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Pharma Notes
Cytochroma (Markham, ON) announces the initiation of a repeat-dose safety and efficacy study of CTAP101 capsules in patients with vitamin D insufficiency, secondary hyperparathyroidism (SHPT) and stage 3 chronic kidney disease (CKD). The newly initiated trial is a randomized, double-blind, placebo controlled, multisite study designed to evaluate the safety, efficacy, pharmacokinetics, pharmacodynamics and tolerability of CTAP101 capsules, administered at various daily doses, in approximately 60 patients. The endpoints in this study will include vitamin D status, adverse events, physical and clinical laboratory assessments, and changes in serum calcium, phosphorus and intact parathyroid hormone (PTH).
Endo Pharmaceuticals
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(Newark, DE) and Bion-
iche Life Sciences Inc.
(Belleville, ON) announce the enrollment of the first patient in the second Phase 3 clinical trial of Urocidin™. The trial is a randomized, active-controlled, open-label, multi-center study with a blinded endpoint assessment designed to compare UrocidinTM with mitomycin C in the intravesical treatment of patients with BCG recurrent or refractory non-muscle invasive bladder cancer. It is estimated that 450 patients will be enrolled for this new trial at approximately 120 clinical sites worldwide.
Stem Cell Therapeutics
Corp. (Calgary, AB) has been granted a patent by the U.S. Patent and Trademark Office -U.S. Patent 7,884,072- entitled, “Prolactin induced increase in neural stem cell number.” The claims contained within this issued patent cover methods of using an effective amount of prolactin to increase stem cell numbers in mammals suspected of having neurological diseases.
Helix BioPharma Corp.
(Aurora, ON) has received approval for its investigational new drug (“IND”) application from the United States Food and Drug Administration to perform its planned U.S. Phase I clinical safety and tolerability study of its lung cancer drug candidate L-DOS47. L-DOS47 is the Company’s first therapeutic immunoconjugate drug candidate under development based upon its novel DOS47 platform technology, which is designed to modify the microenvironmental conditions of cancer cells in a manner that leads to their destruction. L-DOS47 is intended to offer an innovative approach to the first-line treatment of inoperable, locally advanced, recurrent or metastatic non-small cell lung cancer. The FDA has completed its review of the IND and concluded that it is acceptable for Helix to proceed with the study, contingent on Helix providing an amended version of the clinical protocol beforehand, reflecting several minor changes that FDA stipulated during the course of the IND review. The company will now proceed with its remaining pre-study logistical preparations, including preparation and filing with the FDA of the required clinical protocol amendment, with a view to commencing clinical site initiation and patient recruitment activities late spring to early summer of this year.
Dalton Pharma Servic-
es, (Toronto, ON) a privately owned Canadian pharmaceutical services provider to leading pharmaceutical companies, has entered into a Manufacturing Services Agreement with Oncovir Inc., a specialty pharma company based in Washington DC, dedicated to the development of nucleic-acidbased clinical therapies for cancer, infectious, immune, and degenerative disorders. Dalton Pharma Services will provide API manufacturing and aseptic fill/finish services under cGMP, for Oncovir’s collaboration with the Cancer Vaccine Acceleration Fund (CVAF), a joint initiative between the Cancer Research Institute (CRI) and the Ludwig Institute for Cancer Research (LICR). CVAF has completed a new investment agreement with Oncovir, Inc., to enable the production of Oncovir’s immune activator Hiltonol®.
Warnex Inc. (Laval, QC) announces that its Warnex Medical Laboratories division will serve as a central laboratory across Canada for Genzyme Canada Inc.’s (Ottawa, ON) Lysosomal storage disorders testing program. This program will assist physicians in diagnosing Fabry (male), Gaucher, Pompe and Mucopolysaccharidosis Type I (MPS I) diseases.
Medwell Capital Corp.
(Edmonton, AB) announces that it has committed to invest up to $2,000,000 in
Mimetogen Pharmaceuti-
cals Inc. (Montréal, QC), a privately-held, clinical-stage company developing a treatment for dry eye disease. Medwell Capital participated in a Series B equity financing with existing Mimetogen investors, iNovia Capital, MSBi Valorisation and VIMAC Milestone Medica. As part of the financing, Nitin Kaushal, executive vice president of Medwell will join the Mimetogen Board of directors. Medwell Capital will also assist Mimetogen and the current investors with advisory services in potential partnering and financing efforts. Mimetogen’s lead drug candidate for the treatment of dry eye disease, MIM-D3, is a small molecule mimetic of nerve growth factor (NGF). NGF is a naturally occurring protein in the eyes that is responsible for the maintenance of corneal nerves and epithelium, mucin and tear production.
