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TosohThe cusTomermagazine
Tosoh Bioscience
no
.#01
2
011
40 Years of sec
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02ediTorialdear readerdear reader, welcome to the latest issue of the Tosoh Bioscience customer magazine. first of all, we would like to thank everyone for the support and concern that you have shown with regard to the series of events set off by the massive earthquake in Japan. We would like to inform you that no injuries, no damage, and no change in radiation levels from those recorded before the earthquake, were reported from our production site, the nanyo manufacturing complex, which is located more than 1000 kilometers away from the quake‘s epicenter. The motto of this issue is 40 years of size exclusion chromatography (sec) at Tosoh. it is featuring some aspects of the scientific and technological expertise of Tosoh Bioscience in sec technology. The development of the first TsKgel gPc column in 1971 has set the course for a long and proud history of size exclusion chromatography at Tosoh. The hPlc application of this issue with the title ‘sec method for cellulose acetates of wide ds-range‘ was established on an ecosec gPc system at the deutsches Kunststoff-institut in darmstadt by the group of dr. Wolfgang radke. The ‘tips & advice’ section also deals with sec applied for the analysis of protein aggregates with light scattering detection and was provided by Judith Vajda, who recently joined the Tosoh Bioscience technical support team in stuttgart.
enJoY reading and sTaY informed.
regina roemling | marKeTing manager
Tosoh Bioscience gmBh
conTenT
Page [02 - 03] ediTorial conTenT 40 Years of sec Page [04 - 05] conference reVieW Page [06 - 07] TiPs & adVice cusTomer aPPlicaTion Page [08] forum neWs & eVenTs
Tosoh Bioscience gmBh
zeTTachring 6 | 70567 sTuTTgarT | T: +49 [0] 711 13257- 0 | f: +49 [0] 711 13257- 89 | [email protected]
J11lP04a
imPressum
Tosoh bioscience anaLYsis Process insTrumenTaTion
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The TsKgel h-type, polymer-based column series for the analysis of organic-soluble polymers and the first instrument for gPc manufactured by Tosoh was introduced to the Japanese market in 1972, only one year after the introduction of the first gPc column. Today the compact, semi-micro gPc system ecosec is representing the 7th generation of sec instrumentation technology. in 1977 the first genetically-engineered recombinant human insulin was produced in E.coli by herbert boyer and became the first medicine produced by recombinant Dna technology to be approved by the FDa. at the same time Tosoh expanded the sec portfolio by a porous, silica-based, high performance gel filtration column line, the TsKgel sw columns. These columns and their successor products became the leading gel filtration hPLc columns used in biotech industry. simultaneous with the increasing number of approved biotherapeutics the use of these columns for aggregate analysis of recombinant proteins grew continuously over the last 34 years. several hundred scientific publications mention TsKgel sec columns and numerous usP/eP/JP standard methods are based on TsKgel sw series columns. 1978 saw the start of the TsKgel Pw-type column line for gel filtration of water-soluble polymers and in 1979 Tosoh started the development of Toyopearl resins for preparative liquid chromatography applied in biopurification. based on polymethacrylate based Toyopearl hw resins a whole range of process resins for downstream bioprocessing have been evolved since then.
over the years, TsKgel h, TsKgel sw and TsKgel Pw-type column lines have undergone continuous improvement through advances in particle synthesis and column technology. major sec technology leaps were the development of multi-pore particle technology, the most elegant way to achieve near linear sec calibration curves, and the introduction of semi-
micro dimensions for high performance sec columns. multi-pore particle technology solves the known problem of peak disturbances/inflection points, which typically occur due to a mismatch of pore sizes when columns with different molecular weight ranges are coupled. Particles produced by multi-pore technology contain a broad range of pore sizes in a single polymeric bead. This innovative approach essentially creates a linear calibration curve within each particle.
Today the range of sec related products comprises of sec/gPc systems, Toyopearl hw process resins, and hPLc stationary phases for both modes of sec, aqueous gel filtration (gFc) and organic gel permeation chromatography (gPc). For aqueous sec TsKgel sw series silica columns for protein analysis as well as polymer based TsKgel Pw columns for the analysis of water soluble polymers are available. TsKgel alpha & superaw columns are suitable for both, aqueous and organic mobile phases. The portfolio for gPc comprises polystyrene based TsKgel hxl, hhr, and superh columns. Latest additions to the sec column lines were TsKgel Pwxl-cP columns for the analysis of aqueous cationic polymers and linear, semi-micro sec columns, the TsKgel supermultiporePw series, produced by multi-pore technology and designed for fast size distribution analysis of water soluble polymers, such as polyvinylpyrrolidones or dextrans. current product development for high performance sec at Tosoh is striving after a further increase in resolution and speed of separation.
