d r u g d i s c o v e r y w o r l d

DDW

www.ddw-online.com

WINTER 2012/13

the quarterly business review of drug discovery & development

preventing pandemicsthe global influenza threat

When new and deadly strains of influenzaappear, their effects can be devastating,since a lack of immunity among the

population can lead to pandemic disease. Therewere three pandemics in the 20th century – in 1918,1957 and 1968 – which killed millions of peoplearound the world. While flu shots have been avail-able since the 1940s, when a virulent new strainappears there is a real scramble to get sufficientdoses of the vaccine ready to protect the population.

A significant problem derives from the fact thatthe vaccine required to give protection changesevery year. Unlike the situation with viruses such asmeasles, the antigen required to give protectionvaries depending on the predominant circulatingstrains of the influenza virus in any given year, as itconstantly mutates to form new strains.Vaccination remains the only realistic weaponagainst a flu pandemic. The antiviral drugsoseltamivir (Tamiflu) and zanamivir (Relenza) thatwere stockpiled by governments in response arenot particularly effective1, whereas vaccination canbe efficacious two-thirds of the time, according toa large meta-analysis published in LancetInfectious Diseases in 20122. While it is clearly lessthan perfect – and the protection figures are evenworse for the elderly – vaccination is still the bestweapon we have against influenza.

H1N1 pandemic underlines capacity challenge The H1N1 ‘swine flu’ strain was first identified inApril 2009, and by June its geographic spread wasso vast that the World Health Organization(WHO) declared it to be the first pandemic of the21st century. By this time, the seasonal flu vaccinefor the year was already in production. (The annu-al make-up of the vaccination is determined by

WHO’s Influenza Surveillance Network, whichmonitors the circulating influenza strains. It usual-ly contains three antigens, two of which areinfluenza A and one influenza B, and while it maybe the same from one year to the next, more com-monly one or two antigens are changed.) An add-on vaccine was hastily prepared to provide protec-tion against the new strain – but the twin problemsof time and capacity meant there were never goingto be sufficient doses in time for everyone whowanted one, despite the pandemic preparednessactivities that were already in place at the vaccinemanufacturers (Figure 1).

Influenza vaccines have historically been egg-based, with the antigen for each dose requiring oneegg to grow, and the whole process taking severalmonths. Capacity is finite, and when the pandemicwas announced most of this capacity was alreadycommitted to producing the normal seasonal vac-cine3. Cell culture is now starting to be used toproduce influenza vaccines, with the first cell cul-ture-derived vaccine, from Novartis, having beenapproved by the FDA in November. This will sig-nificantly reduce the time it takes to make the vac-cine and increase capacity. Cells can be banked andstored for long periods of time, ultimately allowingmanufacturing capacity to surpass that for egg-based influenza vaccines. Furthermore, viruses donot have to be adapted as they currently are foregg-based vaccine product. Finally, vaccines pro-duced in cells do not have the same immunogenic-ity profile as with egg-based, potentially allowing awider patient population to be immunised.

If sufficient vaccine is ever going to be availablefor the entire global population in the case of aninfluenza pandemic, then the global manufactureof influenza vaccine will also have to increase. Inthe US alone, more than 130 million doses were

By Dr NathanielHentz

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Preventing pandemicsOver the years, influenza has taken the lives of millions. While vaccines arehelping to reduce the damage caused by the virus, there is still a major capacityshortfall. Could access to US and European expertise and quality trainingimprove capacity and prevent the next pandemic?

distributed for the 2011-2012 influenza season.Globally, manufacturers produced 620 milliondoses of seasonal trivalent influenza vaccine in20114. At the time of the 2009 H1N1 pandemic,the best estimate was that maybe one billion dosesof a pandemic vaccine could be made. More couldbe made if adjuvants that enable smaller amountsof antigen to be used in each dose were more wide-ly accepted. With a global population now almostseven times the maximum non-adjuvanted capaci-ty, manufacturing capacity was clearly insufficient.

