28 minute read
Pharma Notes
Resverlogix Corp. (Calgary, AB) reports it has entered into an agreement with Shenzhen Hepalink Pharmaceutical Co., Ltd. (Hepalink) for an equity investment and a license of its lead product RVX-208, for all indications in China, Hong Kong, Taiwan and Macau markets. Under the terms of the transaction, Hepalink will subscribe for 13,270,000 Resverlogix common shares and 1,000,000 common share purchase warrants, for aggregate proceeds of approximately C$35 million. Hepalink will also be entitled to nominate one mutually agreed representative for election to the board of directors of the company.
Aeterna Zentaris Inc. has filed an application for a patent (European Patent Office priority application: EP15000132) on a method of manufacturing zoptarelin doxorubicin. The hybrid cytotoxic molecule is the subject of a pivotal ZoptEC (Zoptarelin doxorubicin in Endometrial Cancer) Phase 3 clinical study in women with advanced, recurrent or metastatic endometrial cancer. The molecule combines a synthetic peptide carrier with doxorubicin, a well-known chemotherapy agent. The synthetic peptide carrier is a Luteinizing Hormone Releasing Hormone (LHRH) agonist, a modified natural hormone with affinity for the LHRH receptor. The design of the compound allows for the specific binding and selective uptake of the cytotoxic conjugate by LHRH receptor-positive tumours. Potential benefits of this targeted approach include a better efficacy and a more favourable safety profile with lower incidence and severity of side effects as compared to doxorubicin alone. The patent application, which is entitled “Enzymatic process for the regioselective manufacturing of NFmoc-doxorubicin-14-O-dicarboxylic acid mono esters,” may, if granted, make it difficult for generic manufacturers to produce the compound on a financially feasible basis after the company’s composition-of-matter patent on zoptarelin doxorubicin expires.
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Transition Therapeutics Inc.
(Toronto, ON) through its whollyowned subsidiary, Transition Therapeutics Ireland Limited has exclusively licensed the worldwide rights to a novel small molecule drug candidate (TT701) from Eli Lilly and Company. The molecule, TT701, is a selective androgen receptor modulator that has been shown in a Phase 2 study to significantly increase lean body mass and a measurement of muscle strength in male subjects. This Phase 2 study of 350 subjects also demonstrated additional beneficial effects, including significant fat mass reduction with no significant change in prostate specific antigen (PSA) levels. Transition is evaluating multiple development paths for TT701, including as a new therapeutic option for patients with androgen deficiency. Under the terms of the agreement, Lilly will receive a contingent upfront consideration of up to US$1 million. In addition, Lilly is eligible to receive up to US$100 million in commercial milestones and a mid-single digit royalty on sales of TT701 products should such products be successfully commercialized.
Privately held biotechnology company Northern Biologics Inc. reports it is collaborating with Celgene Corporation. As part of the collaboration, Northern Biologics has received a US$30 million upfront cash payment. It will use the funds to discover and develop first-in-class therapeutic antibodies in oncology and fibrosis from preclinical discovery through human clinical trials. Northern Biologics will also have the right to receive additional future payments that support the advancement of its portfolio. In return, Celgene will have options to in-license drug candidates and acquire Northern Biologics upon conclusion of the collaboration. Northern Biologics was launched in June 2014 by Blueline Bioscience, a Canadian biotechnology incubator backed by venture capital firm Versant Ventures, in partnership with the University of Toronto and University Health Network’s Princess Margaret Cancer Centre. Versant committed a US$10M Series A financing round to Northern Biologics in October 2014.
Cyclenium Pharma Inc. and Haplogen GmbH have signed a research agreement aimed at the discovery of novel pharmaceutical candidates in multiple disease areas. The collaboration brings together two next generation drug discovery platforms: Cyclenium’s CMRT™ Technology-derived QUEST Library™ and associated hit-tolead optimization expertise with Haplogen’s unique haploid genetics technology and expertise in host target identification, validation and screening. The companies will initially focus on certain anti-viral Haplogen targets, with targets in other therapeutic indications being phased-in as the research progresses. For Cyclenium, this is the fifth discovery collaboration established over the past year.