40 Years of eXPerTise in sec
03 TsKgelsec columns
The deVeloPmenT of The firsT TsKgel column for gel PermeaTion chromaTograPhY (gPc) - TsKgel sW - in 1971 had seT
The course for a long and Proud hisTorY of size eXclusion chromaTograPhY (sec) aT Tosoh. aT ThaT Time The lacK
of suiTaBle Tools for The analYsis of Tosoh’s PolYmer ProducTs Was The driVing force Behind The deVeloPmenT of
TsKgel chromaTograPhY columns.
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The scientific committee of the 7th hic/rPc bioseparation conference, headed by alois Jungbauer, university of natural resources and applied Life sciences, Vienna, austria, composed a striking program of 24 lectures, accompanied by poster presentations and a round table discussion. Following its tagline ‘advancements, applications, and Theory in Downstream Processing’ the conference traditionally focuses on bioprocess applications and on fundamental research on hydrophobically influenced modes of chromatography. Today the emphasis of the conference shifted towards hydrophobic interaction (hic) and mixed mode chromatography (mmc), as the importance of reversed phase chromatography for the purification of biological target molecules has declined over the past years.
Traditionally, the conference commences with a keynote lecture on a current trend or a new development in biotechnology. accordingly this year, Florian rüker from the university of natural resources and applied Life sciences, Vienna, austria, a cofounder of f-star, gave an insight into latest developments in antibody scaffolds. f-star is a company developing therapeutic antibodies and antibody fragments based on its proprietary ‘modular antibody Technology’. This technology can be used to engineer additional binding sites into antibodies without changing the natural antibody format. in the keynote lecture two antibody derived constructs were presented, Fcab and mab2. Fcabs - Fc fragments with engineered antigen binding sites - may be used as stand-alone entities (Fcab: Fc antigen binding), representing molecules with the full functionality of antibodies but only one third the size of a complete antibody. Fcabs can
also be used as building blocks in the context of complete antibodies, so-called mab2, which are fully functional bispecific igg molecules displaying antigen binding sites in the Fab arms as well as in the engineered Fc region. a second key note lecture, held by andreas Premstaller, head of technical development at sandoz, Kundl, austria, gave an overview about the opportunities and challenges for biosimilars process development and for the confirmation of biosimilarity by appropriate analytical, biological, preclinical and clinical studies.
The fundamentals sessions provided interesting data on modeling of chromatographic systems for both, hydrophobic interaction and mixed mode chromatography, in order to examine the relevant interactions in these systems. Lectures covered various aspects of biomolecule adsorption onto hydrophobic surfaces e.g. the folding of proteins upon adsorption and refolding upon desorption or surface energetic of protein adsorption. The interplay between ionic and hydrophobic interactions when using multi-modal stationary phases was presented by christian Frech, university of applied science, mannheim, germany. egbert müller, Tosoh bioscience, stuttgart, germany, presented interesting results on the use of mixed electrolyte mobile phases for modulation of binding capacity and selectivity in hic.
a round table discussion of all members of the scientific committee covered various aspects of hic/rPc/mmc related downstream processing, ranging from the interplay between science and industry - e.g. the problems of intellectual property issues preventing the further
7Th hic/rPc BioseParaTion conference in esToril, PorTugalin The PreVious issue of This magazine We announced The 7Th hic/rPc BioseParaTion conference Which Was held
from march 21-24, 2011 aT The hoTel Palacio in esToril, PorTugal. The one hundred aTTendees enJoYed The conference
in a BeauTiful surrounding aT The coasTline of The aTlanTic ocean in fanTasTic sPring WeaTher. The scienTific
Program coVered BoTh, fundamenTal science/engineering and imPorTanT indusTrial adVances and aPPlicaTions.
