mibio 2012 - formulationformulation.org.uk/images/mibio2012/mibio-out.pdf · mibio 2012 might be a...

MIBio 2012Molecular Interactions in Biopharmaceutical Formulations:Can Stability be Rationalised and Predicted?

An event jointly organised by the Formulation Science and Technology Group of theRSC and the BioMolecule focus group of the APS.

www.formulation.org.uk www.rsc.org

www.apsgb.org

www.formulation.org.uk

Introducing The Formulation Science and Technology GroupThe Formulation Science and Technology Group is a subject group of the RoyalSociety of Chemistry, London (U.K.)

It is the leading scientific organisation dedicated to product formulation.

As a charitable organisation, it works for the benefits of its members and to further the awareness of formulation science.

It fosters the advancement of formulation science across scientific disciplines:biology, chemical engineering, physics, and across its many industrial applications:

• Cosmetics

• Pharmaceuticals

• Foods

• Detergents

• Fuels

• Paints

• etc...

It is a point of focus for all industrialists and academics engaged in the practice offormulation science.

The FSTG organises many events during the year for the benefit of its members;conferences, training days, and networking events.

For more information visit: www.formulation.org.uk


Overlookedsomething?Remember when it comes to reducing drug dosing frequency, the natural choice is albumin.

Extend the half-life of your drug from “days to weeks” using the only technologybased on albumin, a natural non-immunogenic plasma protein with a proven andsafe regulatory profile.

Differentiate your drug with Novozymes’ Albufuse® Flex and Novozymes’ Recombumin® Flex - a broadly applicable and easily assessed platform with patent life beyond 2030.

Designed by nature. Improved by Novozymes.

www.daystoweeks.com

ForewordIt is a pleasure to welcome you to the Trinity Centre for this very special event on theformulation of biomolecules.

This years' theme "Molecular Interactions in Biopharmaceutical Formulations: CanStability be Rationalised and Predicted?” builds on MiBio 2011 and explores newaspects of the formulation of biomolecules. Formulation of biotherapeutics, leadingto the development of stable and convenient drug products, is a crucial part of thebiopharmaceutical process. The stability of biological molecules depends on a complexset of molecular interactions; understanding these interactions is critical for the successof the formulation development. MiBio 2012 has gathered world-leading experts fromacademia and from industry to take us from theoretical predictions of protein behaviour,through probing molecular interactions all the way to solving specific formulationissues of drug product candidates. The meeting will be interactive and whether youare a formulation scientist, process development scientist or an academic with interestin molecular interactions, we are convinced that you will find this event extremelyinteresting and useful. We hope you will discover and learn about their work of thespeakers, poster presenters, suppliers and from each other to inspire yours.

The formulation of biomolecules is vibrant field of research as evidenced by thegrowing number of publications and patents and the number of attendees at theMiBio event. This year we can count on 20 posters 9 sponsors and exhibitors andthe best part of 100 attendees, which is well above the event in 2011. The eventbuilds on the success of the first edition last year, and we hope you will enjoy theexhibitors session as well as the peer-to-peer networking that is planned to help usto get to know each other and ensure we do meet new people and make full use ofthe rest of the day.

Thank you to all the authors who sent in abstracts and are contributing to the postersession. We are also very grateful to all the sponsors and exhibitors who are joiningus, without whose support, we could not hold such a meeting.

Allow me to thank as well the committee of MiBio 2012 who have helped make thisevent a success: Tejash Shah and Stephen Harding.

MiBio 2012 might be a one-day event, but discussions you started today can continueon LinkedIn via the FSTG discussion group, so do join the group today and continueyour discussions online. Please note that the slides of the speakers and poster presenters will also be available on the MiBio 2012 website, and on the FSTG website; these can be found at: www.formulation.org.uk.

With all best wishes for a very fruitful day.

Philippe Rogueda and Jan Jezek - On behalf of MiBio 2012


Programme

8:00 Registration opens

9:00 Opening remarks

Session 1: Prediction and probing of molecular interactions

9:15 Taming protein interactionsProfessor Mike Williamson, Sheffield University

9:45 Networking Session

10:45 Use of ITC/DSC for the studies of biomolecular stability and interaction of excipients with proteinsDr Natalia Markova, GE Healthcare

11:15 The importance of early biophysical characterization of antibodies in the lead selection processDr Leonardo Borras, Alcon Labs

11:45 Exhibitors highlight

12:30 Lunch Break, Exhibition and Posters

Session 2: Exploitation of the understanding of molecular interactionsin product and formulation design

14:00 Key Interactions of Polysorbates in Protein FormulationsDr Hanns-Christian Mahler, Roche

14:30 Special aspects in the pharmaceutical development of protein formatsbeyond mAbsDr Susanne Jörg, Novartis

15:00 Coffee break, Exhibition and Posters

15:30 Screen early - fail early: Rapid analytical screens for protein formulationDr Paul Dalby, University College London

16:00 Sequence-based prediction of protein solubilityProfessor Michele Vendruscolo, Cambridge University

16:30 Concluding remarks

16:45 Conference ends



Session 1 Lecture Abstract Taming protein interactions

Prof. Mike Williamson - Dept of Molecular Biology and Biotechnology, University of Sheffield

All proteins have evolved to interact, and in most cases this interaction is with otherproteins. Sometimes the interactions are strong, but more often they are weak (Kd > 10 ?M), especially in the crowded environment of the cell. Such interactionsare hard to detect by many techniques, though usually straightforward by NMR.Weak interactions are characterised by entropy/enthalpy compensation, and in particular by the importance of solvating water. This importance is illustrated by the pervasiveness of the Hofmeister series, which originates from surface tension of the solvent layer around a protein. The effect of osmolytes on proteins can beconsidered in the same way. Protein interactions and solubility are therefore dependent on the strength of the interaction of the protein with solvent. In workingwith concentrated protein solutions, the aim is not to remove protein interactions, but to tailor the surface layer to produce tamable interactions.

[email protected]


Session 1 Lecture Abstract Use of ITC/DSC for the studies of biomolecular stability and interactionof excipients with proteins

Dr Natalia Markova - GE Healthcare

Stability of liquid protein formulation is critical for achieving desired therapeutic effectduring prolonged storage of biopharmaceuticals. Our ability to predict factors criticalto protein stability is limited due to a complex nature of proteins and a broad rangeof chemical and physical factors contributing to protein destabilization. Thereforeoptimization of protein stability and understanding of the stabilizing effects of excipientson a molecular level remains a combination of trial-and-error and rational approaches.Several biochemical and biophysical techniques are often needed to profile andoptimize protein stability. Biophysical techniques provide data on multiple parameterssuch as protein homogeneity, specific binding activity and affinity, thermal stabilityand thermodynamics of protein binding/unfolding throughout the developmentprocess.

Differential Scanning and Isothermal Titration Calorimetry (DSC & ITC) offer genericmeans for assessment of protein stability and investigation of protein-excipientinteractions. DSC provides direct information on the thermal stability of a protein.Data on the mid-point of unfolding transition (Tm) along with the temperature at theonset of thermal unfolding and extent of reversibility of unfolding transition tendsto be a good indicator of the relative stability of proteins. These parameters allowranking of different buffers and excipients in terms of their effect on protein stability.

ITC is used to assess binding affinity as well as specific activity of a therapeuticprotein in a given formulation. In formulation development knowledge of protein-excipientbinding parameters is useful for determining the lowest possible concentration of boundexcipient needed to ‘saturate’ the protein and attain the stabilizing effect. Minimizingthe concentration of excipient in a formulation reduces costs as well as the level ofadditives that will be administered to a patient.

This presentation will give examples of the application of MicroCa™l DSC and ITCto the studies of protein stability and protein-excipient interactions.

[email protected]


Session 1: Lecture Abstract The Importance of Early Biophysical Characterization of Antibodies inthe Lead Selection Process

Dr Leonardo Borras - Alcon Labs (Novartis company)

A critical aspect of the antibody lead selection process is assessing and understandingthe biophysical properties of antibody lead candidates. An early characterizationhelps to reduce or eliminate development issues while allowing for ideal lead selection.We have developed a comprehensive early-stage assessment package that enablesknowledge-based selection of promising lead candidates. This early biophysicalassessment has proven its value in our development process.

