development of separation methods for recombinant ...€¦ · wcx-10 4 x 250 mm; protein-pak hi-res...

Research Article Applied Optics 1

Development of separation methods for recombinantBotulinum neurotoxins, using a statisticalmodelling-based approachSOFIA NUNES1

1Corresponding author: [email protected]

Compiled November, 2017

In the Biopharmaceutical field, the development of separation methods is essential for the release, stabil-ity testing and characterization of therapeutic proteins to ensure purity, efficacy and safety of the clinicalproduct. Size Exclusion (SEC) and Ion Exchange chromatography (IEC) are common separation tech-niques used for aggregate, fragment and charge isoform characterization, being robust qualitative andquantitative methods. SEC separates protein based on size and is used for monitoring aggregate andfragments. IEC uses the protein surface charge to indicate the charge heterogeneity of the molecule.The goal of this thesis is to establish and optimise the Size Exclusion and the Ion Exchange chromatogra-phy methods for different endonegative (inactive) serotypes of recombinant Botulinum neurotoxins. Todevelop these methods, computational Design of Experiments (DoE) were performed to evaluate differ-ent mobile phases, pH, temperature, flow rate, columns and run times, to maximize the peak resolutionand recovery and, also, to minimize peak tailing.The work described in this thesis demonstrates the presence of arginine as a mobile phase additive iscrucial for SEC performance, and that the same optimised conditions can be used for different toxinserotypes. However, IEC development is more product specific, thus different conditions must be devel-oped for the different serotypes. For the serotype studied, pH and temperature were shown to be criticalfactors.Keywords: Botulinum neurotoxins, SEC, IEC, separation methods, DoE© 2017 IST

OCIS codes:

1. INTRODUCTION

In neurosciences, Botulinum neurotoxin as a biotherapeu-tic is used to treat several diseases, such spasticity, cervicaldystonia, hemifacial spasm and blepharospasm [1].

Botulinum neurotoxins (BoNT) are produced by neu-rotoxigenic strains of anaerobic and spore forming bac-teria of the genus Clostridium (Clostridium botulinum,Clostridium butyrricum, Clostridium barati, and Clostrid-ium argentinensis) [2]. This neurotoxin is the most lethalacute toxin known and when ingested in contaminatedfood can interfere with muscle function causing paralysisor death [3]. However, it is a successful therapeutic agent

used for clinical and cosmetic applications [1].The development of BoNT as a biopharmaceuticals

is complex and highly regulated by regulatory bodies,such as the FDA and EMA, require a full understandingof the drug. So, size exclusion (SEC) and ion exchangechromatography (IEC) are important analytical tool [4].SEC is required to identify and measure aggregates andfragments. IEC characterize and identify charge isoformsof the protein therapeutic [5].

Size Exclusion Chromatography (SEC) separatesmolecules based on differences in size and shape. Thebiomolecules pass through a spherical porous particlestationary phase with a specific pore size and diffuse


depending on the molecular size using a mobile phase.High molecular weight species, for example aggregatedbiomolecules, cannot penetrate the pores of the station-ary phase and therefore elute first from the column. Lowmolecular weight species, for example protein fragments,partially or completely enter the porous particles, slowingtheir progression and therefore eluting later [6][4].

To develop a SEC method with good resolution, manyfactors must be considered such as column (particle sizeand uniformity, bed height, diameter and length), flowrate, sample volume and viscosity. The mobile phase com-position, pH and ionic strength are also important but arenot related directly with the resolution. However, theselast factors influence the conformation or biological activ-ity [4][6][7]. Also, an important consideration for a goodSEC method establish is the possible resin-protein interac-tion: electrostatic and hydrophobic interactions. To avoidthat a good choice of the resin and, specially, the mobilephase should be performed. To suppress electrostatic in-teractions the salt or the ionic strength of the buffer canbe optimized. It is important to consider the salt concen-tration because, at low salt concentration, electrostaticinteraction dominates, and, at high salt concentration,the hydrophobic interaction dominates. The reduction ofhydrophobic interactions can also be achieved by addi-tion of organic solvents, such as acetonitrile and alcohol[4][6][8]. Arginine is a common additive used to suppressnon-specific interactions between protein-resin and pre-vent additional aggregation, giving better resolution andhigher aggregation and protein recovery [9][10][11].

Ion Exchange Chromatography (IEC) is a separationmethod based on net surface charge of proteins. The netsurface charge depends on the protein charged groups,each with different pKa values (acid ionization constants).Ion exchange separation occurs by a reversible interac-tion between a charged surface protein and an oppositelycharged chromatography medium [12][13].