04 conference reVieW
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Tosoh bioscience anaLYsis Process insTrumenTaTion
use of some applications or resins for certain target molecules and the duty of industry to share results in order to increase knowledge - to the heretic question ‘does industry still need hic in times of fixed purification platforms?’. There was a consensus that hydrophobically influenced modes will always be part of the purification toolbox but will have to prove their value.
most industrial case studies and applications presented were related to antibody purification processes. in some platform approaches hic or mmc is used after protein a and ion exchange capture/intermediate steps for the final removal of aggregates and residual host cell proteins. Jie chen, Dyax corp., cambridge, usa, who attended the conference for the third time, presented comparison studies for capture step strategies. some case studies with monoclonal antibodies revealed the critical role capture technology choice plays in relation to the corresponding adaption of established platforms methodology and its impact on overall process efficiency and product quality. Tim breece from Xoma, berkeley, usa demonstrated that a hic polishing step in an antibody purification process can also result in an additional viral clearance. several presentations showed how parameter optimization for platform solutions can be supported by applying design-of-experiments (Doe) strategies and high throughput screening (hTs) devices. other aspects of industrial use of hic or mmc, such as industrial scale column packing and new process column technology, were presented. Doe and hTs were also the standard techniques presented in the Quality-by-Design session. according to the ich guidelines the pharmaceutical industry is encouraged to generate a deep and full process understanding of drug product manufacturing. stefan Lohninger from sandoz gmbh, Kundl, austria, presented as an example the characterization strategy for a reversed phase step applied in a peptide purification process. relevant process parameters were classified based on Doe studies and then applied to establish proven acceptable ranges. based on a qualified scale down model the experiments were executed in terms of Doe, on-off studies, worst case runs and a cleaning-in-place (ciP) study.
The Journal of chromatography a will publish a special edition on the conference. in addition to the scientific program the conference offered several opportunities to network with colleagues. The Portuguese landscape, the beautiful conference venue, tasty meals and an excursion to the famous city of Lisbon including a visit to the unesco world heritage Jeronimos monastery and a dinner in the bica do sapato restaurant further contributed to making this conference an unforgettable event for all participants. The biannual hic/rPc bioseparation conference series alternates between europe and the us. Tosoh bioscience is the sole sponsor of this conference series and provides support for logistics and organisation for the scientific committee. The 8th hic/rPc bioseparation conference will be organized by Tosoh bioscience LLc and will take place in the united states in 2013.
The conference WeBsiTe WWW.hic-rPc.org Will KeeP You uPdaTed
here’s WhaT The aTTendees said aBouT The conference:
“VerY good oPPorTuniTies for neTWorKing. good miXTure of academic and indusTrY relaTed
ToPics”
“i haVe aTTended isPe, iBc, PreP eTc. BuT This is BY far The BesT meeTing i haVe aTTended - good
miX of research and indusTrY ToPics, eXcellenT neTWorKing”
“VerY good neTWorKing. good miX academic/indusTrY. good miX TheorY/realiTY”
“Well organized. PleasanT miXTure of eVenTs and TalKs”
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as a matter of their size, mab aggregates produce scattered light more efficiently than they absorb uV light at 214 or 280 nm. nevertheless, light scattering is not a plug and play technique, compared to simple uV detection. it may take several approaches to get the hPLc system including a sec column running.
we would like to provide some advice that may help to improve the signal to noise ratio and the robustness of the complete system. First of all, it is important to keep the whole system clean and to refresh the eluent approximately every 2-3 days. if you want to avoid sodium azide, you will have to change buffers daily. at least for protein applications, we did not detect any noise problems in the scattering signal related to sodium azide. Therefore we recommend adding 0.05 % sodium azide to aqueous buffers in order to prevent bacterial growth. when working with organic solvents, proper degassing of the eluent is highly recommended.
however, any eluent, either organic or aqueous needs to be filtered thoroughly. air bubbles and small particles will not be detected by uV but will disturb the light scattering signal. changing in-line filters regularly, as well as filtering buffers through a 0.1 µm membrane prevent noisy baselines. Detector contamination due to impurities of the sample as such appears frequently. in this case, the system without the column should be cleaned by rinsing it with water containing 1 % sDs for 4-5 hours, followed by a quick purging step with water and an overnight washing step with a mixture of 20 % ethanol 80% water. against persisting noise the system can be flushed with a sample degrading solvent. Proteases for instance might help if you work with proteins.
noise generated by the hPLc column can be reduced by cleaning the column as described in the manual delivered with the column. overloaded columns also release sample molecules independent from retention time, causing a noisy and drifting baseline. The TsKgel swxl series can be cleaned efficiently by flushing overnight with 0.1 m glycine/hcl buffer ph 3.