Cipher Pharmaceuticals Inc.
(Mississauga, ON) reports it has expanded its Canadian dermatology portfolio acquiring the Canadian rights to Vaniqa® and Actikerall® from Almirall S. A., a Spanish pharmaceutical company. Both products have been approved by Health Canada. VANIQA is a prescription cream clinically proven to reduce the growth of unwanted facial hair in women. VANIQA cream is an enzyme inhibitor and works by blocking an enzyme necessary for hair to grow. The product was approved by Health Canada in May, 2001. Actikerall is indicated for the topical treatment of slightly palpable and/or moderately thick hyperkeratotic actinic keratosis, a pre-cancerous patch of thick, scaly, or crusty skin. The product was approved by Health Canada on July 31, 2014 and will be launched by Cipher in the first half of 2015. Under the terms of the agreement, Almirall will receive an upfront payment of CDN$0.45 million and is eligible for certain milestones from product sales in Canada. Almirall will supply the finished product to Cipher.
Aeterna Zentaris Inc. (Quebec City, QC) announces that it has agreed to transfer its discovery library of roughly 100,000 unique compounds to the South Carolina
Center for Therapeutic Discov-
ery & Development pursuant to a just concluded Material Transfer Agreement. The library will be used to discover drug development candidates for the company in the areas of oncology, neurology, endocrinology and women’s health. The center has agreed to conduct screening and pre-clinical activities with respect to the library and will submit to the company at least one development candidate in its areas of therapeutic interest per year during a ten-year period beginning in 2018. The company will receive the right of first refusal to license the development candidates. Should the company decide to further develop a candidate submitted by the center, MUSC will license the compound to the company, and be entitled to a royalty on the net sales of all commercialized products developed from the development candidate. However, should the company decide not to further develop the development candidate submitted by the center, MUSC shall pay the company a royalty on net sales of all commercialized products developed from the candidate.
STEMCELL Technologies Inc.
has signed a license agreement with the Salk Institute for Biological Sciences for the rights to commercialize BrainPhys™ Neuronal Medium. Invented by Cedric Bardy in the lab of neuroscientist Fred H. Gage, this formulation promotes optimal neuronal and synaptic activity while supporting long-term culture of both human pluripotent stem cell (hPSC)derived neurons and primary tissue-derived neurons. BrainPhys™ Neuronal Medium will complement STEMCELL’s portfolio of products for neural cell culture applications, including the NeuroCult™ product line for tissue-derived neural cells, and the STEMdiff™ Neural System for hPSC-derived neural cells.
OncoGenex Pharmaceuticals,
Inc. announced that its wholly owned subsidiary, OncoGenex Technologies Inc., has executed a termination agreement with Teva Pharmaceuticals Ltd. under which OncoGenex will regain rights to custirsen, an investigational compound currently in Phase 3 clinical development as a treatment for prostate and lung cancers. This transfer of rights occurs in connection with the termination of the 2009 collaboration agreement between OncoGenex and Teva. The agreement between the two parties to terminate the collaboration includes a $23.2 million payment from Teva.
The Fight Against Cancer Inno-
vation Trust and its partners announce the formation of Turnstone Biologics Inc., a biotechnology company focussed on developing treatments for cancer that harness the patient’s own immune system. The company represents a unique collaboration between the Children’s Hospital of Eastern Ontario (CHEO) Research Institute, McMaster University, the Ontario Institute for Cancer Research (OICR), the Ottawa Hospital Research Institute, the University of Ottawa and FACIT. The new company hopes to accelerate clinical translation and commercialization of oncolytic vaccine immunotherapies for the treatment of cancers. Turnstone’s lead technology platform, Marabex™, combines the benefits of oncolytic viral therapy with a tumour-targeted vaccine into a single treatment.