Quality challenges put capacity at risk While there may be sufficient capacity in a normal,non-pandemic year to meet demand for seasonalvaccine doses, if one of the manufacturers has aproduction problem, this would adversely affectavailability. This was brought into sharp focus inOctober, when several countries suspended the dis-tribution of Novartis’s seasonal flu vaccine afterprotein aggregates of predominantly haemagglu-tinin protein were found in doses. Rapid evalua-tion showed that the doses were effective and thedoses were released to patients. Dutch vaccinemanufacturer Crucell – now part of Johnson &Johnson – has also been hit by manufacturingissues, withdrawing its Inflexal V vaccine after twoof 32 batches failed quality control tests.

Issues like this highlight the potential problemsthat can result from a quality problem at a man-ufacturing site, and thus the importance of ensur-ing product quality throughout the manufactur-ing process. The majority of flu vaccines are cur-rently manufactured in the US and Europe, where

quality systems are embedded into the manufac-turing culture – and yet quality problems stilloccur. There is a real drive to expand the globalreach of influenza vaccine manufacture as thiswill increase both manufacturing capacity andresponsiveness in the case of a pandemic. Butwhat impact might this have on quality? Andwhat is being done to ensure that quality stan-dards are maintained, and that quality controlprocesses ensure that any sub-standard batchesare not released to patients?

What is being done about vaccine capacity?To address these capacity issues, WHO instituted aglobal action plan for influenza vaccines in 2006.One of its main objectives is to increase productioncapacity for pandemic vaccines without alteringthe capacity for normal seasonal vaccines. The aimis that, by 2015, enough vaccine for two billionpeople should be available six months after thevaccine prototype strain is transferred to manufac-turers. In the longer term, the hope is that suffi-cient vaccine for the entire world’s populationcould be prepared. This is to be done by expandingvaccine capacity, particularly in those regionswhere capacity is lacking or absent, and the devel-opment of high yield technologies to enable surgecapacity in the event of a pandemic.

The project has already had some success – since2006, global seasonal influenza vaccine capacityhad increased from less than half a billion doses tonearly a billion doses by 2010. In addition, morethan a dozen developing countries have beenawarded grants to establish their own influenzavaccine capacity, including Brazil, China, Egypt,India, Indonesia, Romania, Russia, South Koreaand Thailand. Several of these now have their ownvaccines on the market, with the rest in the latestages of development. WHO has also set up tech-nology transfer hubs in the Netherlands for theproduction of inactivated influenza vaccine in eggs,and Switzerland for adjuvant production.

With much of the capacity and technology in theUS and Europe, pharmaceutical manufacturers,academic groups and health authorities in thoseregions are working with WHO to help achieveincreased production. For example, the USDepartment of Health and Human Services’Biomedical Advanced Research and DevelopmentAuthority, or BARDA, has awarded several multi-million dollar grants to WHO to assist developingcountries to foster pandemic influenza vaccinemanufacturing infrastructure, train staff aboutinfluenza vaccine manufacturing and establish and

Figure 1Annual influenza production

timeline

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distribute technologies that will be useful in theevent a pandemic vaccine is required.

As part of the BARDA-WHO partnership, NorthCarolina State University’s BiomanufacturingTraining and Education Center (BTEC) was award-ed a significant grant to assist in the training of per-sonnel from vaccine manufacturing or researchorganisations in WHO member countries. The goalis to provide key staff from these institutions withhands-on instruction in the latest FDA good manu-facturing practices in vaccine production technolo-gy, so that these can be implemented back home toimprove quality systems and quality control withinthe facility.

The unique role of BTECThe BTEC training programme started in 2010,where three cohorts of 12 students each weretrained in the first year. At first, managers fromsome of these vaccine-producing institutionsattended a three-week long course on the funda-mentals of vaccine manufacture. The course pro-vides an A-Z on how to work with eggs and cells,right through the whole processing of the influenzavaccine from production to purification, analysisand the final aseptic fill. The idea was that theywould be able to provide their own people withsome training in vaccine manufacture. While thiswill certainly be helpful, it was clear from the out-set that it would not be sufficient, particularly assome of these institutions were more based inresearch than manufacture, and thus did not havethe background in working under GMP conditions.