[email protected]@alconlabs.com


Session 2: Lecture AbstractKey Interactions of Polysorbates in Protein Formulations

Dr Hanns-Christian Mahler - Pharmaceutical Development & Supplies, PTD Biologics EuropeF. Hoffmann-LaRoche Inc, Basel, Switzerland

Surfactants, such as polysorbates, are frequently used for formulating proteintherapeutics. They protect proteins against interface-induced stresses. This includesprotection against aggregation and precipitation caused by air/liquid interfaces (e.g. shaking), by ice/liquid interfaces (e.g. freezing and thawing) and against adsorption of protein often observed with various surfaces (e.g. manufacturingequipment). This talk will include examples of all these aspects. Polysorbates aremixtures of fatty acid esters and are for the use in pharmaceutical products typicallysynthesized chemically. A number of synthesis side products occur and are stillfound in the final excipient raw material, making polysorbates a critical excipient,with high degree of variability. Polysorbates also provide a number of other challenges. For example, their adsorption behaviour may make them adsorb to certain manufacturing equipment (e.g. filters), they can induce pseudoallergenic reactions in dogs (making preclinical testing of protein formulations challenging) andfinally, they can degrade as bulk and even in the protein formulations over storagetime, due to hydrolysis and oxidation. Degradation products may alter protein stabilityand/or cause findings in drug products that require further attention. This talk willinclude studies done on polysorbate degradation and discuss impact, as well.Interestingly, various hypotheses exist for how polysorbates stabilize proteins. Thismakes the assessment of variability and degradation challenging. Direct interactionwith protein structures has been discussed. Likely, effects of polysorbates are due to their surface-active nature and consequent interactions at interfaces, competingwith proteins and thus leading to indirect stabilization by minimizing protein/interface-interactions.

[email protected]

Session 2 Lecture Abstract Special aspects in the pharmaceutical development of protein formats beyond mAbs

Dr. Susanne Jörg - Novartis Pharma AG, Switzerland

In the past decades, monoclonal antibodies have become the fastest growing area of biopharmaceutical industry. Currently, alternative protein formats – such as FAbs,scFvs, nanobodies, etc - are under development. These new antibody and therapeutic protein formats come along with new challenges in the pharmaceuticaldevelopment, i.e. in the formulation and manufacturing process development of biopharmaceutical drug products.

To meet the quality target product profile of those products, a variety of critical qualityparameters need to be addressed during development studies. Special attention ispaid towards control of formation of soluble aggregates and particulate matter during manufacturing and storage of the finished drug product. The viscosity of theformulated drug product is in a suitable range allowing convenient administration ofthe desired dosage. Furthermore, compatibility of the protein with disposable materials used during preparation and administration to the patient needs to beassured.

In this presentation, case studies from the pharmaceutical development of drugproducts of different protein formats are presented.

[email protected]



Session 2 Lecture AbstractScreen early - fail early: Rapid analytical screens for protein formulation

Dr. Paul A. Dalby - Dept Biochemical Engineering, UCL, London

Biophysical analyses are essential for understanding the effect of formulation excipientson protein stability. However, most are not accessible for small quantities of preciousmaterials or for higher-throughput analyses. Microscale and microfluidic platformsare enabling small quantities of proteins to be rapidly analysed under pre-formulation,stress and bioprocess conditions, to assess their stability. Automatable high-throughputmethods for measuring protein thermostability, tolerance to freeze-drying, solubility,aggregation and precipitation will be introduced. A recently established rapid andaccurate microfluidics-based biophysical analysis of protein stability and ligand interactions will also be discussed.

[1] Aucamp, J.P., Martinez-Torres, R.J., Hibbert, E.G. and Dalby, P.A. (2008) Biotech. Bioeng. 99, 1303-1310. A microplate-based evaluation of complex denaturation pathways: Structural stability of E. coli transketolase

[2] Aucamp, J. P., Cosme, A. M., Lye, G. J. and Dalby, P. A. (2005) Biotech Bioeng. 89 (5), 599-607. High-throughput measurement of protein stability in microtiter plates

[3] Grant, Y., Matejtschuk, P., Dalby, P.A. (2009) Biotech. Bioeng. 104(5), 957-964. Rapid optimisation of protein freeze-drying formulations using ultra scale-down and factorial design of experiment in microplates.

[4] Ahmad, S.S., Dalby, P.A. (2010) Biotechnol. Bioeng. 108(2):322-332.Thermodynamic parameters for salt-induced reversible protein precipitation from automated microscale experiments.

[5] Gaudet, M., Remtulla, N., Jackson, S.E., Main, E.R.G., Bracewell, D.G., Aeppli, G., Dalby, P.A. (2010) Protein Science. 19, 1544-1554. Protein denaturation and protein:drug interactions from intrinsic protein fluorescence measurements at the nanolitre scale.

[6] Ordidge G.C., Mannall, G., Liddell, J., Dalby, P.A., Micheletti, M. (2012) Biotechnology Progress 28(2):435-444. A generic hierarchical screening method for the analysis of microscale refolds using an automated robotic platform.

[7] Grant, Y., Matejtschuk, P., Bird, C., Wadhwa, M., Dalby, P.A. (2012) Biotechnol. Letts. 34(4):641-648.Rapid identification and optimisation of freeze drying formulation using microscale and design of experiment approaches: A case study using granulocyte colony stimulating factor (G-CSF).

[8] Abe, Y., Gor, J., Bracewell, D.G., Perkins, S.J., Dalby, P.A. (2010) Biochem. J. 432(1):101-111. Masking of the Fc region in human IgG4 by constrained X-ray scattering modelling: implications for antibody function and therapy.

[email protected]

Session 2 Lecture AbstractSequence-based prediction of protein solubility

Professor Michele Vendruscolo - Cambridge University

As increasing evidence indicates that the sequences of proteins encode not just theirfolding but also their solubility and aggregation [1,2], there is great interest in identifyingthe amino acid code responsible for the maintenance of solubility. We have beendeveloping computational methods to predict different aspects of the aggregationprocess, including the rates of growth of the aggregates and the regions that promoteaggregation [3-6]. In this context, I will review our current understanding and abilityto control protein solubility on the basis of the physico-chemical properties of theamino acids.

[1] Chiti et al Nature 424, 805-808 (2003).

[2] Vendruscolo et al. Cold Spring Harb. Perspect. Biol. 3, a010454 (2011).

[3] DuBay et al J. Mol. Biol. 341, 1317-1326 (2004).

[4] Pawar et al J. Mol. Biol. 350, 379-392 (2005).

[5] Tartaglia et al. J. Mol. Biol. 380, 425-436 (2008).

[6] Agostini et al. J. Mol. Biol. 421, 237-241 (2012).

[email protected]



MIBio 2012 Sponsors

Actavis UK LtdMail: Whiddon Valley Barnstaple Devon EX32 8NSTel: +44 (0)1271 311 200Web: www.actavis.co.uk

Novozymes A/SMail: Krogshoejvej 36, 2880 Bagsvaerd, DenmarkTel: +45 4446 0000Web: www.novozymes.com

Ozone Detection in Pharmaceutical Containers

Charlotte Bell1, Elaine Martin1, Brendan Fish2

1Biopharmaceutical and Bioprocessing Technology Centre, Newcastle University, UK2GlaxoSmithKline, Barnard Castle, UK

Breaches in container integrity can have serious implications in terms of pharmaceuticalproduct quality and patient safety. Drug recalls have occurred due to compromised container integrity. Not only is this highly undesirable from the patient safety point of viewbut also from the economic perspective of the company, as loss of batches, investigationsinto breaches and damaged reputations can have serious financial implications.

Container integrity testing before product release is therefore critical. One method utilised ishigh voltage leak detection (HVLD), where a high voltage is passed over a glass containerand the change in current measured. Deviations from a nominal value indicate compromisedcontainer integrity. Concern has been raised about the effect of HVLD on pharmaceuticalswith a previous study [1] hypothesising that high voltages resulted in ozone formation inthe container headspace, leading to subsequent degradation of the drug product.Biopharmaceuticals unlike small molecular weight chemically synthesised drugs, havelarge protein molecular structures which are complex in nature. Ozone formation in containers housing these products is of particular concern as evidence exists about thedamaging effects of ozone on protein structures [2, 3]. In addition to this, the manufactureof biopharmaceuticals is more complex than the manufacture of small molecular weightdrugs and hence more expensive, consequently greater financial losses will potentially beincurred should products be damaged.