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by katherine Gillis, Julie clor, kamala tyaGaraJan
Plate-based drug and toxin screening using multiplexed benchtop fl ow cytometry
Drug-based screening, identifi cation of positive ‘hit’ compounds, and evaluation of mode of action require an understanding of how the drug modulates critical cellular parameters and activities which eventually lead to cytotoxicity or alter cell proliferation.1 This is particularly important in oncology research, where combinatorial strategies using drug mixtures can have additive/synergistic effects on proliferation and apoptosis. Given the increased need for assays with statistically-rich informational content and mechanism-based data, drug researchers and toxicologists have turned to fl ow cytometry assays. With its potential for precise and quantifi able comparisons between cell populations, fl ow cytometry enhances dose-response calculations with the statistical power afforded by large sample size.2
The guava easyCyte™ benchtop fl ow cytometry systems (EMD Millipore) enable plate-based compound screening because they require signifi cantly fewer cells per well, and because its powerful InCyte™ software enables multiparametric comparisons of concurrent responses. The addition of a violet laser in the easyCyte™12 instrument permits the simultaneous measurement of up to 12 parameters, offering fl exibility and more powerful analysis in multiparametric screening studies.
This article demonstrates the power of the guava® system and accompanying software to provide enriched data during drug screening processes. Using just two fundamental assays, plate-based screening was performed for various cytotoxic compounds and identifi ed key ‘hits’. The same assays were used to further evaluate select compounds in greater detail with dose-response and time-response studies. These assays also provided information on the mechanism of action of the compounds gambogic acid, celastrol and thimerosal, and their modulation of apoptotic and proliferative responses.
Multiparameter analysis
discovery process. Screening multiple compounds while multiplexing cell health markers leads to many data points and often makes ‘hit’ identifi cation complex. The guava easyCyte™ benchtop fl ow cytometry systems simplify this process by analyzing up to six parameters simultaneously, whether a single parameter from up to six plates, or six parameters from each sample in a plate, as demonstrated here.
The relationship between changes in mitochondrial membrane potential (MMP) and cell health permits the use of a powerful assay for apoptosis. Combining EMD Millipore’s MitoSense Red for assessment of MMP with Caspase 3/7 FAM detection reagent, Annexin V Brilliant Violet™ 421 (BioLegend) and 7-AAD enables multiparametric assessment of apoptosis with simultaneous discrimination of necrotic cells in a single, simplifi ed assay.
Upon treatment with gambogic acid, Jurkat cells showed a decrease in MMP, increased caspase activity, increased Annexin V staining and increased cell death, as expected. Combining multiple cell health markers in the manner described permits discrimination of the stage of apoptosis. For example, not all cells that were positive for Annex-
in V were also found to be simultaneously positive for Caspase 3/7.
Monitoring proliferation
Assessment of proliferation is critical to cell analysis, and is of particular importance when screening for bioactivity or cytotoxicity. Because its expression is required for progression through the cell cycle, measuring expression of the nuclear protein Ki67 has become the prototypical assay for cell proliferation.
A robust Ki67 signal was observed in untreated Jurkat cells. In contrast, a subpopulation of cells treated with the anti-infl ammatory compound celastrol demonstrated the diminished Ki67 expression that is indicative of a reduction in proliferation. Celastrol has been shown to inhibit proliferation in numerous tumor cell types via downregulation of cyclins D1 and E3.3
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Plate-based screening
The assays described above were next applied to the platebased screening of cytotoxic compounds. A panel of 80 cytotoxic, immunosuppressive, anti-proliferative and anti-infl ammatory compounds was obtained from Microsource Discovery Systems, Inc. at a concentration of 10 mM in dimethyl sulfoxide (DMSO). Compounds were diluted in complete growth medium to either 10 μM or 40 μM. Plate setup included negative controls containing 0.2 per cent DMSO, and positive controls treated with 1 μM staurosporine, a known protein kinase inhibitor.
The population percentage altered for each parameter was compared in heat map format using the InCyte™ software. The software allows for quick identifi cation of ‘hit’ compounds and comparison of all parameters simultaneously, as shown in the pie graphs and population percentages in Figure 1. While the number of hits between the 10 μM (1A) and 40 μM (1B) plates look similar, the percentage of cells showing higher percentages of effects varied, as shown by the darker shades of blue in hit wells.