Isotechnika Pharma Inc.
(Edmonton, AB) announces that Lux Biosciences, Inc. (Jersey City, NJ) has commenced its Phase 3 trial using voclosporin for the treatment of non-infectious uveitis, a leading cause of vision loss and long-term disability. The study is a six-month randomized trial of voclosporin versus placebo in patients with active non-infectious intermediate, posterior, or pan-uveitis. This Phase 3 trial will involve 150 patients in North America and Europe. Lux is conducting this additional Phase 3 trial as outlined in the Complete Response Letter received in August, 2010, from the FDA. In 2006, Isotechnika granted Lux worldwide rights to develop and commercialize voclosporin for ophthalmic diseases. In return, Isotechnika will receive development milestones payments, as well as royalties on net sales. Voclosporin (branded as Luveniq™ by Lux) was accepted for review by the European Medicines Agency (EMA) in March 2010. The product has received Orphan Drug designation in both Europe and the U.S. for the treatment of non-infectious uveitis.
Canadian specialty pharmaceutical company Paladin Labs Inc. (St. Laurent, QC), announces that Health Canada has approved Abstral®. Abstral® is a novel, rapidly-disintegrating, sublingual (under the tongue) formulation of fentanyl, a well-established opioid used for the management of episodes of breakthrough pain experienced by cancer patients who are already receiving opioid analgesics for chronic pain. Paladin obtained the Canadian rights from ProStrakan Group plc in December 2008.
Great People. Great Chemistry.
Reply card #4410
Cascade ultrafiltration system for high throughput therapeutic protein purification
By Mukes H M A y A ni T H er AP ure Bio PHA r MA i nc.
INtRoDuCtIoN therapeutic proteins (tPs) are purified using RIPP (Recovery, Isolation, Purification and Polishing) scheme in the biopharmaceutical industry. Recovery and isolation steps are performed using relatively low resolution methods such as microfiltration, depth filtration, precipitation and centrifugation. For purification, high resolution methods such as adsorption and chromatography (e.g. protein A affinity, hydrophobic interaction or ion exchange) are employed. usually, these purification methods are slow and expensive resulting significant burden on downstream processing and higher cost of the products.1
figure 1
Major conventional membrane separation processes used in biopharmaceutical industry.
Tank Solvent
Buffer
Membrane module
Tank Diafiltrate Retentate
Membrane module
Tank Permeate
Membrane module
Ultrafiltration membranes are integral part of biopharmaceutical industries and have been used at several stages to separate macromolecules from smaller solutes - such as salts, water, sugars, amino acids and proteins.2 In downstream processing, they are typically applied for protein concentration (i.e. the removal of solvent from solutions of macromolecules), desalting and diafiltration (i.e. the removal of salts and other low molecular weight compounds), buffer exchange (i.e. the exchange of solvent molecules), and in some cases, for fractionation (i.e. the separation of one protein from another) (see Figure 1). However, these operations are aimed for rapid processing using single or multiple membrane unit(s) configured in traditional manner. Although the membrane making technology has been improved over the years, the benefit in purification has not been seen as a consequence of the conventional manner of application. The limitation of sharp membrane cut-off has been seen as the limiting factor for suitability of membranes in protein purifications. However, this limitation can be offset by combining multiple membrane units. It has been found that multiple membrane unit can be operated to achieve better overall resolution of a target protein and thus purification performance can significantly improved.3-6 The purity and recovery could be complemented to chromatographic purification in some niche applications.7 The author has investigated suitability of cascade ultrafiltration systems for several systems4,5 and made specific observations in regards to fundamental characteristics of ultrafiltration cascade systems and suitability at larger scale.
What is cascade ultrafiltration system?
A system of ultrafiltration membrane units, integrated to make a bigger unit in which separation characteristics of individual units are inter-dependent. The membranes are configured to make an overall system which is suitable to achieve a desired objective of protein purification. The overall cascade ultrafiltration system becomes compact, low-energy consuming, scalable and provides operational flexibility.
