quality and allowed to proceed to the binding assay with high confidence (Figure 8, red wells). An adjoining sample which shows a broadened monomeric peak, indicative of some oligomers and perhaps low levels of additional particulates tens of nanometers in size may be classified as intermediate quality and allowed to proceed but with a warning flag as to confidence in the results (Figure 8, blue wells). A sample exhibiting significant particulate content in the micron-size range can be assumed to be either contaminated or highly prone to aggregation and prevented from continuing on to the binding assay (Figure 8, black wells).

Additional Benefits
As an added bonus, DLS inherently determines diffusion coefficients which are helpful in assessing mass transfer effects: the mass-transfer-limited reaction rate in SPR is k_m=0.98(D⁄h)^2⁄3(f⁄bx)^1⁄3, and the diffusion layer thickness is d=D⁄k_m, where D is the diffusion coefficient, h and b the height and width of the SPR flow cell, respectively, x the distance from the flow cell entrance and f the flow rate.¹

Another unique feature of the DynaPro Plate Reader II is the built-in, high-magnification camera which snaps a picture of each well after taking a DLS measurement. These images, stored and shown with the associated DLS data, are especially helpful as diagnostics. A review of the images is useful for determining why the black-classified data in Figure 8 looks bad: has the sample precipitated, or perhaps the well inadvertently was not actually loaded with sample? Figure 9 presents additional examples of sources of poor data identified by camera images.

Finally, the same microwell plates utilized in the DynaPro may be transferred to a spectroscopic plate reader for additional confirmation of content and quality.

Sensitivity
Yes, every instrument and technique has its limitations on sensitivity. The lower limit of robust detection for the DynaPro is 0.125 mg/mL lysozyme (M = 14.4 kDa). Since the intensity of light scattered by macromolecules is proportional to molar mass, the sensitivity is inversely proportional to molar mass, translating to a lower limit of 0.0125 mg/mL of a 150 kDa IgG. Sensitivity to aggregates follows the same trend, i.e., a 100 nm R_h aggregate consisting of approximately 5000 IgG monomers will be indicated at a concentration of ~ 2 ng/mL.

Even if the primary sample concentration is below the limit of detection, DLS is still a useful test of solution quality since it will indicate with excellent sensitivity the ‘nanocrud’ content– sub-micron particulates and large protein aggregates that are detrimental to the binding analysis and microfluidic system. The same analysis should also be used to assess dilution buffers employed in SPR to create a series of ligand concentrations.

Conclusions

The selection of candidate molecules with the potential for optimal therapeutic effect and patient benefit depends on a reliable target binding screen, as performed with SPR or BLI. This requires, in turn, assuring that the solutions utilized in the analysis are of good quality. Poor quality samples impact the data quality adversely and hold the potential for fouling flow cells and microfluidic channels.

High-throughput dynamic light scattering with the DynaPro Plate Reader II is readily implemented in the screening work flow to classifying solutions as 1) high quality, offering maximal confidence in the interaction analysis; 2) intermediate quality, suitable for measurement with caution in relying on the results; and 3) low quality, not suitable for analysis and potentially fouling the measurement device. Adding an HTDLS pre-screen can prevent much of the uncertainty and productivity loss associated with variable ligand quality, leading to more reliable binding data and confidence on the final candidate selection.

After identification of the most promising candidates, the DynaPro is also widely used in pre-formulation and candidate developability studies to assess aggregation, conformational and colloidal stability.^2,3 See www.wyatt.com/DLS for additional information.

References
1. Karlsson et al. Methods 1994, 6, 99-110.
2. Saito et al. Pharmaceutical Research 2013, 30(5), 1263-1280.
3. Razinkov et al. Current Drug Discovery Technologies 2013, 10(1), 59-70.

About the Author
Daniel Some, PhD. is a Principal Scientist with Wyatt Technology Corp.

“Biomarkers to better predict disease progression will be a valuable tool to provide more personalized care for patients suffering from CKD,” says Dr. Adeera Levin, CanPREDDICT lead investigator and head of UBC Division of Nephrology. “If developed as a blood test that corresponds to different patient trajectories, it will offer clinicians a better way of knowing which patients need more intensive medical attention and management.”

Dr. Levin adds that a prognostic test will enable clinicians to more aggressively manage CKD in patients expected to rapidly decline, potentially delaying end-stage renal disease. Patients whose kidney function is predicted to stay stable or improve can be monitored less frequently, saving health care dollars.