From the results shown in Figure 1, autofl uorescent compounds and those treatments causing less than 20 per cent change compared with negative controls were eliminated to focus on compounds that showed cytoactivity at either concentration. Twenty compounds induced cell response at 10 μM while one additional compound induced response at 40 μM only. Some compounds, such as gambogic acid, induced a large change in MMP, with a lower percentage of cells being Annexin V positive and even fewer showing Caspase 3/7 activity. These results were consistent with published reports that gambogic acid induces apoptosis specifi cally in tumor cells while having less impact on normal cells.4-6
Other compounds, such as the anti-tumor agent juglone,7 induced a change in mitochondrial depolarization and an almost identical degree of caspase response at 10 μM, with changes in Annexin V binding only occurring at the 40 μM concentration. Several compounds showed large impacts on MMP changes with a lower percentage for the other markers, while others demonstrated impact on all assays in parallel. From these screening results, a subset of compounds was selected for dose and time-response analysis.
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figure 1 heat map results output from incyte™ software showing four-parameter data for cell health impact of 80 compounds on Jurkat cells.
each well is mapped to a four-quadrant circle with color intensity refl ecting the percentages of cell population positive for loss of mitochondrial membrane potential (top left quadrant), annexin v staining (top right quadrant), caspase activation (bottom left quadrant) and cell death (bottom right quadrant). drug concentrations used were 10 µm (panel a) and 40 µm (panel b).
IR provides more universal accuracy in protein quantifi cation.
figure 2 dose response of Jurkat cells to celastrol treatment.
100
n Percent of Populatio
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0 0.05
0.5 5 µM Inducer
Dose and time-response experiments
The mechanism of celastrol, a known antioxidant and anti-infl ammatory compound, was investigated by evaluating its dose-dependent impact on Jurkat cells. Population percentages for cell health markers at various concentrations are plotted in Figure 2. The degree of MMP decrease and Annexin V staining was similar, with a lower percentage of cells showing caspase activity across the concentration range. The loss of cell proliferation increased with increased celastrol concentration. Proliferation recovery was observed at the two highest concentrations, but this can likely be attributed to the high percentage of cells exhibiting cell death at the same concentrations. The study demonstrated that treatment with celastrol caused mitochondrial depolarization, apoptosis and proliferation loss at lower concentrations than those causing any signifi cant cell death.
To assess the effect of increasing compound exposure time on cell health, Jurkat cells were treated with gambogic acid, an antitumour compound, and thimerosal, an antifungal and antiseptic compound, for
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figure 3a and figure 3b time course for Jurkat cell response to gambogic acid and thimerosal.
100 100
80
60
40
20 n Percent of Populatio
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0hr 3hr 6hr 16hr 24hr Time
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Jurkat cells were incubated with 10 µm gambogic acid (3a) or 2.5 µm thimerosal (3b) for 0, 3, 6, 16 or 24 hours and either stained with a cocktail of cell health markers or evaluated using a ki67-based proliferation assay.
various durations. Time-dependent responses to gambogic acid (Figure 3A) demonstrated mitochondrial and apoptotic impacts. Results obtained with gambogic acid demonstrate that a rapid and signifi cant loss of MMP occurred at earlier time points when compared to other cell health markers tested. Annexin V- and Caspase 3/7-positive population percentages showed a steady increase at three and six hours with maximal percentages at 16 hours. Signifi cant cell death and proliferation loss were not seen until 16 hours.
Time-dependent responses to thimerosal underscored different cell health impacts (Figure 3B). Even with short incubation times, thimerosal caused almost complete depolarization of the mitochondrial membrane by three hours. At three hours, this was also accompanied by signifi cant changes in Annexin V binding, with a continued increase at six, 16 and 24 hours. A high percentage of cells showing caspase activity and cellular death was not seen until the six-hour time point, with maximal caspase activity attained at 16 hours. Low levels of proliferation inhibition were seen starting at six hours, with a gradual decrease in proliferation over time. While equivalent cell death and proliferation loss responses were seen with gambogic acid, an increased percentage of cells displayed death responses to thimerosal.