Now, more advanced courses are being run, andinstead of managers the attendees are people who areactively involved in the production of vaccines. Inaddition to the first course on the fundamentals ofcGMP influenza vaccine manufacturing, two furthercourses are now being run on advanced processes forinfluenza vaccine manufacturing, one looking atupstream aspects and the second downstream. Theobjective is to get detailed knowledge of best prac-tices from the US into the hands of operators inplaces without a history of vaccine manufacture. Ifthe number of countries where good quality vaccinesare made is increased, then WHO’s goal of raisingthe global capacity and spread of influenza vaccinemanufacture is more likely to become a reality.

In addition to training scientists from around theworld the process of vaccine manufacturing underGMP conditions, BTEC also focuses on QC funda-mentals such as basic statistics, data analysis, trou-bleshooting, instrument calibrations and evenpipetting, while using state-of-the-art instrumenta-tion. The trainees can alter their pipetting tech-

nique and measure the effect on accuracy and pre-cision. The underlying importance of this pro-gramme is that emphasis on quality permeatesthrough all levels of scientists within vaccine man-ufacturing process. Training normally occurs in thespecific area that the scientist works. For example,QC training is typically reserved for QC scientists,but through this unique training programme atBTEC, scientists that normally work upstream(virus production) or downstream (virus process-ing and purification) have an opportunity to learnthe QC side. By using this approach the impor-tance of quality is instilled into all manufacturingareas, with the assumption that the quality of theproduct will ultimately improve.

Quality control in vaccine manufactureOne of the key issues that the BTEC courses coveris quality control. Fundamentally, QC ensures thata product is what it says it is, and meets all qualityand purity standards. In terms of influenza vaccinemanufacture, while the assays that are currentlyrequired by the regulators are fairly unsophisticat-ed, they are labour-intensive and require care.

SRID AssayPotency is measured using the single radialimmunodiffusion, or SRID, assay5-8. The antigenis introduced on to the centre of a gel containingan antibody to that antigen. The antigen diffusesthrough the gel, creating a precipitin ring that con-tinues to grow until it reaches equilibrium, aprocess that can take hours to overnight. The

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May 2012 – Trainees learn SDS-PAGE for assessing influenza vaccine purity and identity in theQC/analytical lab of the BARDA-sponsored Fundamentals of cGMP Influenza VaccineManufacturing course at BTEC

diameter of the precipitin rings are proportional tothe concentration of hemagglutinin (HA) on thesurface of the antigen presenting vaccine product.To estimate potency, the samples rings are com-pared to those created by reference standards(provided by approved agencies such as CBER andNIBSC) and whether this meets required specifica-tions. Although ring diameter measurement isautomated through imaging software, the wholetesting process is manual, and there is little oppor-tunity for automation. The gels are prepared asneeded at the time of the analysis. Furthermore,the gels are prepared with antibodies to the par-ticular influenza strains announced each year.Finally, this assay requires an overnight incuba-tion, followed by staining, destaining and dryingsteps. While it does give an indication of thepotency of the antigen, the variability of the assayis quite high and could use improvement.However, because SRID measures the effectivenessof a vaccine by examining the interaction betweenthe antigen (influenza) and its corresponding anti-body, it is representative of what is actually goingon in the body. While it does not give any infor-mation about whether an immune response can beelicited, it does show whether or not the antigencan be recognised by an antibody.

Titre determination A second test for potency or titre determinationsis the haemagglutination assay, an influenza-spe-

cific protein quantification assay9. HA is the pri-mary antigen protein present on the surface ofinfluenza viruses that causes red blood cells toclump together, or agglutinate. The influenzasample is incubated with a dilute solution con-taining any one of several species of red bloodcells (eg, chicken, turkey, horse, guinea pig, rabbitor sheep), and the virus dilution at which aggluti-nation starts is identified. Agglutination is thepoint where the solution is visually cloudy orhazy. However, this has to be determined by eye,and most often the agglutination point is notstraightforward. There are intermediate phasesbetween not- and fully-agglutinated. For exam-ple, a donut-shaped ring will form during partialagglutination, where the ring diameter can pro-vide an estimate of potency by comparing ringsizes. In this case, the dilutions near the point ofagglutination are typically further diluted toallow a better estimate of potency. Since the testis typically conducted visually, the accuracy isextremely dependent on the skill of the operator.