In the study carried out by McGinley at al [1], ozone was measured indirectly through theconversion of a chemical probe, 2,3-dimethyl-2-propene, to acetone which was detectedby GC headspace chromatography. To further investigate their hypothesis, it is desirableto look into other methods for ozone detection This research seeks to develop a robustmethod to identify whether ozone is detectable within containers undergoing HVLD.

Ozone detection methods used in other applications will be considered and adapted forthis application including: UV absorbance of ozone directly at 258nm and a method utilising potassium indigo trisulfonate which displays reduced absorbance at 600nm afterexposure to ozone. Gas tight syringes will be used as sampling and reaction vessels toensure the precision of the method according to recommendations made in previousstudies [4]. Operational parameters of the HVLD system to be investigated include voltageand number of passes of a vial through the machine to understand their effect on ozoneformation. If ozone can be identified and the amount quantified, then steps will be takento mitigate ozone formation in containers.

1. McGinley, C.M., et al., Degradation of Drug Product Caused by a Leak Detection Instrument, in AAPS Annual Meeting & Exposition 2007: San Diego.

2. Cataldo, F., On the action of ozone on proteins. Polymer Degradation and Stability, 2003. 82(1): p. 105-114.

3. Sharma, V.K. and N.J.D. Graham, Oxidation of Amino Acids, Peptides and Proteins by Ozone: A Review. Ozone: Science & Engineering, 2010. 32(2): p. 81-90.

4. Chiou, C.-F., B.J. Mariñas, and J.Q. Adams, Modified Indigo Method For Gaseous And Aqueous Ozone Analyses. Ozone: Science & Engineering, 1995. 17: p. 329-344.


Poster Abstracts


An orthogonal approach to pre-formulation and stability studies.The SGS M-Scan Centre of Excellence in Biophysical Analysis.

Michael S. Caves, Marisa C. Barnard, Inigo Rodriguez-Mendieta, Mark I. MillichipSGS M-Scan Limited, 2-3 Millars Business Centre, Wokingham, Berkshire, RG41 2TZ

In the process of natural selection there has been a trade-off between optimal functionalityand structural stability of proteins. Most proteins are optimised for functionality, ratherthan structural stability, and therefore often denature over a relatively short timescale atambient temperature. For this reason, the generation of information on protein higher orderstructure is essential during the development, manufacture, and shelf-life estimation ofprotein-pharmaceutical products. Biophysical techniques are perfectly suited to such a task.

On this subject, ICH guideline Q6B states: ‘The ultraviolet and visible absorption spectraare determined as appropriate. The higher-order structure of the product is examinedusing procedures such as circular dichroism, nuclear magnetic resonance (NMR), or othersuitable techniques, as appropriate’.

This brief mention of biophysical analysis contrasts with the increase in understanding,over the last few decades, of the importance of biophysical characterisation to predictionsof biopharmaceutical behavior. This, along with the increasing importance of biosimilarsto the global pharmaceutical industry, is leading to changes in the emphasis of regulatoryauthorities on higher order structure analysis, as demonstrated by recent EMA and FDArecommendations on the analysis of biosimilars.

The response of SGS M-Scan’s Wokingham laboratories to these trends was to developthe first GxP-compliant circular dichroism (CD) analysis service in the United Kingdom, andthe company also has established GxP compliant absorbance spectroscopy, differentialscanning calorimetry (DSC), analytical ultracentrifugation (AUC) and SEC-MALS services.SGS M-Scan is now expanding its range of services to include other biophysical techniques,such as FTIR, fluorometry and dynamic light scattering (DLS).

We endeavor to illustrate the application of biophysical techniques to pre-formulation andstability studies. CD, FTIR, fluorescence and DSC are flexible techniques that accuratelyreport on the conformational and thermal stability of proteins. Such techniques can beused in conjunction with aggregation-characterising techniques such as SV-AUC, DLSand SEC-MALS to comprehensively characterise protein-pharmaceuticals under a widerange of conditions.

Our poster illustrates two separate stories. We demonstrate SGS M-Scan’s particle sizingcapabilities by assessing BSA with SEC-MALS, SV-AUC and DLS. The importance of anorthogonal approach, combined with operator expertise, to the sizing of entities withinprotein samples is demonstrated. We also carry out a comparability assessment on twodifferent IgG formulations. CD, absorbance spectroscopy, fluorescence spectroscopy,FTIR and DSC are all used to characterise the two IgG formulations, with only a minorityof these techniques demonstrating a difference between the samples, further evincing theimportance of orthogonality to the assessment of biotechnological products.

MiBio 2012

Challenges in the measurement of protein zeta potential

Jason CW Corbett, Mark R Pothecary, Paul Clarke, Michael Kaszuba, Ciaran MurphyMalvern Instruments Ltd., Enigma Business Park, Malvern, Worcestershire, WR14 1XZ

Dynamic Light Scattering (DLS) is a widely implemented technique for characterization ofproteins and their formulations; allowing not only the determination of molecular size butalso an assessment of stability. In recent years, the stability of proteins in solution hasattracted significant scientific and commercial interest, due to the exponential growth inthe use of proteins as biotherapeutic drugs. Since knowledge of formulation stability andbehaviour are paramount for delivery of a safe and commercially viable product, thedemand for tools that can characterize these formulation properties has increased.Protein mobility - measured using electrophoretic light scattering (ELS) - is one propertythat has been identified as a promising indicator of formulation stability, viscosity andbehaviour.

While biologic drugs are often formulated at high concentrations, e.g. 100mg/ml, thedesire to make measurements early in the biopharmaceutical development cycle meansthat measurements at lower concentrations are also desirable, with a view to predictingbehaviour at formulation concentrations.

Acquiring protein mobility measurements requires the application of an electric field to thesample - a physical process that itself can cause damage to the protein by stimulatingaggregation. Consequently, the resultant mobility measurements reflect that of the aggregatemolecules rather than the protein of interest.

Accurate and high quality measurements of protein mobility require an instrument withsufficient sensitivity to measure both the low count rates and low electrophoretic mobilitiesassociated with dilute protein solutions, as well as a measurement technique that reducesthe risk of aggregate generation.

This poster describes the work carried out by scientists at Malvern Instruments, whichshows that much of the aggregation that occurs during electrophorectic measurementstakes place at the electrodes. It also describes a new measurement technique inventedand patented by Malvern Instruments, the Diffusion Barrier Method, which protects theprotein sample by isolating it from the cell electrodes, and increases the reliability of thedata generated from the measurement.


Poster Abstracts


An atomic force microscopy based method for biopharmaceutical stability determination

O. Croad1, D. J. Scott2, C. J. Roberts1, S. Rigby-Singleton3, P. M. Williams1, and S. Allen1

1Laboratory of Biophysics and Surface Analysis, School of Pharmacy, The University of Nottingham, UK2School of Biosciences, The University of Nottingham, LE12 5RD, UK; 3Molecular Profiles Ltd., 8 Orchard Place, Nottingham Business Park, NG8 6PX, UK

The stability of an active pharmaceutical ingredient (API) within a biopharmaceutical productis of utmost importance during formulation development, manufacture, storage and use.The stability measurement approach developed herein exploits the ability of an atomic forcemicroscope (AFM) to detect interaction forces between individual molecules. By recordingforces on and/or between protein functionalized surfaces, the stability of various proteinscould be determined when subjected to both physical and chemical denaturing conditions.The nature of the technique allows minute quantities of the API to be used, and provides apotential approach that could be employed for the rapid determination of appropriate formulation conditions.

Here, we demonstrate the first key step in developing this approach using bovine pancreaticinsulin (BPI); a clear relationship between the adhesion of an AFM probe to a protein coatedsurface, and the degree of protein unfolding within a sample. Excellent agreement betweenthe AFM derived data and that obtained for the protein in bulk solution also demonstratesthat the AFM experiments provide information that is reflective of the behaviour of the protein in solution. With further development this approach may be used to screen forstability in a wide variety of biopharmaceutical formulations, and further the understandingof the link between API stability and aggregation propensity.