Figure 2 shows a schematic of a typical three-stage cascade ultrafiltration used for fractionation of two proteins in continuous manner. The membrane 1 (designated as M1) completely retains protein A and membrane 3 (designated as M3) to product B. The membrane 2 (designated as M2) preferentially transmits protein B while it has molecular weight cut-off (MWCO) smaller than M1. The feed is introduced into the cross flow loop across M2 for fractionation of A and B. The permeate from M2 is sent to retentate cross flow circuit across M3, whereas the controlled flow stream from the cross flow loop across M2 is sent into the cross flow across M1. The permeate from M1 is introduced into the cross flow circuit across M2 as product B recovery stream. The permeate drawn from M3 is essentially free of protein B and function as in situ generated sweeping buffer, which is introduced into the cross flow
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figure 2 cascade ultrafi ltration confi guration for fractionation of two proteins.4
Overall retained product A Overall permeate product B
M2
M1 M2 M3
Feed In situ sweep buffer
M1 = Membrane 1, M2 = Membrane 2, M3 = Membrane 3
across M1 for recovery of product B from overall retentate. The bleed fl ow drawn from the retentate cross fl ow circuit of M1 is the overall retentate product A while that drawn from retentate fl ow circuit of M3 is an overall permeate product B. In this design, the fl ow ratio of the overall retentate fl ow to permeate fl ow as well as value of fl ow into M1 fl ow circuit from M2 cross fl ow circuit can have signifi cant effect on purifi cation and productivity. These parameters can be optimized to achieve optimal purifi cation.
Why cascade ultrafi ltration system give better resolution?
Each ultrafi ltration membrane constituting cascade system can be confi gured and operated at a permeate fl ux corresponding to its high selectivity in the pressuredependent part of the fi ltrate fl ux curve. Thus, it can provide high resolution purifi cation while maintaining the inherent high throughput and high yield characteristics of conventional ultrafi ltration. Hence, the possibility of protein separation without being limited to relative size of proteins is a sharp contrast to the previous limit of having to separate solutes that differ by more than ten-fold in size, a limit which would no longer be required. An example is presented in reference 4, where a protein having molecular size 14.3 kDa (lysozyme) is purifi ed from 17 kDa size proteins in continuous manner.
It provides the opportunity to combine several different separation steps into a single scalable unit operation, such as simultaneous purifi cation and concentration. The integration of membrane modules provides the opportunity to generate buffer insitu and could recycle it for economy and better separation. In some cases, recycled buffer dilutes proteins and reduces concentration polarization as well as membranefouling effects. By using different MWCO membranes, and/or surface charge properties of membranes, one can effectively improve resolution in a single-unit set up. Also, cascade provides the opportunity to carry out the separation process in batch, continuous, or semi-batch mode, depending on our requirements. It is also suitable for the fractionation of two proteins with negligible dilution.
How cascade ultrafi ltration operation is different from conventional ultrafi ltration?
Preferably, cascade ultrafi ltration units are operated in constant fl ux mode to achieve higher separation factor between two protein fractions. As the protein compositions are different in different membranes constituting cascade system, the permeate fl ux, i.e. permeate fl ow need to be optimized to achieve higher retention or permeation of specifi c protein component across each membranes. Usually, permeate pumps are employed to achieve constant permeate fl ux while use of cross fl ow pump limits effect of concentration polarization or fouling over the membrane surface. centration of lysozyme.
J. Chromatogr. Sci. 2010;347:150-158. 5. Mayani M, Mohanty K,
Filipe C, Ghosh R. Continuous fractionation of plasma proteins HSA and HIgG using cascade ultrafi ltration systems.
Sep. Purif. Technol. 2009:70:231-241 6. Mohanty K, Ghosh R.
Novel tangential-fl ow countercurrent cascade ultrafi ltration confi guration for continuous purifi cation of humanized monoclonal antibody. J. Membr. Sci. 2008;307:117-125. 7. Lightfoot EN. Can membrane cascades replace chromatography? Adapting binary ideal cascade theory of systems of two solutes in a single solvent. Sep. Sci. Technol. 2005;40:739-756.
What are limitations of cascade ultrafi ltration?
The system needs optimization of operating conditions and membrane confi guration. For continuous processing, the system works better when protein concentration is as low as one to two per cent. However, the in situ generation of sweeping buffer can be of signifi cant importance in maintaining low protein concentration in the fl ow circuits. Also, the system operational complexity is more as a consequence of permeate and retentate pumps.
References
1. van Reis R, Zydney AL.
Bioprocess membrane technology. J. Membr.