Conclusion
Multiparametric analysis at a singlecell level provides comprehensive mechanistic and cell health information, which greatly facilitate assessment of cytoactive compounds. These studies showed that platebased screening using the guava easyCyte™ 12 benchtop fl ow cytometer enabled uncomplicated comparison of mechanisms of action of diverse compounds while also providing the sample-conserving benefi ts of microcapillary fl ow cytometry. Specifi cally, gambogic acid induced signifi cant and rapid mitochondrial depolarization, celastrol acted by inhibiting proliferation and inducing apoptosis, and thimerosal triggered rapid cell death. The ease of these multiplexed assays, coupled with plate-based microcapillary cytometry and the heat mapping functionality in the InCyte™ software, enables researchers to obtain insights into how compounds modulate apoptotic and proliferation processes and their relationship to mitochondrial dysfunction and cell death pathways.
References
1. Wesierska-Gadek, J., Gueorguieva, M., Ranftler, C., Zerza-
Schnitzhofer, G. (2005, Sep.) A new multiplex assay allowing simultaneous detection of the inhibition of cell proliferation and induction of cell death. J Cell Biochem,1;96(1):1-7. 2. Lombardo, T., Anaya, L., Kornblihtt, L., Blanco, G. (2012) Median effect dose and combination index analysis of cytotoxic drugs using fl ow cytometry. In Flow
Cytometry - Recent Perspectives,
M.Sc. Ingrid Schmid (Ed.) ISBN: 978-953-51-0626-5, InTech, DOI: 10.5772/38214. 3. Kannaiyan, R., Manu, K.A., Chen,
L., Li, F., Rajendran, P., Subramaniam, A., Lam, P., Kumar, A.P.,
Sethi, G. (2011, Oct.) Celastrol inhibits tumor cell proliferation and promotes apoptosis through the activation of c-Jun N-terminal kinase and suppression of PI3 K/
Akt signaling pathways. Apoptosis, 16(10), 1028-41. 4. Shi, X., Chen, X., Li, X., Lan, X.,
Zhao, C., Liu, S., Huang, H., Liu,
N., Liao, S., Song, W., Zhou, P.,
Wang, S., Xu, L., Wang, X., Dou,
Q.P., Liu, J. (2010, Jan. 1) Gambogic acid induces apoptosis in imatinib-resistant chronic my-
eloid leukemia cells via inducing proteasome inhibition and caspase-dependent Bcr-Abl down regulation. Clin Cancer Res., 20(1):151-63. 5. Zhao, L., Guo, Q.L., You, Q.D.,
Wu, Z.Q., Gu, H.Y. (2004) Gambogic acid induces apoptosis and regulates expressions of Bax and
Bcl-2 protein in human gastric carcinoma MGC-803 cells. Biol.
Pharm. Bull., 27, 998–1003. 6. Li, X., Liu, S., Huang, H., Liu, N.,
Zhao, C., Liao, S., et al. (2013)
Gambogic acid is a tissue-specifi c proteasome inhibitor in vitro and in vivo. Cell Rep., 3(1), 211–22. 7. Xu, H.L., Yu, X.F., Qu, S.C.,
Zhang, R., Qu, X.R., Chen, Y.P.,
Ma, X.Y., Sui, D.Y. (2010, Oct. 25) Anti-proliferative effect of
Juglone from Juglans mandshurica Maxim on human leukemia cell HL-60 by inducing apoptosis through the mitochondria-dependent pathway. Eur. J. Pharmacol., 645(1-3), 14-22.
Katherine Gillis, Applications Scientist, katherine.gillis@emdmillipore.com; Julie Clor, Applications Scientist, julie.clor@emdmillipore. com; Kamala Tyagarajan, R&D Manager, kamala.tyagarajan@ emdmillipore.com
To see this story online visit
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by ken cook,frank steiner,mauro de Pra
Development of Ultra-fast pH-Gradient Ion Exchange Chromatography for the Separation of Monoclonal Antibody Charge Variants
Introduction
The pre-formulated buffers for pH gradient, introduced by Thermo Fisher Scientifi c, have greatly simplifi ed the development of ion exchange chromatography (IEX) of monoclonal antibodies (mAbs). Three features make this simplifi cation possible.