Figure 1: The effect of solution urea concentration on the adhesion frequencywith an insulin coated tip. Each bar represents the percentage of the measurements that showed adhesion at a particular urea concentration.

Presenting author: [email protected]

MiBio 2012


Biosensor and chromatography based strategies for high throughputmeasurement of protein self-association during formulation development

Krisztina Kovacs-Schreiner1, Olatomirin Kolade1, Brendan Fish2, Daniel G. Bracewell11Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, UCL, UK2GlaxoSmithKline, Barnard Castle, UK

In protein formulation, solution conditions and high protein concentrations are responsiblefor the self-association that leads to aggregation. By detecting self-association behaviourusing the second virial coefficient (B22), process parameters can be modified in order toavoid aggregation. The task of B22 determination is made all the more challenging by theweak interactions responsible for self-association. In the literature, the most commonapproach to B22 measurement is static light scattering (SLS), which is currently notamenable to rapid screening. To this end, self-interaction chromatography (SIC) and abiosensor system (SPR – surface plasmon resonance) were investigated. Both techniquescould be adapted to a high throughput approach and required limited sample volume. The challenges and value that each technique offered to quantification of self-association is discussed.

Poster Abstracts


Protein Stabilisers: New Opportunities from Patent Expiry

Arthur P. Lallement- CambridgeIP Ltd, Cambridge, UK

Hypothesis: The first patents protecting biopharmaceutical formulation stabilisers havealready expired, potentially opening new opportunities.

Background: The biopharmaceutical industry emerged in the 1980’s. Protein stability was an early topic for research as the tridimensional configuration of protein can be quicklymodified by its isotonic environment and various interactions with surrounding molecules.To address the stability of proteins in pharmaceutical formulations, formulations includingmolecules stabilising biopharmaceutical therapeutic agents were patented very early.

Methods: Biopharmaceutical formulation patent literature published before 1995 has beenreviewed using CambridgeIP proprietary methodology. Several databases have been crossreferenced, including but not limited to INPADOC and Boliven®. The analysis has been conducted with CambridgeIP proprietary search platforms Red Eye and Boliven®.Interviews of biopharmaceutical and industry experts have been conducted.Boliven® systems have been used to develop technology matrices informing semi-automatedand expert-validated analysis of the patent space to generate trend information and identifyrelevant examples.

Results: The first patent claiming the stabilisation of a biopharmaceutical formulation (Title “Cytomegalovirus attenuation method and vaccine “) was filed in 1974 and mentionsthe use of sorbitol or degraded gelatine to stabilise the vaccine. The APIs mentioned in patents published before 1995 claiming the stabilisation of a proteinare interferon, Fibroblast Growth Factor (FGF), platelet factor, insulin, interleukin and antibodies. Schering and Takeda were the first major players in this IP field and their patents have beengranted. Takeda developed formulation with glucan sulphate to stabilise FGF, while Scheringdeveloped formulations with amino acids to stabilise an interleukin. Other early players whohad granted patents protecting stabilisers for FGF formulations include the Californian bio-pharmaceutical company Scios Nova Inc. As of the end of 1994, 11 granted patents were published; most of these are now expired.

Conclusion: The early protein stability patent assets of Schering, Takeda and Scios Novaare now expired and their technology is potentially reusable for new developments. Most ofthe expired patents protected FGF, interferon and interleukin plus various antibodies. Thelegal status of key early protein stability patents are described in the study, includingwhether the patents were granted, not granted and whether they have expired.

MiBio 2012

Figure1: Granted publication trend analysisprior to 1995

Figure2: Top assignees - Overall patentpublications prior to 1995


The Development of a Systematic Coarse-Grain Model for Polymer Drug Encapsulation

Robbie Mackenzie1, Charles Laughton1, Martin Garnett1, Cameron Alexander1, Jonathan Booth2

1School of Pharmacy, University of Nottingham, UK2AstraZeneca, Macclesfield, UK

Many drug delivery systems use polymers to carry drugs or provide slow release properties.They can increase circulation time, provide targeting and reduce drug side effects. However,there is currently difficulty in achieving high levels of drug loading into the polymer carriers.Often there is no pattern to the success of a drug polymer combination in terms of itsencapsulation efficiency.

We are using molecular dynamics simulation methods to predict the structures of poly (glycerol adipate) (PGA) loaded with a variety of model small-molecule drugs, and to analyse the drug-polymer interactions . Specifically we have modelled the nanoprecipitationof hydrophobic PGA in water containing drug molecules. With insights from molecular simulations we can analyse and fine tune aspects of the polymer at the atomic level to work towards higher encapsulation efficiency.

Accurate atomistic models are needed to give us the detail required for the rational optimisation of the chemical structure of the polymer, but from a computational perspective,they are too time consuming and resource intensive. Thus, a coarse-grained model of thepolymer and drugs is being created that is systematically designed from atomistic simulations. In this way we can reduce the degrees of freedom in the system while retaining a high level of accuracy in our model.

A number of techniques are being used to create the coarse-grained model on the basis ofsmall-scale ‘gold standard’ atomistic simulations: Boltzmann inversion using the VOTCAsoftware package [1] can parameterise both bonded and non-bonded interactions for thepolymer. A drug melt can be used to parameterise non-bonded interactions for the drugmolecules. The existing MARTINI coarse-grained force field [2] covers the interactions of thesolvent molecules with themselves and the solute.

This rigorous approach, using a range of state-of-the-art molecular modelling techniques,will enable us to create the most accurate in silico representation of polymer drug encapsulation. Results from this model should reduce the level of trial and error in design optimisation, and improve the efficiency of current polymer drug delivery systems.

[1] V. Rühle, C. Junghans, A. Lukyanov, K. Kremer, D. Andrienko, Versatile Object-oriented Toolkit for Coarse-graining Applications, J. Chem. Theory. 2009, 5 (12), pp 3211–3223

[2] S.J. Marrink, H.J. Risselada, S. Yefimov, D.P. Tieleman, A.H. de Vries. The MARTINI Forcefield: Coarse grained model for biomolecular simulations. JPC-B, 111:7812-7824, 2007.

Poster Abstracts


Crystals as formulation form for proteins

Nuno Madeira do Ó, Snow StolnikUniversity of Nottingham, University Park, NG7 2RD, Nottingham

Protein delivery continues to prove a challenge to the pharmaceutical industry. One of the major challenges for the production stage of protein formulations is to preserve theirphysicochemical stability and biological activity during exposure to processing conditions.Protein aggregation currently presents a major problem in development of protein therapeutics.Different approaches have been adopted to overcome this issue, including chemical modifications such as conjugation, addition of excipients to the liquid formulation andfreeze-drying. In protein delivery research, protein crystals have been considered as analternative to creating depot formulations. The crystalline form offers an increased stabilityof protein when compared to the corresponding amorphous substance, less susceptibilityto degradation, and the elimination of aggregation issues, consequently making proteincrystals a potentially interesting option. However, the studies on protein crystallization havemainly been concerned with the production of for X-ray crystallography rather than producingcrystals in conditions suitable for a delivery formulation.

This work aims to produce a crystal population under (i) conditions suitable for direct formulation administration (i.e. using GRAS materials) and (ii) applying the quick solventevaporation method based on batch protocol that allows the production of a great numberof small crystals and is simple to scale up.

The results show that the method generated crystals for several tested proteins (lysozyme,thaumatin, insulin and pepsin) using GRAS excipients in the crystallisation buffer. NuclearMagnetic Resonance (NMR) and Fourier Transformed Infrared spectroscopy (FTIR) indicatethat the spectra in general follow the starting material, the NMR peaks being more ‘pronounced’. Interestingly the use poly-vinyl pyrrolidone (PVP) in the crystallization bufferresulted in significant reduction in the size of the crystals obtained (86% smaller than thecontrol population; eg 3.3 µm compare to 23.2 µm, respectively.) The solubilisation of thecrystals from a dried form, with and without PVP, was shown to be fast; the crystals weresolubilised 5-10 seconds after the addition of water. Current studies are focusing on theassessment of biological activity of the proteins release from the crystals and on expandingthe list with further examples.