Sci. 2007;297:16-50. 2. Ghosh R. Protein Bioseparation Using Ultrafi ltration: Theory,
Applications and New
Developments, London,
Imperial College Press/
World Scientifi c Publishing Pte Ltd., 2003. 3. van Reis R, Gadam S,
Frautschy LN, Orlando S,
Goodrich EM, Saksena S,
Kuriyel R, Simpson CM,
Pearl S, Zydney AL. High performance tangential fl ow fi ltration. Biotechnol.
Bioeng. 1997;56:71-82. 4. Mayani M, Filipe CDM,
Ghosh R. Cascade ultrafi ltration systems –
Integrated processes for purifi cation and conMukesh Mayani (Ph.D., MCIC) is Research Scientist (Drug Development) at Therapure Biopharma Inc., a Canadian CDMO and involved in developing downstream processes for purifi cation of complex therapeutics. He has several years of experience in drug manufacturing and bioseparations engineering. He holds the Ph.D. degree in Chemical Engineering from McMaster University, Canada and M.Tech. from Indian Institute of Technology, India.
Learn more about High throughput Protein Purifi cation on our Whitepapers Web Portal at
www.bioscienceworld.ca
By dA vid k norr 1 , Z A c HA ry vA n d en Heuve L 2 , M A rc Be BA n 1
High throughput purification of human IgG using the Agilent Bravo 96AM and AssayMAP protein A cartridges
AbStRACt to address a growing desire for robust, high-throughput chromatography capability, Agilent technologies has developed a 96channel pipetting head to enable microchromatography on the bravo liquid handling platform using AssayMAP protein purification cartridges from biosystem Development. the system can operate using direct fluid displacement for chromatography applications, or air displacement for conventional liquid handling. Early results using the system to purify immunoglobulin from artificially constructed samples show the system can purify between 1 and 100 µg of target protein when presented in backgrounds as high as 10 mg/ml of non-specific protein. Recovered protein quantities accurately reflect input quantities, and these results are repeatable over a number of purifications using used or un-used cartridges.
Introduction
The rise of biologics as important pharmaceutical agents has driven an ever-increasing demand for protein analysis methods that are precise, sensitive, and amenable to high throughput. These methods are used to analyze complex samples from throughout the biopharmaceutical discovery process, from research samples taken from protein expression systems to biological samples present in serum to production cell lysates and cell culture supernatants. In typical workflows, HPLC and immunoassays are used to affinity purify specific proteins from complex samples and quantify using absorbance, fluorescence, or chemiluminescence. While these methods have been successfully used for years, a significant challenge has been in adapting these methods to the demands of high throughput analysis where concerns of sample capacity and reagent use are prominent.
The Agilent Bravo with AssayMAP® technology is a simple, precise, and high-throughput platform for microscale purification and preparation of polyclonal or monoclonal antibody (MAb) from bioprocess samples. The 96AM liquid handling head utilizes AssayMAP cartridges with the same protein A chemistry as traditional affinity HPLC and adds advanced liquid handling of the Agilent Bravo to enable protein purifications from complex matrices. This automated platform allows for a full range of high throughput, highly parallelized liquid handling operations, including precision flow rate control to enable true chromatographic separation of the target molecule in low sample volume. The result is a robust system for multiplexing up to 96 samples in a single run.
Here we present results showing system performance purifying human immunoglobulin G (hIgG) from samples with different concentrations of background protein.
Materials and Methods
Reagents: IgG from human plasma (#16-16-090707, Athens Research & Technology, Athens, GA, USA) was diluted to 4 mg/ml in PBS (Sigma). Fish gelatin from cold water fish (FGel, Sigma, #G7041) was reconstituted in PBS at a stock concentration of 20 mg/ml. Bound protein was eluted in 100 mM glycine (pH 2.5, Fisher Scientific, #G46). Phosphate buffered saline (PBS) was diluted from 10X stock (Sigma, #P5493). Protein A purification cartridges were obtained from BioSystem Development.
Instrumentation: Experiments were carried out using a standard Agilent Bravo with a 96AM liquid handling head and an Agilent 96-channel wash station and pump module controlled by VWorks software. Absorbance values were collected at 280 nm using a Varioskan Flash multimode reader (Thermo). Electrophoresis was performed using Agilent Protein 230 chips (Agilent Technologies # 5067-1518) run on an Agilent 2100 Bioanalyzer with 2100 Expert software.
figure 1
1
0.8
0.6
0.4
02 0.2
0
0 0.5
FGel background (mg/ml) (mg/ml) 0 5 10
1
1.5 IgG(mg/ml) 2 2.5
Absorbance values of eluates from Protein A cartridges loaded with hIgG and different concentrations of FGel. R2 values for the three different sample sets, 0, 5, and 10 mg/ml background protein, were 0.9997, 0.9998, and 0.9992 respectively Average values for each concentration were calculated from 8 readings. Error bars indicate standard deviation. Superimposed eletropherogram traces from Protein 230 chips. 166 kDa peak indicates expected position for hIgG. Arrows labeled M indicate upper and lower protein ladder markers.