While these assays are variable and not particu-larly sophisticated, they are accepted by the regu-lators to demonstrate vaccine potency, despite awidespread recognition of their limitations.Alternative tests are being developed that will bemore amenable to automation and thus have lesslikelihood of variability, as well as being faster torun because of the potential for high throughputtechniques being applied to them.

May 10, 2011 – Traineesparticipate in a hands-on

activity in the intermediatescale upstream processing labduring the BARDA-sponsored

Fundamentals of cGMPInfluenza Vaccine

Manufacturing course at BTEC

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SDS-PAGEA third test relies on separation of proteins in a gelmatrix to provide identity and purity characteris-tics10. In this case, SDS-PAGE (sodium dodecyl sul-phate – polyacrylamide gel electrophoresis) is usedto separate proteins based on molecular weight. Bycomparing the sample banding pattern to a refer-ence standard, the identity and purity can be con-firmed. For further confirmation, Western blottingis used where the proteins are transferred to a mem-brane, which is then immunostained, allowinganother level of specificity. Although SDS-PAGEand Western blotting are well-established tech-niques, they are largely manual and semi-quantita-tive as best, leading to high variability.

Potential for automation and minimisationBTEC is addressing these deficiencies by workingon ways to automate the primary vaccine analyses.First, HPLC is being investigated for hemagglu-tinin content or potency11-12. In this technique,HA and its subunits are separated and quantitatedwith a high degree of precision and accuracy.Furthermore, HPLC is already automated fromsample injection through data analysis, thusaddressing the manual liabilities realised by SRIDand the hemagglutination assay. The second tech-nique is an intermediate solution – still carryingout the current style of haemagglutination assaybut using a plate-based technology to supplantvisual reading and interpretation. Instead, a spec-trophotometer could be used to look at turbidity ata particular wavelength. Potency would be esti-mated by comparison to fully agglutinated andnon-agglutinated samples. Removing the humansubjectivity and replacing it with a data-driven sys-tem would minimise the potential for variabilityand improve accuracy.

Finally, microfluidic platforms are being investi-gated as an orthogonal approach to SDS-PAGE.Specifically, microfluidics platforms such as theAgilent 2100 Bioanalyzer or the BioRad Experionautomated electrophoresis systems are well-suitedfor QC operations in that the sample separation,detection and data analysis are all conducted in alab-on-a-chip environment which is both automat-ed and enclosed. Furthermore, the data are cap-tured as electropherograms, allowing peak migra-tion times to be more accurately determined andthe peaks areas are integrated, allowing improvedquantitation in terms of accuracy and precision.

The ability to automate QC tests like thesewould not only speed up testing and improve accu-racy and precision by reducing manual manipula-

tion and interpretation, it would also ensure sam-ple traceability. If an operator misplaces a samplein a particular well because they are working withhundreds or even thousands of samples, it can neg-atively impact the integrity of the QC process. Byautomating QC procedures sample mislabelling ormisplacement in a rack are greatly reduced.Automation allows pipette tips to be tracked, forexample, even for more manual-style assays suchas haemagglutination. More modern assays basedon HPLC or some form of plate reader will alsohave an indexing capability, with data available fordownload and computer analysis. The data aremore robust, particularly in terms of sample iden-tification, with every sample tracked and identifiedcorrectly throughout.

Automated liquid handling systems are anotherway in which indexing can be ensured. Whereeach sample is coming from is certain, as is whereit is going. Samples of batches coming off themanufacturing floor have to be fully traceable,with multiple samples of the same batch destinedfor different assays. Critical decisions are made onthe results of these assays, and if a sample does notmeet the required standards the batch does notmove forward to the next stage of the manufac-turing process, and will likely have to be thrownaway. Data quality is of the utmost importance.Clearly, patient safety is the overriding driverbehind the whole QC process, so any substandardbatches must be eliminated. However, false posi-tives must also be avoided for economic reasons –bad data that incorrectly indicates that a batchdoes not meet the required standards could costmillions of dollars.