MiBio 2012


Use of Dynamic Mechanical Analysis and Dynamic ScanningCalorimetry in the Determination of Critical Temperature Parameters for the Stabilization of Freeze-dried Biologicals

Kiran Malik, Chinwe Duru & Paul MatejtschukNational Institute for Biological Standards & Control (NIBSC) – A Centre of the Health Protection Agency,

Blanche Lane , South Mimms, Potters Bar , Herts. EN6 3QG. United Kingdom.

Biological materials are often unstable for long term liquid storage and a lyophilized formatis commonly selected. An understanding of the thermal properties of biological materialsand their formulants is critical to their successful freeze drying. Differential ScanningCalorimetry is often used but can be unrevealing for complex formulations (Gearing et al2010). In this study we compared the use of DSC and Dynamic Mechanical Analysis for the determination of glass transition values for formulants commonly used in freeze driedformats. The influence of changes in formulation on the glass transition value has also beenstudied by sequential modification of each component in order to assess its impact.Selection of formulation so as to achieve a raised glass transition value is important for theoptimisation of freeze drying processes for biological materials.

Data presented in the poster indicated that Tg’ values were detected in multi-componentsamples more readily by DMA than by DSC although the transition values are usually athigher temperatures.

[email protected]

Poster Abstracts


Biacore™ system, in determination of biomolecular activity

Dr. Natalia Markova - GE Healthcare BioSciences AB, Uppsala, Sweden.

Label-free technology solutions impact discoveries all the way from basic research to finalQC of a biotherapetic agent, by providing information on protein interactions, functionalityand stability.

Implementation of a testing strategy for biological products using a combination of SPRbiosensors and microcalorimetry has the potential to significantly improve both productcharacterization as well as overall productivity. Label-free assays for the determination offunctionally active molecules can be used for multiple applications throughout the entirebio-therapeutic and vaccine workflows, such as:

• Protein concentration analysis in QC• Optimizing protein purification conditions • Batch-to-batch comparability and stability studies • Product release testing meeting GxP requirements

This poster will focus on easy and time-efficient sample preparation for in-depth evaluationof antibodies, and how improved and unrivalled sensitivity is allowing analysis of more biologically relevant binding behaviour, including analysis of binding to immobilized cells,allowing accurate evaluation and optimization of biotherapeutic formulations.

MiBio 2012


Dry powder therapeutic mAb formulations with enhanced temperature stability

F. McNamee1, W. Gebbie1, K. Davidson1, J. Partridge1, J. Vos1, J. Abate2, C. Kirchhoff2, B. D. Moore1

1XstalBio Ltd, Thomson Building, University Avenue, Glasgow, G12 8QQ, U.K2Pfizer Global Research & Development, 700 Chesterfield Parkway West, Chesterfield USA

There is an increasing demand for differentiated strategies for formulating biotherapeuticsto deliver stable and convenient drug products that can be conveniently implemented intoa biopharmaceutical process.

XstalBio have developed platform technologies for stabilizing a wide range of biotherapeuticswithin a dry powder format. By using a rapid isothermal precipitation process, in conjunctionwith stabilizing additives, dry powder products can be prepared for a range of administrationstrategies, including inhalation, high concentration subcutaneous injection (>200 mg/mL)and sustained release.

The aim of this study was to optimise dry powder formulations of a human monoclonalantibody to achieve a highly extended shelf-life.

Materials & Methods - Monoclonal human antibody, herein termed PFCP1, was obtainedfrom Pfizer Inc, Chesterfield, St. Louis. PFCP1 is a fully human monoclonal antibody specific for human cytotoxic T lymphocyte-associated antigen 4 (CTLA-4, CD152). PFCP1dry powders were prepared by coprecipitation of an aqueous mixture containing histidinebuffered antibody, concentrated glycine coprecipitant and PSA (L-arginine & L-glutamicacid), into either propan-2-ol or 2-methyl-1-propanol.

An accelerated stress study was then carried out in which PFCP1 dry powders werestored at 40ºC at uncontrolled humidity for a standard period of ~47 weeks.

After temperature stressing, the PFCP1 dry powder was reconstituted back into buffer ata target protein concentration of 1 mg/mL, and monomer content was measured by size-exclusion chromatography, using a Tosoh TSKGel G3000 SWXL 7.8 mm ID x 30 cm column.

Results - All of the stressed samples retained ≥95% monomer content. SamplePFCP1_56_6 retained 98.5% monomer - a loss of only 1.1% over 47 weeks at 40ºC.

The bioactivity of the PFCP1 samples was tested in a PFCP1 specific ELISA; the resultsshowed that bioactivity was also not compromised by the solvent coprecipitationprocess.

Conclusions - Human monoclonal antibodies can be readily formulated using XstalBio technology by incorporating precipitation stabilizing additives (PSA). Coprecipitation leadsto finely-divided dry powders, which can be rapidly reconstituted back into aqueous, torelease the monoclonal antibody in monomeric form. Moreover, conversion to this dry format imparts excellent thermostability to the mAb.

Applications to Biotherapeutics - Recent developments of this technology have providedpowders that can be rapidly reconstituted to produce very high concentration mAb solutions(>200 mg/mL), deliverable through a 27 gauge needle, with acceptable glide force andosmolality.

[email protected]

Poster Abstracts


Biacore™ system, in determination of biomolecular activity

Mei-an SungdeltaDOT Ltd, London BioScience Innovation Centre, 2 Royal College Street, London, NW1 0NH

Capillary Electrophoresis is a powerful and versatile analytical tool that can be used toaddress many aspects of bio-processing analytics. deltaDOT's High PerformanceCapillary Electrophoresis (HPCE) system benefits from its core Label Free IntrinsicImaging technology (LFII® system), which allows improved resolution, quantification and reproducibility. The lack of labels also frees LFII® system to perform universal analysis on a wide variety of analytes. This allows the system to monitor the bioprocessfrom inception to QA/QC and finally stability and formulation studies, looking not just at proteins, peptides and DNA but also at small molecules such as amino acids or evenionic salts present in the growth media as well as molecular interactions such protein-protein,protein-small molecule interactions. The poster will describe case studies where deltaDOT'sHPCE system is used to assess batch-to-batch consistency, stability studies and thedegree of protein folding of formulated vaccine products.

MiBio 2012


Molecular interactions of heat-induced colloidal intermediates of wheyproteins alter the biological activity of enzymatic hydrolysates

I.B. O’Loughlin1,2, B.A. Murray1, P.M. Kelly1, A.A. Robinson3, R.J. FitzGerald2, and A. Brodkorb1* F. McNamee1, W. Gebbie1, K. Davidson1, J. Partridge1, J. Vos1, J. Abate2, C. Kirchhoff2, B. D. Moore1

1XstalBio Ltd, Thomson Building, University Avenue, Glasgow, G12 8QQ, U.K2Pfizer Global Research & Development, 700 Chesterfield Parkway West, Chesterfield USA

Whey is recognised as a source of functional food ingredients benefiting human wellbeing andmolecular interactions in whey proteins may play a substantial role in the bio-functionality ofbreakdown products. The effects of heat-induced denaturation and resulting colloidalaggregate behaviour of whey protein isolate (WPI) solutions on angiotensin-I-convertingenzyme (ACE) inhibition and allergenicity of substrates was determined before and afterenzymatic hydrolysis and subsequent fractionation. The molecular interactions of thewhey proteins were monitored by chromatography (reversed-phase and size-exclusion)and electrophoresis (native- and SDS-PAGE). WPI solutions (100 g L-1 protein w/w) weresubjected to different heat-treatments ranging from 50 to 80 ˚C. Heated and non-heatedsolutions were then hydrolysed to a target degree of hydrolysis (DH) of 5 % with a commercially sourced enzyme preparation with high proteolytic activity in whey.

Colloidal aggregates of WPI were formed as a result of di-sulphide interchange andnon-covalent molecular interactions upon heat-treatment. These aggregates were subsequently readily hydrolysed – the course of which was followed by light and confocallaser scanning microscopy (CLSM). Individual protein fractions in WPI showed differencesin their aggregation behaviour and these constituent proteins also displayed differences intheir rate of proteolysis. Heated WPI samples (P < 0.01) were hydrolysed more quicklywith reaction times reduced by 50% and the generation of a greater percentage of thesoluble low-molecular weight material < 1 kDa. The associated physical changes weremarked by declining colloid particle size, viscosity and surface hydrophobicity as hydrolysis progressed.