Finally, there are some issues concerning the system flow rate. always change flow rates gradually. equilibrating the column with 4-5 column volumes at operational flow stabilizes the baseline. You might improve signal to noise ratio by increasing the system flow as long as the pressure limits are not exceeded. small flow rates do not only generate peak broadening due to diffusion, but also result in a noisy baseline. These tips are ought to provide a reliable light scattering signal.
in fact, static light scattering is a multi-detector measurement. Therefore the concentration source, which is usually either uV absorption or the refractive index, is also important. in case of uV absorption at 214 nm
(e.g. for mab aggregate detection) you have to be aware that sodium azide absorbs at wavelengths in the low two hundreds. hence we recommend using the ri-signal as source of concentration, also because the refractive index increment is approximately the same for all proteins. nevertheless the ri-signal does have disadvantages, too. it is prone to solvent peaks, which disable measurements of molecules with the same retention time. solvent peaks can be reduced by preparing samples in daily refreshed system solvent. similar to the solvent peak, the injection peak is not related to the sample itself. The injection peak can be decreased by slowing down injection speed.
in general and as a conclusion of our experiences, the light scattering instrument is the first to indicate any unsteadiness in the system. system suitability tests with a standard sample can be used to check the system performance before starting an analysis sequence. once the performance is ensured, an additional light scattering detector provides valuable information about the presence and structure of protein aggregates and the monomer itself as shown in the chromatogram of a mab aggregation analysis below.
sec-uV-ri-lighT scaTTering analYsis of a monoclonal anTiBodY
on TsKgel g3000sWxl: ri (greY), uV (280 nm, Blue) & 90° ls (red)
imProVing sec analYses WiTh lighT scaTTering deTecTionsTaTic lighT scaTTering in comBinaTion WiTh size eXclusion chromaTograPhY is a ValuaBle Tool for VerifYing PuriTY
of monoclonal anTiBodies (maBs) as such or as a quicK checK While The doWnsTream Processing TaKes Place.
Besides fluorescence deTecTion, lighT scaTTering is one of The mosT sensiTiVe meThods To deTecT ProTein aggregaTes.
06 TiPs & adVice
0 2 4 6 8 10 12 14 16
LB 90°
A 280
RI
time in minutes
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Tosoh bioscience anaLYsis Process insTrumenTaTion
sec provides a convenient way for the characterization of molar masses and molar mass distribution of synthetic and natural polymers. however, a crucial balance of the polarities of the analyte, the solvent and the stationary phase is required for a reliable sec method. since Ds of cellulose derivatives alters the polarity, solubility and aggregation behaviour, the sec methods found in literature are usually valid only within a narrow Ds-range. Published sec methods usually apply ThF or DmF modified by the addition of Libr as eluents on styrene-divinylbenzene (sDV) columns. however, often neither the degree of substitution nor the Ds-range applicable for the given chromatographic conditions are specified. The only sec system to be suited for cellulose acetate over a wide Ds-range (Ds range 0.4-1.2) uses pyridine/water on a grams column. This application note reports on sec-separations of high molar mass cas for cas of higher Ds in the range Ds = 1.5-3.
most samples were prepared by partial saponification of a commercial ca sample having a Ds of 2.6. other samples were of industrial origin. a Tosoh biosystems ecosec-system equipped with a maLLs detector (Dawn DsP, wyatt Technology, santa barbara, usa) was used. The stationary phase was a gram linear XL column (10 µm particle size, 30 x 0.8 cm, Pss Polymer standards service, mainz, germany). Dmso was used as the eluent. The low refractive index increment of cellulose acetates in Dmso (0.024-0.044 mL/g, depending on Ds) does not allow for reliable molar mass determination by sec-maLLs. The very sensitive ecosec ri, however, could easily detect those samples reliably. however, the light scattering instrument revealed aggregation if no or only low Licl concentrations were used. aggregation was not observed at Licl concentrations above 50 mmol/L. some of the samples required higher salt concentrations for dissolution. Figure 1 shows a comparison of the chromatograms of three cas of different Ds, which were prepared from the same base material. The elution volumes and the peak shapes of the samples are nearly identical, indicating that no degradation occurred during saponification. This has also been confirmed by molar mass determination using light scattering in a different solvent with higher sample contrast (dn/dc). The close agreement in the chromatograms of the samples can therefore be regarded as an indication of nearly identical calibration curves of the cellulose acetates despite differences in Ds. The fourth sample of
Ds = 1.72 is a commercial sample. it is presented in order to show that the close agreement of the chromatograms is not due to an insufficient chromatographic resolution. The independence of the calibration curves on Ds is advantageous. it will allow reliable molar mass determination of cellulose acetates without worrying about variations of sec elution volume due to differences in Ds.