The choice of the IEC stationary phase and mobilephase gradient depends on the isoelectric point of theprotein. If the solution pH is higher than the pI, theprotein will be negatively charged and therefore bindto a positively charged anion exchanger. If the solutionpH is lower than the pI, the protein will be positivelycharged and therefore bind to a negatively charged cationexchanger. The anion exchanger has positively chargedfunctional groups linked to the matrix, so the more nega-tively charged variants elute later than the less negativelycharged variants. On the other hand, the cation exchangerhas negatively charged functional groups linked to thematrix, so the more positively charged variants elute laterthan the less positively charged variants. The analyte re-tention depends on mobile phase pH, ionic strength, andcounter ion additives. The interactions between station-ary phase and the protein varies along the mobile phasegradient that alters the charged state and solubility ofthe analyte or/and ionization state of stationary phase[13][14][15].

Mass spectrometry can be used to identify charge mod-ifications due to the increase or decrease in mass of a basicor acidic isoform relative to the unmodified protein. Thereare three ways to analyses therapeutic data: intact mass;reduced mass (Middle) and digestion for peptide map-ping (Bottom). The intact mass give information of theoverall heterogeneity of the protein, but not provide thetype of modification or location. The reduced mass pro-vides information of the light chain and heavy chain oranothers fragments confirming the distribution gave byintact mass and detecting where is the sequence modifi-cation (LC or HC). The peptide mapping identifies smallmodifications or no mass changes in the intact protein,giving a better understanding of the modifications on theprotein [16].

2. DEVELOPMENT METHODOLOGYThe main strategy of this project was to use the power ofDesign of Experiments (DoE) to define the main methodfactors and interactions that influence resolution, peaktailing and recovery and predict optimized conditionsfor improved resolution. The DoE approach can evalu-ate multiple factors at the same time with reduced runsthan the conventional ‘one factor at a time’ approach. Theapplication of DoE in chromatography method develop-ment provides a detailed mathematical analysis of thesystem and resolution of the problem with minimal, well-planned experiments that avoid time-consuming trial anderror methodology [19] [20]. For the DoE and respectiveanalysis two statistical software packages were used: JMPand Minitab. The flow diagram for SEC and IEC methoddevelopment and optimization is on fig.1.

Fig. 1. Simplify SEC and IEC development methodol-ogy.


3. EXPERIMENTAL

A. SEC

The SEC experimental procedure included: mobile phasepreparation, sample preparation, ULPC run and data anal-ysis.

Different mobile phases where prepared with L-Arginine monohydrochloride, sodium chloride, sodiumphosphate monobasic anhydrous, Sodium phosphatemonobasic anhydrous and ultra-pure water. Always, ad-justing the pH and filtering through a 0.22µm filter.

Sample preparation was the dilution of the type BoNTin PBS if required. The blank was mobile phase.

The sample was introduced in Water Acquity UPLCsystem to be separated with the column Waters AcquityBEH SEC 200 1.7µm, 4.6 x 150 mm.

The Empower 3 was the software used to preform theseparation and process the data.

B. IEC

The IEC experimental procedure include: mobile phasepreparation, sample preparation, ULPC run and data anal-ysis.

For IEC, it was required a buffer A and buffer B (forthe gradient). Different buffer A where prepared withMES monohydrated, MES sodium salt, Trisodium Citrate,Tris base, Tris HCl, Sodium phosphate dibasic anhydrous,Sodium phosphate monobasic anhydrous and ultra-purewater. The buffer B was the buffer A preparation plussodium chloride. The adjust of the pH and filter througha 0.22µm filter was always performed.

Sample preparation was the dilution of the type BoNTin PBS if required. The blank was buffer A.

The sample was introduced in Water Acquity H-ClassUPLC system to be separated with the columns: PropacWCX-10 4 X 250 mm; Protein-Pak Hi-Res Q 4.6 x100mm5µm; Sepax Proteomix SEX 4.6 x 100mm 5µm.

The Empower 3 was the software used to preform theseparation and process the data. For the last study of theinfluence of temperature and pH on IEC profile, it wasused Autobled Plus.

C. IEC fractionation and MS

For the IEC fractionation, the method was described onfig. 2. To buffer exchange the fractionated samples, 100mM Tris pH 7.6 was used. The samples where collectedfrom IEC column as represented on fig. 9.

For mass spectrometry, buffer A was a solution offormic acid and buffer B was formic acid with acetoni-trile solution. The equipments were: Synapt G2Si (QTOF)from Waters, UPLC Acquity H-class and column ProteinBEH C4 300Å 1.7µm 2.1 x 50 mm. The data was analyzedby MassLynx.

Fig. 2. IEX fractionation conditions.