Hydrolysates from heat-denatured WPI possessed reduced allergenicity and increasedACE-inhibition bio-activity compared to respective un-treated control fractions.Chromatography and mass-spectroscopy (LC-MS/MS) highlighted differences in the peptide profiles and the identified sequences of the samples.

We postulate that the heat-induced alteration of the conformational state of the whey proteins and consequent generation of colloidal intermediates results in greater susceptibility to certain proteolytic activities. This study demonstrates the substantialeffect that heat-induced protein molecular changes can have on enzymatic hydrolysiswhich may subsequently lead to an improved bio-functional attributes.

The Food for Health Ireland project at Teagasc Moorepark (www.fhi.ie) is supported byEnterprise Ireland under grant number CC20080001.

* Corresponding author, P: +353-25-42422, F: +353-25-42340, E: [email protected]

Poster Abstracts


Computational Pharmaceutics - How to Apply Molecular ModellingTechniques in Drug Delivery

Defang OuyangAston Pharmacy School, School of Life & Health Sciences, Aston University, Birmingham, B4 7ET, UK

Computational pharmaceutics combines experimental approaches and computer modellingtechniques to explore the mechanisms of drug delivery and then to develop novel drugdelivery systems. Three examples are discussed: siRNA delivery, cyclodextrin complexesand solid dispersion.1-5 For siRNA delivery, molecular dynamics (MD) simulation can provide us with a physical view of gene-polymer complexation. For cyclodextrin-drugcomplexes, MD simulation can provide the structure, dynamics and energetics ofcyclodextrin-drug complexes. For solid dispersions, MD simulations can help us explorethe physical state between polymer and drug which strongly relate to the physical stability of solid dispersion. As we show with these examples, molecular modelling canprovide a powerful method for the investigation of the mechanism of drug delivery at themolecular level.

1. Ouyang, D.; Shah, N.; Zhang, H.; Smith, S. C.; Parekh, H. S., Reducible disulfide-based non-viral gene delivery systems. Mini Rev. Med. Chem. 2009, 9 (10), 1242-1250.

2. Ouyang, D.; Zhang, H.; Herten, D. P.; Parekh, H. S.; Smith, S. C., Flexibility of short-strand RNA in aqueous solution as revealed by molecular dynamics simulation: are A-RNA and A '-RNA distinct conformational structures? Aust. J. Chem. 2009, 62 (9), 1054-1061.

3. Ouyang, D.; Zhang, H.; Herten, D. P.; Parekh, H. S.; Smith, S. C., Structure, dynamics, and energetics of siRNA-cationic vector complexation: a molecular dynamics study. J. Phys. Chem. B2010, 114 (28), 9220-9230.

4. Ouyang, D.; Zhang, H.; Parekh, H. S.; Smith, S. C., Structure and dynamics of multiple cationic vectors-siRNA complexation by all-atomic molecular dynamics simulations. J. Phys. Chem. B 2010, 114 (28), 9231-9237.

5. Ouyang, D.; Zhang, H.; Parekh, H. S.; Smith, S. C., The effect of pH on PAMAM dendrimer-siRNAcomplexation - Endosomal considerations as determined by molecular dynamics simulation. Biophys. Chem. 2011, 158 (2-3), 126-133.

MiBio 2012


Investigation of the Pharmacokinetic Properties of Aspirin Analogues

B.Rai, C.J.Perry School of Applied Sciences, University of Wolverhampton, Wolverhampton, UK.

Colorectal cancer is the second leading cause of cancer-related deaths in the westernworld. There is evidence to suggest that aspirin can prevent colorectal cancer. It was foundthat aspirin and the aspirin analogue, Benzoyl aspirin (PN51) are toxic to colorectal cancercells [1].

The pharmacokinetic studies of PN514, Hexanoyl aspirin (PN506) and m-bromobenzoylsalicylate(PN524) can determine whether or not these potential anti-cancer agents are suitable fororal administration. Hence the rate at which these analogues hydrolyse will be studied.

Thin layer chromatography (TLC) and infrared spectroscopy (IR) techniques were used totest the purity of the analogues.

UV spectroscopy was used to follow the rate of hydrolysis in a range of buffers (pH 0-13). The full pH rate profile ofPN514 was determined. The profile followed the sameshape as the classic aspirin rate profile. The rates foundwere also very similar at any given pH to that of the rate forAspirins. This suggests that the results obtained are bothaccurate and reliable. The pKa (determined by titration)obtained (4.15) strongly agrees with the pH rate profile produced as both decrease and increase in rate is seeneither side of the pKa. The changes in rate are due to different rate determining steps becoming dominant at varying pHs. For example from pH 5 to 3, PN514 is reactingto OH- ions but when the H+ ion concentration increasesconsiderably at pH 2.2; Specific acid catalysis takes over asthe dominant reaction which causes the rate to increase asH+ ions increase therefore k= [H+][Ester].

PN514 was more prone to acid catalysis. This may be due to the benzene ring in its structurewithdrawing electrons from the carbonyl group on the ester. This causes the dipole presentto increase. The overall effect is that of the polarity increases making the molecule moresusceptible to specific acid catalysis (and polar molecules such as water). Aspirin has amethyl neighboring the carbonyl group. The methyl donates electrons to the carbonylgroup, this decreases its dipole and the overall polarity also decreases making aspirin lessprone to attack by H+ ions.

As the differences in rates seen between PN514 and aspirin are very small it can be saidthat PN514 may be suitable for oral administration.

However it is important to state that only when the mechanism of action in the way in whichaspirin and aspirin analogues prevent colorectal cancer is fully understood can a suitabledosage form be applied.

[1] Deb, J., Dibra, H., Shan, S., Rajan, S., Manneh, J., Kankipati, C., Perry, C. and Nicholl, I. (2011) Activity of Aspirin Analogues and Vanillin toward a Human Colorectal Cell Line. Oncology Reports, 26, pp.557-565

Poster Abstracts


Protein-protein interactions and understanding aggregation in drug formulation and long term storage

Dorota RobertsManchester Institute of Biotechnology, CEAS, the University of Manchester, Manchester, UK

Unpredictable aggregation in bioprocessing and formulation of biotherapeutics is responsiblefor reducing yield and increasing loss of material. Some progress has been made towardscost efficient formulation, but each biotherapeutic has different aggregation propertieswhich makes it difficult to target the cause of aggregation without long term and costlyresearch.

Light scattering is a technique which, in combination with methods assessing structuralproperties of proteins such as differential scanning fluorimetry (DSF), can provide a desirabledatabase of aggregation prone biotherapeutics. In this study, we focus on assessing thechanges of antibody structure and the native-native self-association, which both - in time -lead to the formation of larger insoluble aggregates present in the formulated medicines.Here, we have examined a range of antibody solutions in pH range between 4 and 8 and forvarying ionic strengths.

Additionally we provide an insight into the light scattering measurement for rapid screeningof weak non-specific repulsions or attractions between proteins. The method, which we arecurrently develop using a model protein, uses composition gradient multi angle light scattering(CG-MALS) instrumentation to apply automated continuous mixing (ACM) technique toacquire the osmotic second virial coefficient (B22). The ACM technique allows for fastscreening of B22 for multiple solvent conditions within a short time, and reduces the sampleconsumption.

DSF data of a monoclonal antibody indicate that the conformational stability is independentof ionic strength from 0 to 100 mM and pH values between 6 and 8. However, we find self-association and aggregation is greatest at pH 8 and low ionic strength, while increasingionic strength or reducing pH lower the aggregation. The behaviour is consistent withaggregation being driven by electrostatic attraction. The results are rationalized in terms ofantibody electrostatic properties.