sec meThod for cellulose aceTaTes of Wide ds-rangecellulose aceTaTe (ca) is one of The mosT imPorTanT cellulose deriVaTiVes. iTs main aPPlicaTions are The use in
memBranes, films, fiBers, PlasTics, and filTers. The chemical and PhYsical ProPerTies of These deriVaTiVes are
sTronglY influenced BY The degree of suBsTiTuTion (ds) and The molar mass and molar mass disTriBuTion. Therefore,
There is a sTrong need To characTerize These ProPerTies.
07 cusTomer aPPlicaTion
6 7 8 9 10 11 12
V/mL
1.2
1.0
0.8
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⇒⇒figure 1: sTacKed oVerlaY of chromaTograms of cellulose ace-
TaTes of differenT ds: ds (greY: ds = 2.45, red: ds = 2.09, green: ds = 1.59)
deriVed from The same Base maTerial BY ParTial saPonificaTion and a
commercial samPle (Blue: ds = 1.72).
⇒auThors: heWa oThman ghareeB1, Wolfgang radKe1 & PeTer Kilz2
[1] deuTsches KunsTsToff-insTiTuT, schlossgarTensTr. 6, 64289 darm-
sTadT [2] Pss PolYmer sTandards serVice gmBh, in der dalheimer Wiese 5,
55120 mainz
⇒⇒reference:
[1] soo-Jung lee, clemens alTaner, Jürgen Puls and Bodo saaKe, carBo-
hYdraTe PolYmers 54 (2003) 353-362
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‘forum ProzesschromaTograPhie‘ 2011 in sTuTTgarT
meeT Tosoh Bioscience aT TradeshoWs and conferences or Join one of our renoWned WorKshoPs
uPcoming eVenTs
June 19 - 23 | 2011 hPLc 2011 | buDaPesT [hungarY] June 27 - 29 | 2011 18Th arbeiTsTagung miKromeThoDen in Der ProTeinchemie | marTinsrieD [germanY] ocT. 11 - 13 | 2011 bioTechnica 2011 | hannoVer [germanY] noV. 21 - 22 | 2011 noVia hPLc Tage 2011 | FranKFurT [germanY]
Trainings | WorKshoPs
seP. 21 - 22 | 2011 Forum ProzesschromaTograPhie | sTuTTgarT [germanY]
neWs & eVenTs | meeT Tosoh Bioscience
08 chromaTograPhYWorKshoPs
The adVanced chromaTograPhY WorKshoP goT a neW name. The adVanced chromaTograPhY WorKshoP in german
language differenTiaTed from The Well KnoWn Basic WorKshoPs on Process chromaTograPhY in iTs formaT. The
adVanced course ProVided a Broad VarieTY of lecTures aBouT releVanT ToPics of doWnsTream Processing BuT no
PracTical hands-on eXPerimenTs. To BeTTer reflecT iTs characTer of a Kind of sYmPosium on Process chromaTograPhY
iT has Been renamed To ‘forum ProzesschromaTograPhie’. This eVenT Will TaKe Place in sTuTTgarT in sePTemBer.
The program will comprise of one and a half days of various lectures combined with a short excursion including dinner. The lectures will be presented by recognized speakers from academia and industry involved in process development and downstream processing. in 2009 the advanced workshop provided insight into the following topics: purification of monoclonals and other therapeutic proteins, practical aspects of column packing, high throughput resin screening, process development using mixed mode resins, process characterization and validation, process control, new resin developments and continuous chromatography. Presenters came from biopharmaceutical industry, such as rentschler biotechnologie, sandoz, boehringer ingelheim, from technology suppliers, such as atoll, chromacon and Tosoh bioscience, and from academia, such as the boKu Vienna.
The program for the ‘Forum Prozesschromatographie‘ 2011 is not yet completed but it will again combine current topics in downstream processing, such as high throughput screening and platform approaches for specific targets with praxis-related presentations. it is our aim to provide another high level event for advanced chromatographers involved in process development, validation, and scale-up.
The Tosoh Bioscience WeBsiTe WWW.TosohBioscience.com Will
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