The fi rst feature is that the buffers can cover a pH range from 5.6 to 10.2; this pH window enables the characterization of mAbs with a wide range of isoelectric points, providing a global pH gradient ion exchange screening method that will accommodate the majority of therapeutic mAbs.
The second feature is that, if a linear solvent gradient is programmed in the pump, the actual pH gradient produced in the column will be linear as well. Although this sounds trivial, scientists involved in pH gradient studies know how diffi cult it is to develop buffer formulations capable of fulfi lling this requirement. The advantage of a genuine linear pH gradient is that the method can be confi dently fi netuned merely by narrowing down the pH range around the mAb and its variants, thus allowing for the adjustment of the gradient time according the resolution requirements.
The third feature is the mobile phase preparation: the pre-formulated pH buffers only needs to be diluted by a factor of 10 in deionized water, and the mobile phase is ready to use. Compared to a salt gradient ion exchange chromatography method, where no generic screening can be easily designed, and where method development goes through the rather tedious preparation of several buffers at different pH values, the time and effort invested in method development are substantially reduced.
High resolution pH gradient separations are obtained with 30 minute gradients and relatively long columns, such as the Thermo Scientifi c™ MAbPac™ SCX-10 column, 10 μm, 4 × 250 mm.1 Fast separation of mAb variants were demonstrated by using the MAbPac SCX-10 column, 5 μm, 4 × 50 mm with no signifi cant loss in resolution2 on a Thermo Scientifi c™ UltiMate™ 3000 BioRS system. In this case, the pH gradient from 5.6 to 10.2 was completed in 7.5 minutes, and the resolution between variants was still satisfactory, despite the short analysis time.
In this work, we show how to push the throughput of pH gradient IEX even further. To achieve this, a MAbPac SCX-10 RS column, 5 μm, 2.1 × 50 mm was operated on a new Thermo Scientifi c™ Vanquish™ UHPLC system. The Vanquish UHPLC is a fully biocompatible system, suitable for the analysis of intact proteins. The combination of low gradient delay volume and high precision gradient formation makes it the ideal system for high throughput analysis with gradient elution. Here the system was used with the default confi guration and a total system gradient delay volume of 175 μL. Fast charge variant separations of 5 mAbs are shown.
figure 1
Separation of 5 mAbs with a generic 0−>100 %B in 10 minutes at 0.45 mL/min (method #1 Table 1). Samples are as follows: A) bevacizumab, B) cetuximab, C) infl iximab, D) trastuzumab, E) mAb A.
Experimental
Vanquish UHPLC, consisting of: • Vanquish System Base (P/N VH-S01-
A) • Binary Pump H with Default Mixer (P/N VH-P10-A) • Split Sampler HT (P/N VH-A10-A) • Column Compartment H (P/N
VH-C10-A) • Diode Array Detector HL (P/N
VH-D10-A)
Chromatographic Conditions
Column: MAbPac SCX-10 RS, 5 μm, 2.1 × 50 mm (P/N 082675) Buffers: Thermo Scientifi c CX-1 pHGradient buffer A (pH 5.6) 125 mL (P/N 083273) CX-1 pH-Gradient buffer B (pH 10.2) 125 mL (P/N 083275) Mobile Phase A: CX-1 pH-Gradient buffer A (pH 5.6) diluted 10x in deionized water Mobile Phase B: CX-1 pH-Gradient buffer B (pH 10.2) diluted 10x in deionized water Column Compartment Temperature 30 °C, forced air
Detector and Conditions
Detector: LightPipe™ 10 mm Standard Flowcell (P/N 6083.0100) Detection Wavelength: 280 nm Data Acquisition Range: 5 Hz (for fl ow rate ≤ 0.5 mL/min) and 50 Hz (for fl ow rate ≥ 1.0 mL/min) Response Time: 2 s (for fl ow rates ≤ 0.5 mL/min) and 0.1 s (for fl ow rates ≥ 1.0 mL/min)
Data Processing
Software: Thermo Scientifi c™ Dionex™ Chromeleon™ 7.2 Chromatography Data System (CDS)
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mab name concentration (mg/ml) injection volume (µl)
bevacizumab 25 1
cetuximab 5 4
infl iximab 10 4
trastuzumab 21 2
mab a 21 2
Results and Discussion
One of the benefi ts of using CX1 buffers for the pH-gradient is the simplifi ed method development and optimization. This is due to the fact that the pump running a linear solvent gradient will result in a linear pH gradient in the column. This is not the case for most of home-made buffer formulations, which would produce a non-linear pH gradient in response to a linear programmed gradient. A non-linear pH gradient makes method optimization diffi cult due the uncertainty of the actual effects of any changes in the programmed gradient.