Although automated liquid handlers greatlyincrease the precision of assays, inaccuracy in liq-uid transfer processes can also be a major source ofassay error13. While the trade-off between qualityand productivity will never truly be resolved,advances in liquid handling quality assurance areeasing that struggle. The Artel MVS®Multichannel Verification System is the only tech-nology able to verify the accuracy and precision ofeach tip of an automated liquid handler in onerapid experiment. This allows laboratories to morefrequently verify the performance of their instru-mentation so that liquid handling errors can beidentified and removed before they impact uponassay results. The MVS is based on a techniqueknown as ratiometric photometry, which producesmeasurements that are traceable to internationalstandards, allowing comparability of all liquidhandling devices regardless of model, location ornumber of dispensing channels.

References1 Jefferson, T et al. BMJ 2009,339, b5106.2 Osterholm, MT et al. LancetInfect. Dis. 2012, 12, 36.3 Hickling, J and D’Hondt, E. Areview of productiontechnologies for influenza virusvaccines, and their suitabilityfor deployment in developingcountries for influenzapandemic preparedness. WorldHealth Organization Initiativefor Vaccine Research, GenevaSwitzerland, Dec. 20, 2006, 1-34.4 Partridge, J and Kieny, MP.Global production capacity ofseasonal influenza vaccine in2011. Vaccine, 2012, article inpresshttp://dx.doi.org/10.1016/j.vaccine.2012.10.111.5 Potency Determination ofInactivated Influenza VirusVaccines by the Single RadialImmunodiffusion (SRID)Method. U.S. Food and DrugAdministration/CBERDocument ID: 0003337/21/2011.6 Wood, JM et al. Journal ofBiological Standardization, 5(1977) 237-247.7 Williams, MS et al. Journal ofBiological Standardization, 8(1980) 289-296.8 Vaccines for Human Use –General Considerations. USPharmacopeia 1235, 2011.9 Killian, ML (2010). Chapter7: Hemagglutination Assay forthe Avian Influenza Virus. In E.Spackman, Methods inMolecular Biology, Vol. 436:Avian Influenza Virus (pp. 47-52). Totowa, NJ: Humana Press.10 Shaw, ML et al. PLoSPathog, 4(6), 2008, e1000085.11Kapteyn, JC et al. Vaccine27, 2009, 1468–1477.12 Lorbetskie, B et al. Vaccine29, 2011, 3377–3389.13 Hentz, N. SLAS tutorial2012, 02,06.

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Ultimately, our best chance of preventing aninfluenza pandemic in future will involve the devel-opment of more effective vaccines as well as speedymanufacture, increase in number of doses and pre-venting spread at the origin. Universal vaccines arebeing investigated as these could give much broad-er protection against different influenza strains,and remove the need for annual vaccinations.However, these are still some way in the future,and until then the need for increased global capac-ity for influenza vaccine manufacture remains.Training operators from new facilities in modernquality control methods and current analyticaltechniques, and adoption of robust quality sys-tems, will be vital if the goal of being able to man-ufacture sufficient vaccine for the world’s entirepopulation is to be achieved. DDW

Dr Nathaniel Hentz is the Assistant Director of theBTEC Analytical Lab at North Carolina StateUniversity. Prior to this current role, Dr Hentzserved as an independent consultant working withArtel offering guidance on their efforts towardautomated liquid handling quality control withinhigh throughput screening laboratories. Dr Hentz’stenure in the HTS industry includes nearly twoyears as Senior Research Investigator at Bristol-Myers Squibb in Wallingford, CT where his teamsupported the fully-automated screening systemswithin the Lead Discovery group. Prior to BMS,Dr Hentz enjoyed seven years at Eli Lilly RTP

Laboratories in North Carolina. During his tenureat Lilly RTP, Dr Hentz was responsible for Tier 1ADMET screening in 2004 and led the NewTechnologies group. He received his PhD in ana-lytical chemistry from the University of Kentuckyin 1996 and joined Lilly as a postdoctoral scientistthe same year.

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June 22, 2011 – Trainee uses Artel PCS system to practise pipetting technique as part of theQC/analytical lab during the BARDA-sponsored Fundamentals of cGMP Influenza VaccineManufacturing course at BTEC