4. RESULTS & DISCUSSION

A. SECA full factorial design with a centre point in Minitab (20runs) was chosen to evaluate 4 factors:

- Sodium phosphate concentration: 20-200 mM;- Sodium chloride concentration: 0-300 mM;- Arginine concentration: 0-300 mM;- Serotype: 2 and 3.The main response evaluated was USP tailing.The analysed data of the DoE are on fig. 3 and 4.

Fig. 3. Main Effects plot for USP tailing factor.

The arginine concentration is the factor that has moreimpact on the USP tailing factor response. The increasingarginine causes a clear reduction of the USP tailing. Inthe presence of high arginine concentration, the variancein sodium phosphate or NaCl concentration had a lowimpact on the response. However, without arginine, thehighest concentrations of sodium phosphate and NaClimproved the peak tailing. The serotype is the factorwith the least effect on tailing, confirming the possibil-ity of using the same HPSEC method for the differentserotypes. This was also confirmed by visualization of thechromatograms.

By the response surface for USP tailing and recovery, itwas concluded that the sodium phosphate concentrationshould not be too low or too high, so to have reduced


Fig. 4. Interactions plot for USP tailing factor.

tailing and better recovery the sodium phosphate concen-tration range should be around 100-150 mM. The tailing isalways low and the total area is higher when the arginineconcentration is high.

To a better understanding of the mobile phase impact, itwas performed size exclusion of 3 weeks degraded BoNT3 (25°C). The mobile phases assessed were 115Mm sodiumphosphate, 300mM NaCl and 300Mm L-Arginine (Minitaboptimal composition); 115Mm sodium phosphate and300mM NaCl (to confirm the importance of L-Arginine inthe mobile phase) and 50Mm sodium phosphate, 200mMNaCl and 200Mm L-Arginine (used in previous Ipsenwork). The results are showed on fig. 5.

Fig. 5. Comparison of elution profiles for differentsodium phosphate, NaCl and L-Arginine concentration.Zoom view of overlay chromatograms for degradedBoNT 3 during 3 weeks.

These results confirm the presence of arginine is essen-tial to achieve better resolution, less tailing and baselinedrift and reduce the retention time of BoNT 3. Also, thereis an improvement in the results when all the compoundsare present at higher concentrations. It should be notedwith the increased of arginine is observed an increasein aggregate peak area. The same event was describedby Arakawa, demonstrating that arginine improves thereliable quantification of aggregates [19]. The data alsodemonstrates that the arginine not promote aggregation,

preventing the protein binding to the column but cannotdissociate irreversible bound protein. Consequently, it canbe concluded the same approach because the amount ofmonomer eluting is not decreased by the arginine (around97 percent). This can be justified because the argininehas a preferential binding of unbound protein, which isin equilibrium with the surface bound protein. Conse-quently, arginine shifts the equilibrium binding towardthe dissociated state [11][19].

Once the optimal mobile phase conditions were con-firmed (115mM sodium phosphate, 300mM NaCl and300mM L-Arginine), the 25ºC heat stressed material ofBoNT 2 and 3 during one, two and tree weeks were ana-lyzed. BoNT 2 is stable at 25ºC, because no aggregationincreasing was observed. However, BoNT 3 aggregationincrease during the time, demonstrating degradation overthe time at these conditions. So, the optimised HPSECmethod is able to analyse degraded samples and is there-fore suitable as a stability indicating assay for BoNT 2 and3.

To conclude the HPSEC development, the method wasqualified to demonstrate it is robust, linear and precise.Prior to starting the qualification, the NaCl concentrationwas reduced to prevent reduction of the column life time.Arginine at 400mM was also evaluated to demonstratemethod robustness around the 300mM arginine limit withBoNT 3. The reduction of NaCl concentration to 200mMgenerated a USP tailing of 1.336 and a USP resolution be-tween the main peak and following peak of 7.213. Theseresults supported the use of reduced NaCl concentration.No difference was observed between 300mM and 400mMarginine, which demonstrated robustness around the argi-nine concentration. A linear regression analysis (R2) oftotal peak area vs. concentration was calculated to be0.9992 over the range of 0.05-0.5mg/mL of sample. Theprecision (repeatability) of the assay was calculated as aPercent Relative Standard Deviation of six replicate injec-tions of sample. The Percent Relative Standard Deviationfor total peak area and monomer peak area, monomer per-cent peak area and monomer retention time were lowerthan five percent. Overall the results show the HPSECmethod is suitably precise. The final SEC methods isshowed on fig. 6.

B. IECTo develop an IEC method for BoNT 2 a statistical mod-elling approach using Design of Experiments was used tosave time and material. Before starting with DoE, it wasrequired to define the conditions and factor ranges.