It is recommended to perform a generic screening from pH 5.6 to 10.2 when the pH at which a given mAb elutes is not known. In this work, the generic screening was run in 10 minutes at 0.45 mL/min. As it can be seen in Figure 1 in some cases, satisfactory separation of the charge variants was achieved during the fi rst run. This was the case for cetuximab and infl iximab: several charge variants could be resolved with suffi cient resolution.
After the generic screening, the method development efforts should be aimed at decreasing the analysis cycle time and at the same time improving resolution. To achieve this goal, two parameters were modifi ed, namely the pH range and the gradient slope. Here we used gradient slope based on gradient volume, i.e. ∆(%B)/VG, where %B is the amount of B eluent and VG is the volume of mobile phase delivered by the pump during the gradient. A narrower pH window allowed for a reduced run-time, whereas a shallower gradient slope provided better resolution.
The gradient slope of the generic screening between pH 5.6 and 10.2 was 22.2 (%B)/mL; the improved and faster analysis were obtained with gradient slopes of 8 (%B)/mL and 10 (%B)/mL. Details of the conditions are listed in Table 1. Figures 2a, 3a, 4a, and 5a, show the results obtained by this approach. The number of resolved variants was larger than in the initial screening. Gradient time was fi ve minutes, hence half of the time of the initial screening.
The following step was used to develop fast analysis cycles compatible with high throughput. With this approach we aimed for a 2.5 minute gradient time or lower, and total analysis time of less than four minutes, including column re-equilibration. Data are shown in Figures 2b, 3b, 4b, 5b, and 6b. The purpose was to develop a method suitable for high-throughput that can run at least 300 samples a day. High fl ow rate was used for this purpose. A high fl ow rate allows running short gradients with relatively shallow gradient slopes; the shallow slope is required to preserve selectivity between charge variants. Additionally, column equilibration, which is directly dependent on the volume of mobile phase fl owing through the column, is reached quicker. MAbPac SCX-10 RS column is pressure rated up to 7,000 psi (~ 480 bar), therefore
figure 2
Separation of bevacizumab with different gradient conditions. Chromatogram A condition #2 Table 1; chromatogram B condition #3 Table 1.
Separation of cetuximab with different gradient conditions. Chromatogram condition A #4 Table 1; chromatogram condition B #5 Table 1
figure 3
table 1 description conditions used for the separation of charge variants of the different monoclonal antibodies.
method # figure Gradient range (%b) Gradient ph range Gradient time (min) flow rate (ml/min) Gradient slope* (%b/ml)
1 1 0-100 5.6-10.2 10.0 0.45 22.2
2 2a 20-40 6.5-7.4 5.0 0.50 8.0
3 2b 23-35 6.7-7.2 2.5 1.00 4.8
4 3a 10-35 6.1-7.2 5.0 0.50 10.0
5 3b 10-35 6.1-7.2 2.5 1.00 10.0
6 4a 20-40 6.5-7.4 5.0 0.50 8.0
7 4b 18-27 6.4-6.8 0.8 1.20 9.4
8 5a 35-60 7.2-8.4 5.0 0.50 10.0
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figure 4
Separation of infl ixumab with different gradient conditions. Chromatogram condition A #6 Table 1; chromatogram condition B #7 Table 1.
figure 5
Separation of trastuzumab with different gradient conditions. Top chromatogram condition #8 Table 1; bottom chromatogram condition #9 Table 1
it can be operated at high linear fl ow rate. In this work, we used fl ow rates up to 1.2 mL/min.