MiBio 2012


Microscale freeze-dried and liquid formulations of biotherapeutics to investigate the relationship between forced degradation and long-term shelf life

Researcher: Mathew Robinson1

Supervisors: Dr. Paul Dalby1, Dr. Paul Matejtschuk2

Advisors: Dr. Adrian Bristow2, Dr Colin Longstaff2, Dr. Dan Bracewell11Department of Biochemical Engineering, University College London (UCL), London, UK2National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, UK

Protein aggregation and loss remain a costly problem in the processing of biotherapeuticsand whilst several studies have optimised certain unit operations (e.g. centrifugation, filtration and chromatography), the formulation and lyophilisation processes remains relatively neglected. Currently, there are two approaches to improve drug stability and shelflife: 1) protein engineering and 2) formulation optimisation. This study will characterise thestability of two biotherapeutics – a cytokine (~20kDa) and an enzyme (~60kDa) in aqueousformulations. The data (melting curves) presented is produced from a high-throughput, lowsample volume, rapid screening apparatus: the Optim-1000 (Avacta Ltd). This machinemeasures static light scattering (a means of characterising aggregation) and intrinsic protein fluorescence (a means of characterising conformational changes in structure), astemperature is ramped from 15°C to 90°C at 1°C/min. The maximum required sample volume is 9 µl and the minimum protein concentration is 0.1 mg/ml, making the Optim-1000an attractive alternative to the conventional techniques for assessing stability.

The resulting melting curves have shown that, for 0.5 mg/ml enzyme, the most stabilisingbuffer is 10 mM sodium acetate (pH 3.5), which gives the enzyme a primary Tm (meltingtemperature) of 58°C. It was also found that addition of some excipients (e.g. 2.5% w/vmannitol) marginally stabilises the enzyme further (Tm ~59°C) whereas some excipients (e.g.20 mg/ml glycerol) destabilises the enzyme within the buffer (primary Tm ~20°C). The dataproduced fits very well to sigmoid curves, as indicated by adjusted R2 values which arevery close to 1. The technique was also applied to the cytokine. Out of the conditionspresented here, the most stabilising buffer for the cytokine is 50 mM sodium citrate (pH 3.5)with a primary Tm of approx. 60°C. In future work, this technique will need to be verifiedwith other equipment, e.g. DLS, DSC and SEC chromatography in order to successfully justifyits accuracy and potential to replace the slower techniques in rapid formulation screens.

Poster Abstracts


Thermal characterization of protein based sterile ocular implants

Garima U. Sharma1, Kiran Malik2, Ashkan Khalili1, Teresa Barata3, Paul Matejtschuk2, Peng Khaw1, Steve Brocchini11UCL School of Pharmacy and NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye

Hospital & UCL Institute of Ophthalmology, London, UK2Standardization Science, National Institute for Biological Standards and Control, Health Protection Agency,

Potters Bar, UK3UCL School of Pharmacy

Glaucoma is the leading cause of irreversible blindness worldwide. It causes increase in intra ocular pressure (IOP) which leads to optic nerve damage and subsequent blindness.Glaucoma filtration surgery (GFS) is a procedure that creates a fistula to allow the outflow ofaqueous humor thus delaying the progression of the disease. Scarring is the main reason forclosure of the fistula resulting in failure of the surgery. This leads to increase in IOP causingprogression of the disease and subsequent loss of vision. There is no licensed treatment tocontrol the scarring after GFS. Current practice is to use cytotoxics off-label. These havelimited efficacy and significant devastating side-effects including leakage from the surgicalsite with the potential for infection. Bevacizumab (Avastin), humanized monoclonal antibodyagainst VEGF- A, has shown the potential to control scarring when administered into thesubconjunctiva following GFS. However, it requires multiple injections as the drug clears tosystemic circulation rapidly. Therefore, there is a need for a dosage form that would prolongthe local residence time of the protein. Bevacizumab has been developed as an implantabletablet for ocular use by our group. This provides prolonged release of the antibody in thesubconjunctival space. An in vivo study showed that this formulation is biologically activeand prolongs the survival of the bleb in a rabbit scarring model. Tablet fabrication requires afreeze drying step which can cause aggregation of the protein and loss of activity. The aim ofthis study is to characterize the thermal properties of the excipients of the antibody formula-tion during freeze drying.

Differential Scanning Calorimetry (DSC) (Q2000, TA instruments) was used to measure theTg’ and Tg of the excipients alone and in combination with the antibody. The method usedfor the determination of Tg’ was to equilibrate the sample at -60°C and increase the temperature up to 10°C at a rate of 10°C/min. To determine the Tg of the formulation, modulated DSC was used by heating the sample at 2°C/min with the modulation of 1°Cfor every 60 seconds. Freeze Drying Microscopy (FDM) (Lyostat 3) was used to study thecollapse temperature (Tc) for the excipients and formulation.

DSC experiments showed the sub-ambient glass transition temperature (Tg’) for Avastin tobe -28.2°C and that of trehalose to be -29°C. One excipient in the tablet is hyaluronic acid(HA). Evaluation of HA from a clinically used pharmaceutical formulation showed a meltingendotherm at -23°C. This can be attributed to the presence of buffer salts present in the HAformulation. The knowledge of these transition temperatures is useful to design the mostsuitable freeze drying cycle for our tablet formulation. Performing the primary drying at -30°C(below the Tg’) may help to increase the stability of the freeze-dried protein.

In conclusion, solid dosage forms of antibodies are being developed for ocular use. The Tg’of Avastin was found to be -28°C. DSC and FDM can be used to characterize the excipientsand to understand the thermal events that occur during freeze-drying and hence decide thefreeze drying cycle.

MiBio 2012


The Evaluation of High Throughput Apparent Solubility andConformation Assays as Predictive Tools for Early Stage Formulation Development

Harriet Crawley-Snowdon1*, Neil Mody2, Malgorzata Tracka1

1 MedImmune Department of Formulation Sciences in Cambridge, UK2 MedImmune Department of Formulation Sciences in Gaithersburg, USA* University of Kent

Protein insolubility and hydrophobicity associated with conformational change has thepotential to adversely impact drug product quality, activity, immunogenicity and manufacturability. Therefore, high throughput screening assays are required to identify relative solubility and conformational stability early on in formulation development.

Many techniques to determine protein solubility are well described in literature and commonly used in industry. The experimental approach used in this study involves thetitration of mAb with Polyethylene Glycol (PEG) in 96 wells plate. The mAb and PEG interaction results in a PEG-induced precipitation event, mainly via an excluded volumemechanism. Qualitative conclusions may be indentified for rank order correlations, whileapparent solubility results are based on data extrapolations.

Fluorescence is well established technique that can provide valuable information about proteins conformational changes and therefore about molecule propensity to aggregation.The primary indication for the fluorescence assay is to establish mAbs conformational stability during early stage formulation development. This assay investigates the selection of buffer and excipients promoting mAb solution stability under thermal stress. As a result,qualitative conclusions can be made to identify stabilizing and destabilizing formulation conditions.

This poster focuses on method development and method optimization for both assaysrespectively. For solubility assay various PEG sizes and protein:PEG ratios were investigatedleading to optimization of the method with 1 mg/ml starting concentration of mAbs andtitration with PEG8000 over the range 2-16%. Then, wide application range of the assaywas explored e.g. clones screening, buffer optimization conditions, and excipients selectionin formulation development. During fluorescence assay method development following dyeswere investigated: 1-Anilinonaphthalene-8-Sulfonic Acid (ANS), 9-(2-Carboxy-2-cyanovinyl)julolidine (CCVJ), 9-(2,2-Dicyanovinyl)julolidine (DCVJ), Sypro Orange andProteostat Aggregation kit. The solubility of ANS, CCVJ, and DCVJ were examined. Thedyes stability in pH range 2-9 was also tested. As a result ANS and DCVJ were picked forfurther method development. Then, other assay parameters such as incubation time andprotein:dye ratio were tested. Finally method applicability to stressed sample was verified.

These high throughput screening assays identify two product characteristics: apparent solubility and conformational stability early on in formulation development and lead toreduction of development cycle time.

Poster Abstracts


Inhalable formulations loaded with therapeutic protein constructs

F. van de Manakker1*, A.W. Weber1, K. Baumann2, and J. Ferré2

1FeyeCon Carbon Dioxide Technologies, Rijnkade 17A, 1382 GS, Weesp, The Netherlands2Arquebio S.L., Campus Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain

Nowadays, treatments of respiratory diseases, such as lung cancer and tuberculosis, arenot always effective and often lead to adverse side effects. This is generally related topoor drug bioavailability, inactive drug metabolites and/or high, toxic drug concentrationsoutside the required place of action. This poster highlights a proof of concept for a uniquelocal respiratory treatment to achieve higher therapeutic efficacy and a more site-specificmode of action.