The first approach was the use of weak cation exchange.Several factors were changed like buffer, pH, samplebuffer exchange. Unfortunately, none of the solutionsshowed good separation due to the instability of BoNT2 at those conditions. Consequently, a new strategy wasimplemented which started the IEC development fromthe beginning.

It is known that BoNT 2 is stable at pH 6.5 at 100Mm


Fig. 6. Final HPSEC conditions for BoNT 2 and 3

salt concentration and the pI is 6.3 therefore a basic pHbetween 7 and 8.5 for the mobile phase was chosen. Itwas also considered that the toxin has to be stable at thecolumn loading pH which is slightly more basic due tothe Donan effect. To minimize this effect, the buffer con-centration shouldn’t exceed 50mM and the choosen buffershould have a pKa equal to the desired pH ± 0.5 to achievea sufficient buffer capacity. Based on this, Tris was selectedas the buffer due to its pKa (25 ºC) of 8.07. Consequently,Strong Anion Exchange was chosen because the bufferpH is more basic than the toxin pI, so the biomolecule netcharge is negative. The term strong means that the chargedensity on resin surface is maintained over a broad pHrange, and therefore these resins retain their selectivityand capacity over a wide pH range [14][15]. The first AEXcolumn chosen was Protein-Pak Hi-Res Q 4.6x100mm5µm that has a quarternary ammonium group with a pKaof 10.5. The other factors and choices are described morein the deep on the thesis.

It was performed a salt gradient 0-100 percent in 40min to understand where the protein eluted in the mobilephase gradient. It was observed one peak between 12-14percent of salt with poor separation and baseline. Withthis information and, after gradient slope optimisation,the gradient choosen was 0-25 percent of salt in 40 min.

The definitive screening DOE was generated in JMPto understand what are the main factors that affect reso-lution, recovery and peak number (18 runs). The factorswere:

- Flow rate: 0.3-0.5 ml/min;- pH: 7.5-8;- Temperature: 25-30 degree Celsius. Previous study

showed that the BoNT 2 is not stable at temperature higherthan 30 degree Celsius.

- Tris concentration: 20-30 mM;- Sodium chloride concentration: 0.5-1 M.The results show that temperature and pH are the most

relevant factors as on fig. 7.The custom design was used to further understand

temperature and pH as factors and build a response sur-

Fig. 7. Fit Definitive Screening

face (12 runs). Also, Auto Blend Plus was used to preparethe different buffers automatically. the new ranges were:

- pH: 7.5-8.5;- Temperature: 15-25 degree Celsius.

Fig. 8. Resolution (left) and Total Area (right) ResponseSurface estimated by Stepwise minimal BIC

By fig. 8, for resolution, the critical factor is pH of themobile phase. From the response surface, pH around 8give a good resolution. The predictive response for reso-lution is a good model that described well the variance ofthe data and it is not over-fitted. However, for the totalare is difficult to find a good model to fit on the data, soit is less reliable. This model, show the combination oflow temperature has the highest recovery. However, forthe middle pH, 25 ºC give highest area. This results wereconfirmed by visually analysis of the chromatograms.

The final IEX method is described on fig. 2, after, usedfor IEX fractionation.

C. IEC fractionation and MSIEC fractionation coupled with mass spectrometry wasused to characterize and potentially identify the BoNT 2isoforms. The IEX profile for BoNT 2 is described on fig. 9and it was collected the five fractions.

It was identified two main isoforms of BoNT 2. Theinitial hypothesis for the two isoforms was due to the pres-ence or absence of residues Gly-Ile-Arg from the LC whichthe first peak is the isoform with GIR and the second with-out GIR. The reason for that hypothesis is because, duringthe activation step of BoNT, trypsin cleaves the inactivesingle polypeptide BoNT chain between Arg 421 – Lys 422to generate the di-chain made of a 50kDa light chain anda 100kDa heavy chain linked by a disulfide bond. Thelight chain is then cleaved again between Lys 418 - Gly


Fig. 9. IEC fractionation for BoNT 2 - zoom chro-matogram

419 which removes GIR [3][20]. This phenomenon is welldescribed by the fig. 10.

Fig. 10. BoNT 2 activation by Trypsin

The Intact mass results discard this hypothesis becausethe two isoforms have a similar MW that both correspondto the form without GIR as shown in fig. 11. So, somefuture work includes peptide mapping of the fractions tounderstand the small modifications responsible for gener-ating the two different isoforms.