The chromatographic pattern between methods at moderate and high fl ow rate was preserved. The separation capabilities of different methods were compared based on the resolution between charge variants. Since in several instances peak pairs were overlapping, and it was not always possible to measure peak width at half height or at the baseline, here we used resolution based on statistical moments. Resolution was calculated directly by Chromeleon 7.2 CDS according to the formula:
where t2/R and t1/R are the retention times of the more and less retained peak respectively, and μ2/2 and μ1/2 are the related second moment.
In some cases, the ultra-fast separation approach was accompanied by some resolution loss. This is the case of trastuzumab, where average resolution loss was ~ 3 per cent. In the case of the complex variants pattern of cetuximab, the average resolution loss at high fl ow rate was ~ 13 per cent. Infl iximab resolution decreased by ~ 11 per cent, however separation of the fi ve main charge variants and two minor ones was achieved in one minute. This impressive result was obtained by running the column at 1.2 mL/min with a 0.8 min gradient time. In the case of bevacizumab, the ultrafast separation approach even yielded to ~ 4 per cent improved resolution. The better resolving power can be explained by a slightly narrower pH range and a shallower gradient slope. mAb A was analyzed only with the generic screening and high fl ow rate (1 mL/min) method. The highthroughput method provided 19 per cent better resolution on average of fi ve charge variants.
Conclusion
The ultra-fast charged variant separations described here are achieved because of several advances in chromatography techniques. The mechanism of pH gradient chromatography lends itself to the use of shorter, faster columns. The availability of high pressure rated small particle size ion exchange columns are a perfect match to pH gradient methodology. The commercial buffer formulations used here form a linear gradient
figure 6
Separation of mAb A with different gradient conditions. Chromatogram A condition #1 Table 1; chromatogram condition B #10 Table 1.
which allows intelligent optimization of the methods. Finally, there is the use of the new Vanquish UHPLC system which has extremely low delay volumes, high precision gradient formation and a totally inert fl ow path.
Ultra-fast separation that requires total analysis cycle in the order of two minutes, including column reequilibration and injection time, enables users to run more than 1,400 samples during 48 hours continuous operations. To allow unattended tasks with such large amount of samples, the Vanquish UHPLC system can be extended with the Vanquish Charger Module. This can host up to 9,000 samples in a thermostatted environment, and transfer them to the Vanquish Autosampler.
table 2 overview of the resolution between charge variants at different conditions. resolution was calculated by chromeleon cds using statistical moments.
mab Gradient time (min) 1-2 2-3 3-4 4-5 5-6 6-7
bevacizumab 5 0.94 0.87 1.81 - - 2.5 1.00 0.89 1.89 - - cetuximab 5 0.79 1.14 1.00 1.13 1.28 0.98 2.5 0.53 1.05 0.94 0.93 1.20 0.91 infl iximab 5 0.68 1.56 1.33 0.87 - 0.8 0.59 1.40 1.30 0.70 - trastuzumab 5 1.25 1.14 1.70 0.87 - 2.5 1.27 1.11 1.63 0.79 - mab a 10 0.14 1.31 0.94 1.26 - 5 0.32 0.96 1.22 2.02 - -
References
1. Thermo Fisher Scientifi c. Thermo Scientifi c Application Note 20784, A Novel pH Gradient
Separation Platform for Monoclonal Antibody (MAb) Charge
Variant Analysis, AN20784_E,
Sunnyvale, CA, 2013. 2. Thermo Fisher Scientifi c. Thermo Scientifi c Application Note 20946, A Fast and Robust Linear pH Gradient Separation Platform for Monoclonal Antibody (mAb) Charge Variant Analysis,
AN20946_E, Sunnyvale, CA, 2014.
Ken Cook,Thermo Fisher Scientifi c; Frank Steiner,Hemel Hempstead, United Kingdom; Mauro De Pra,Thermo Fisher Scientifi c, Germering, Germany
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