In the approach presented, a glycosidic prodrug is first systemically administered in vivo.Subsequently, stabilized particles containing specific cell-targeting _-galactosidase-basedprotein constructs are inhaled. Once present in the lung, the targeted protein constructslocally convert the glycosidic prodrug into the active drug leading to site-specific drugaction.

In a Dutch-Spanish collaborative project of the companies FeyeCon and Arquebio, inhalable formulations containing a fusion protein construct consisting of the enzyme _-galactosidase and a RGD sequence, well known for its tumor and epithelial cell recognizing and adhesive abilities, have been developed. Combining Arquebio'sexpertise in development and production of the protein construct using state-of-the-artfermentation and ultrafiltration processes with FeyeCon's know-how in particle formationusing pressurized carbon dioxide (CO2) technologies has led to a high quality, widelyapplicable inhalation product.

Protein-loaded, inhalable particles were successfully obtained in high mass yields (typically 70-90 %) by applying mild particle formation techniques based on high pressureCO2 technologies. Studies demonstrated full preservation of the protein construct's biological activity during the particle production process, excellent particle inhalationproperties, and lung-specific prodrug conversion in a murine respiratory model.

In conclusion, by the application of mild CO2 based particle formation technology, sensitive fusion protein constructs have successfully been processed into high qualityinhalable microparticle formulations for effective and site-specific treatment of lung disease. The relevant protein production and particle formation processes can be scaled up and validated to fulfill GMP conditions, required for future pre-clinical and clinical tests.

*Corresponding author: [email protected]

MiBio 2012

Exhibitors Contact DetailsAvacta Analytical LtdMail: Unit 651, Street 5, Thorp Arch Estate, Wetherby, LS23 7FZ, UKTel: +44 (0) 844 414 0452Web: www.avacta.com

GE Healthcare UK LtdMail: Amersham Place, Little Chalfont, Bucks, HP7 9NA, UKTel: +44 (0) 7785 228 807Web: www.ge.com

Malvern InstrumentsMail: Enigma Business Park, Grovewood Road, Malvern, Worcs, WR14 3JE, UKTel: +44 (0) 1684 892 456Web: www.malvern.com

NanosightMail: Minton Park, London Road, Amesbury, SP4 7RT, UKTel: +44 (0) 1980 676 060Web: www.nanosight.com

Protein SimpleMail: 3040 Oakmead Village Drive, Santa Clara, California, USATel: +001 (408) 510-5500Web: www.proteinsimple.com

SGS M-Scan LtdMail: 2-3 Millars Business Centre, Fishponds Close, Wokingham,

Berkshire, RG41 2TZ, UKTel: +44 (0) 118 989 6940Web: www.sgs.com

T A InstrumentsMail: 730-740 Centennial Court, Centennial Park, Elstree, Herts, WD6 3SZ, UKTel: +44 (0) 20 8238 6100Web: www.tainstruments.com


MIBio List of Participants

DelegatesMohammed Aleheidib, NAVPC, [email protected]

Jamshed Anwar, University of Bradford, [email protected]

Anjali Apte, GlaxoSmithKline, [email protected]

Andreas Arnell, Lonza Biologics, [email protected]

Teresa Barata, Engineering & Physical Sciences Research, [email protected]

Karen Beech, The University of Nottingham, [email protected]

Charlotte Bell, Newcastle University, [email protected]

Barbara Bolgiano, NIBSC, [email protected]

Jens Breinholt, Novo Nordisk A/S, [email protected]

David Brockwell, University of Leeds, [email protected]

Jens Bukrinski, Novozymes Biopharma, [email protected]

Oliver Croad, The University of Nottingham, [email protected]

Lyn Daintree, CrystecPharma, [email protected]

Nicholas Darton, Arecor Ltd, [email protected]

Paul Davis, Mologic Ltd, [email protected]

Robert Dennehy, GlaxoSmithKline, [email protected]

Barry, Derham, Arecor Ltd,[email protected]

Jennifer Drew, GlaxoSmithKline, [email protected]

Nathalie Fa, GlaxoSmithKline, [email protected]

Neil Feeder, CCDC, [email protected]

Alexander Golovanov, University of Manchester, [email protected]

Andrea Goncalves, University of Nottingham, [email protected]

Andy Johnson, Immunocore Ltd, [email protected]

Richard Johnson, Upperton Limited, [email protected]

Natalie Kazimierczak, Kerry, [email protected]

Hanne Kinnunen, University of Bath, [email protected]

Krisztina Kovacs-Schreiner, University College London, [email protected]

Arthur Lallement, CambridgeIP, [email protected]




DelegatesFloriane Laurent, University of Bath, [email protected]

Robbie Mackenzie, Nottingham University, [email protected]

Andrew MacLeod, XstalBio Limited, [email protected]

Joao Francisco Vilhena, Madeira Do O, The University of Nottingham, [email protected]

Nuno Francisco, Madeira Do O, The University of Nottingham, [email protected]

Kiran Malik, NIBSC, [email protected]

Fiona McNamee, XstalBio Limited, [email protected]

Ciaran Murphy, Malvern Instrumentation,[email protected]

Mariam Nuhu, University of Manchester, [email protected]

Roderic O’Keeffe, Ipsen Biopharm Ltd, roderic.o’[email protected]

Gbolahan Oladiran, GlaxoSmithKline, [email protected]

Ian O’Loughlin, Teagasc, [email protected]

Derek Prater, Mundipharma, [email protected]

Balraj Rai, University Of Wolverhampton, [email protected]

Jascindra,Ravi,NPL,[email protected]

Hans Rogl, Boehringer Ingelheim Pharma GmbH & Co. KG, [email protected]

Yousif Sabeel, Central Medical Supplies, [email protected]

Garima Sharma, UCL School of Pharmacy, [email protected]

Lee Smith, GreyRigge Associates Ltd, [email protected]

Mei-an Sung, deltaDOT, [email protected]

Wei Tian, Glide Pharma, [email protected]

Malgorzata Tracka, MedImmune, [email protected]

Shahid Uddin, Medimmune Ltd, [email protected]

Frank, van de Manakker, FeyeCon Carbon Dioxide Technologies, [email protected]

Christopher van der Walle, MedImmune Ltd, [email protected]

Elizabeth Weaver, Ipsen Biopharm Ltd, [email protected]

Roger Wu, GlaxoSmithKline, [email protected]

Ahmed Yasin, GlaxoSmithKline, [email protected]


SpeakersLeonardo Borras, Alcon Labs, [email protected]

Paul Dalby, UCL, [email protected]

Susanne Jorg, Novartis, [email protected]

Hanns-Christian, Mahler, Roche,[email protected]

Natalia Markova, GE Healthcare, [email protected]

Michele Vendruscolo, University of Cambridge, [email protected]

Mike Williamson, Sheffield University, [email protected]

OrganisersStephen Harding, University of Nottingham, [email protected]

Jan Jezek, Arecor, [email protected]

Philippe Rogueda, Watson pharma, [email protected]

Tejash Shah, GlaxoSmithKline, [email protected]

Exhibitors & SponsorsMiles Ashton, Protein Simple, [email protected]

Marisa Barnard, SGS M-Scan Limited, [email protected]

Michael Caves, SGS M-Scan Limited, [email protected]

Colin Crowley, SGS M-Scan Limited,[email protected]

Jas Mahey, T A Instruments, [email protected]

Catherine McDonnell, GE Healthcare UK Ltd, [email protected]

Chris Miller, Avacta Analytical Ltd, [email protected]

Carolyn Mullen, Malvern Instruments, [email protected]

Lisa Newey-Keane, Malvern Instruments, [email protected]

Pierre Peotta, Nanosight, [email protected]

Anders Vagno Pedersen, Novozymes, [email protected]


mibio 2012 - formulationformulation.org.uk/images/mibio2012/mibio-out.pdf · mibio 2012 might be a...

Documents