Such a test will also be extremely valuable for the research and drug development community.

“Chronic kidney disease is a strategically important research area for AstraZeneca, knowing which patients with this disease are likely to face rapid decline and which ones are not, is a major hurdle in clinical trials for new therapeutics,” Dr. Peter Greasley, director clinical research at AstraZeneca explains. “More than 80 per cent of AstraZeneca’s portfolio across small and large molecules has a personalized healthcare approach and biomarkers identified in this collaboration will enable patient stratification that in turn may reduce the time, number of patients, and costs needed for such trials.”

CKD is a major global public health issue. One in 10 adults have some degree of kidney function decline, and CKD is estimated to affect three million Canadians.

The  announcement was made by Canada’s Minister of Innovation, Science and Economic Development Navdeep Bains, alongside Canadian Minister of Agriculture and Agri-food Lawrence MacAulay and Prince Edward Island Premier Wade MacLauchlan.

In Charlottetown, the Natural Products Canada (NPC) will receive $14 million over five years, in support of its work to establish Canada as a global leader in the development and marketing of natural products.

In Montréal, the Centre for Commercialization of Cancer Immunotherapy (C3i) will receive funding of $15 million over five years to develop, translate and commercialize cancer immunotherapy.

“Today’s CECR recipients reflect the impressive depth and quality of research conducted here in Canada in two vital areas: innovative cancer therapies and ground-breaking natural alternatives to products already on the market,” said Bains. “The Government of Canada is proud to support this important stage on the research spectrum: getting products out of the lab and into the market, so that they can begin to improve the lives of Canadians.”

The two recipients were chosen from the most recent competition in the Centres of Excellence for Commercialization and Research (CECR) program. The CECRs match clusters of research expertise with the business community, facilitating the development of products and technologies at a stage in the commercialization process where it is otherwise difficult to attract private-sector investment.

“Our research ecosystem needs to be balanced in a way that supports the constant interchange between discovery and innovation,” said B. Mario Pinto, president, Natural Sciences and Engineering Research Council, and chair, Networks of Centres of Excellence Steering Committee. “Commercialization centres satisfy a very specific need, by providing a solid connection between the ideas generated by foundational research and the expertise that can take the most promising ideas towards commercialization.”

The NPC, headquartered in Charlottetown, includes key partners in PEI (PEI BioAlliance as NPC Atlantic), Saskatchewan (AgWest Bio as NPC West), Ontario (Ontario Bioscience Innovation Organization as NPC Ontario), and Quebec (Institute for Nutrition and Functional Foods as NPC Quebec). The federal contribution will be matched by over $10 million from industry and other sources, for total funding of over $24 million over the next five years.

Rory Francis, executive director of the PEI BioAlliance, expressed his gratitude to the federal government for seizing the opportunity to bring together a unique national team to help academic centres and early stage companies develop and commercialize new products for global markets.

“Natural Products Canada will align the existing expertise of universities, scientific research organizations, small and medium sized enterprises, multi-nationals, the investor community, and government partners in accelerating time to market for promising early stage technologies,” said Francis. “This is an amazing opportunity for the Canadian bioscience sector and a very exciting platform for the next stage of growth of the PEI Bioscience Cluster, ” he said.

The Prince Edward Island Bioscience Cluster has had a strong focus on natural products for health applications for over a decade. Of the 44 companies in the PEI Cluster, more than 30 work on natural product-related technologies. Revenue earned by PEI bioscience companies surpassed $200 million in 2015.

C3i, which is based in Montréal, will accelerate access to innovative cancer immunotherapies for patients. Operating out of the Hôpital Maisonneuve-Rosemont’s Research Centre, C3i will be a one-stop shop for the development, translation and commercialization of ground-breaking cancer treatments.

The CECR program currently funds 23 centres, working in areas that include information and communications technologies, health, natural resources and energy. Created in 2007, the program invests $30 million per year in Canadian innovation.

“We’ve found that the transition from healthy to cancerous blood stem cells happens in clear, compartmentalized steps,” said Mick Bhatia, principal investigator of the study and director of the McMaster Stem Cell and Cancer Research Institute. “We’ve identified two steps in that staircase.”

In a paper published by the scientific journal Cancer Cell, the researchers detailed how they were able to fingerprint myelodysplastic syndromes (MDS), a state for blood cells that turns into acute myeloid leukemia (AML) cancer in approximately 30 per cent of patients. The study demonstrates that early and accurate prediction of this aggressive cancer is possible.