Fig. 11. Theoretical and measured MW for BoNT 2 (-)

5. CONCLUSIONIn the biopharmaceutical field, development of an ana-lytical tool box to fully characterize the drug substanceand control the quality and purity of the product, batchto batch, is mandatory. To build the tool box several meth-ods are required including size exclusion chromatography

and ion exchange chromatography. These methods werealready established for BoNT 1, nevertheless, the develop-ment for BoNT 2 and 3 were, also, essential.

The aim of this thesis was, therefore, developingHPSEC and IEC methods for BoNT 2 and 3. HPSEC isused to measure aggregation and IEX measures chargeisoforms. With this purpose, a statistical based approach,including Design of Experiments, was firstly used in Wrex-ham site, which allowed faster method development anda key understanding of the main factors and how theirinteractions impacted outputs, such as peak tailing, reso-lution and recovery.

HPSEC development was performed with full factorialdesign. This confirmed the importance of arginine in themobile phase, needed to suppress the hydrophobic andelectrostatic interactions between the neurotoxins and theresin. Furthermore, the DoE demonstrated that HPSECconditions can be used for both BoNT 2 and 3.

The separation of degraded BoNT 2 and 3 (incubationat 25 ºC) by the final HPSEC method revealed that thetechnique is appropriate as a stability test, able to detectaggregates and fragments. Likewise, it was also shownwith degraded BoNT 3 that arginine improved aggregaterecovery.

The HPSEC was validated by linearity and repeatabil-ity tests, confirming that the HPSEC assay developmentwas successful and is suitable to measure the purity levelsof BoNT 2 and 3. The final method comprises the fol-lowing conditions: • Column: Water Acquity BEH SEC200 1.7µm, 4.6 x 150mm; • Mobile phase: 115mM sodiumphosphate, 200mM NaCl and 300mM arginine at pH 7.0± 0.1; • Injection volume: 10µ; • Column temperature:25 ºC; • Detection: UV at 214 nm for detection of verylow molecular weight peptides and 280 nm for quantifi-cation; • Flow rate: 0.2 mL/min; • Running time: 12min;• Samples concentration: validated to 0.05-0.5 mg/mL;• Gradient: isocratic. An IEC method was developedfor BoNT 2, based on strong anion exchange. During thedevelopment, it was shown that BoNT 2 is not stable attemperatures higher than 30 ºC, likely due to denatura-tion.

The definitive screening design evaluated the flow rate,NaCl and Tris concentration, pH and temperature, in or-der to identify the main factors and the relationship toresolution and recovery. The pH and temperature wereconfirmed as key factors for optimal IEX separation.

The custom design generated a response surface forresolution and total area in relation to temperature andpH. The results indicated that, for a maximized resolution,the most important factor is the pH between 7.8 to 8 asoptimal range for IEX separation. However, highest recov-ery is achieved with the combination of low temperatureand a pH close to 7.5. But, it should be noted that the areamodel was poor, therefore, the combination of pH closeto 8 and a temperature of 25 ºC gave the best resolutionand acceptable recovery for BoNT 2.

The final method comprises the following conditions: •


Column: Protein-Pak Hi-Res Q 4.6x100mm 5µm; • Mobilephase A: 30 mM Tris pH 8.0 • Mobile phase B: 30 mM Tris,0.5M NaCl pH 8.0 • Injection volume: 20µL • Columntemperature: 25 ºC; • Detection: UV at 214 nm; • Flowrate: 0.3 mL/min; • Running time: 60min; • Samplesconcentration: 0.5 mg/mL; • Gradient: linear 0-25 percentsalt in 40 min. The obtained IEX results are satisfactory,but should be qualified to demonstrate it is a suitablemethod to quantify the isoforms of BoNT 2.

IEC fractionation and intact mass results refuted the hy-pothesis that the source of the different isoforms is due tothe presence of the GIR residue on the light chain. There-fore, peptide mapping must be performed to characterizethe different isoforms.

A. Future workFor HPSEC method, intermediate precision must be per-formed and the final conditions have to be confirmed withendopositive (active) material.

The next steps for IEC development could be, inter alia:the comparison between the results of pH 7.5 and 15 ºCversus pH 8 and 25 ºC; the salt gradient optimisation stepcould also be further evaluated; and, another possibility,is to screen different strong cation exchange and weakanion exchange columns.

In this study, the IEX was performed with salt-gradients. However, elution using a pH-gradient could beevaluated. The utilization of pH-gradients could provideadvantages, such as resolution improvements; lower thesalt concentration in collected fractions and the possibilityto correlate isoelectric point with elution profiles [21][22].

As already mentioned, peptide mapping for the dif-ferent isoforms is a near future work to understand thesource of the different BoNT 2 charge isoforms.

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