AML is the most common type of leukemia in adults, and about 1,300 Canadians are expected to develop the disease each year.

Bhatia’s research team found when they deleted one version of the important GSK-3 gene, the other version of the gene became active but remained non-cancerous. However, when the second version of the gene was also deleted, AML cancer began.

To test this, Bhatia’s team collaborated with Italian researchers at the University of Bologna to apply these initial findings to human blood samples that had been previously collected from patients with MDS, some of whom eventually developed AML. McMaster researchers did a retroactive study, and demonstrated that gene expression analysis of patient blood samples was accurate in predicting which patients would develop AML and which would not.

“This discovery improves our ability to identify which patients with MDS will develop AML,” said Bhatia. “However, our next step is to go beyond better predictive measures for the development of a blood cancer, and use this predictive gene expression as a target for drugs to prevent AML from developing altogether. This will be part of a new era of genetic-based drug discovery.”

The research was funded by the Canadian Institutes of Health Research and the Canadian Cancer Society Research Institute.

In a show of support, Canadian Prime Minister Justin Trudeau was on hand for the launch announcement held at the MaRS tower in Toronto, where the new stem cell facility will be located.

“The health of Canadians is a priority…we believe that supporting this new, world-class facility will have significant benefits for innovative health-related technology in Canada and around the world. It will also generate new jobs and make Ontario an even stronger competitor in the bio-tech industry,” he said.

CCRM and GE say they will welcome partners from pharma, biotech and cell therapy industries to bring this initiative to life.

According to stakeholders, the wide-scale deployment of cell-based therapies could bring enormous economic and social potential for transforming the course of incurable diseases. Moreover, the global market for cell-based therapies is expected to surpass the $20 billion USD mark by 2025, with an annual growth rate of 21 per cent. The main targets for cell-based therapies are high impact disease areas with significant unmet need, including cancer, heart disease, neurodegenerative diseases, musculoskeletal disorder and autoimmune diseases.

Cell therapy’s pace of development and clinical outcomes have, in many cases, exceeded expectations. Industrialization, technological innovation, and systemic support are now required to maintain this momentum. The centre will provide cell therapy companies with facilities and expertise to help establish manufacturing processes that can produce the large cell numbers required for clinical and commercial use. Located in Toronto’s hub of stem cell science, bioengineering, and clinical trials activities, the centre will work with its industry partners to introduce new technologies to solve emerging technical challenges and bridge gaps in current and future workflows.

Kieran Murphy, CEO of GE Healthcare’s Life Sciences business, commented: “It is increasingly clear that cell therapies and regenerative medicine will transform healthcare globally, but successful industrialization is now crucial to widespread adoption. This new centre will enable us to work with cell therapy companies to push beyond existing technical limits and problem-solve. Toronto’s concentrated and collaborative clinical infrastructure, combined with the strong guidance of the internationally-renowned CCRM, make it an ideal location for the centre.”

Michael May, president and CEO, CCRM, added: “We have built a strong industry consortium of nearly 50 companies to help drive a collaborative approach to realizing the potential of regenerative medicine. GE Healthcare already plays a leading role in that consortium and the company’s deep knowledge of the bioprocessing industry, combined with its global scale and health care insights, makes it the ideal anchor partner for the new centre. We greatly appreciate FedDev Ontario’s support in making this crucial initiative happen. Both partners are essential to the centre’s success.”

Quebec City, QC – Medicago, a company specializing in the development and production of plant-based therapeutic proteins and vaccines, says it will contribute one-third of the financing for a new Class 3 (CL3) containment  laboratory to be located at Université Laval’s Infectious Disease Research Center.

In addition to Medicago’s $500,000 investment, the city and the research centre’s university hospital foundation will provide the remaining funds required to launch laboratory operations.

“We are very pleased to support this local project of importance for Quebec City,” said Andy Sheldon, president and CEO of Medicago.

A CL3 lab is an indispensable tool for conducting pre-clinical studies to advance the research and development of vaccines and other pharmaceuticals. The difficulty of access to CL3 laboratories frequently requires researchers to use resources located outside of Quebec and Canada, often in Europe or the U.S. According to Sheldon, the new facility will help to fill this void.

“This laboratory will contribute to the development of a centre of excellence of an international calibre, and create significant value for the biotechnology sector,” he said.

Sheldon made the initial commitment to the project on 24 October 2014, on Medicago’s behalf, to provide one-third of the amount required to launch the laboratory’s operations.