Run-to-run reproducibility of cartridge-based purification of hIgG. Six purifications were run using 96 cartridges and the same sample input plate containing three different concentrations of hIgG along with 10 mg/ml FGel. between each run the sample plate was rotated 180° (relative rotation is indicated next to each sample in the legend). Average absorbance values for each input concentration (24 samples) are given including standard deviation. Runs five and six used a separate set of cartridges.
PBS at a flow rate of 33 µl/s, then equilibrated by aspirating an additional 50 µl of PBS at 0.41 µl/s. Cartridges were loaded by aspirating 50 µl of sample at a rate of 0.1 µl/s, then washed by aspirating 50 µl of PBS at a rate of 0.41µl/s. Cartridges were dismounted and then the probes were washed 3 times with PBS. Human IgG was eluted by aspirating 50 µl of elution buffer into the probes, mounting the cartridges, then dispensing the syringe contents at a rate of 0.1 µl/s into a 96-well microtiter plate (Greiner µv Star, # 675801). Following elution, the cartridges were flushed with additional PBS, dismounted and the syringes washed.
Linearity was measured by creating 3 sets of 2-fold serial dilutions of hIgG prepared with final concentrations of FGel at 0, 5 and 10 mg/ml. HIgG concentrations for each set were 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 and 0.015625 mg/ml. Each dilution set was introduced to the system using a 12-well sample reservoir (Porvair Sciences, Ltd., # 390012) with each well corresponding to a column of 8 syringes. A single concentration was placed into each of 8 wells and 1X PBS was added to a ninth well as a control. Following purification as outlined above absorbance values for each set of dilutions were averaged and plotted as shown in Figure 1.
Purity was determined using the Bioanalyzer to run sets of un-purified and purified products from individual cartridges. Four µl of sample was mixed with 2 µl of non-reducing denaturing solution, heated, then diluted with 84 µl of de-ionized water and loaded onto an Agilent Protein 230 chip which was run as described in the Agilent Protein 230 Kit Guide (P/N G2938-90054).
Figure 2 shows the super-imposed output from four lanes.
To examine reproducibility and robustness of the assay, a set of samples was constructed with 0.1, 0.4, or 2 mg/ml hIgG in a background of 10 mg/ml FGel. A sample plate then was prepared such that each dilution filled 4 columns (i.e., A1 – H4, A5-H8, or A9-H12). This plate was used during six successive purification runs. Between each run, the sample plate was rotated 180° so that cartridges used to purify high or low amounts of hIgG in one run would be used for the most different concentration in the next run, and so on. After four runs, the cartridge box was replaced and two more runs were performed.
Results and Discussion:
Performance of the system was examined by purifying different concentrations of hIgG that were presented in different backgrounds of a contaminating protein. Linearity of the response was nearly perfect throughout the 1 to 100 µg binding capacity of the cartridges. Purified products co-migrated with hIgG during capillary electrophoresis. In addition, purification results were consistent over multiple assays, and for different cartridges.
figure 2
[FU] 500
400
300
200
100
0
15 20 25 30 35 40 [s]
figure 3
1.2
1
0.8
0 A 28 0.6
0.4
0.2
0 Run 1 1 fwd Run 2 rev Run 3 fwd Run 4 rev Run 5 fwd Run 6 rev
0.1 0.4 IgG starting concentration (mg/ml) 2
Results in fi gure 1 illustrate ability of the 96AM platform to purify protein directly proportional to input of specifc target presented. Yields of hIgG were linear throughout the range of target binding capacity for the cartridges. This performance was obtained even with nonspecifi c backgrounds as high as 10 mg/ml, which approximates that seen with many biological samples. Purifi ed samples examined using electrophoresis under native conditions maintained the expected major peak at approximately 166 kDa while non-specifi c material was minimized. Sample-tosample results throughout six purifi cation runs showed excellent agreement in the amount of eluted product. Results were similar for new and used cartridges, regardless of whether they had been used to purify widely different amounts of target in a previous run. This indicates robustness in both the protocol and the hardware, including cartridge production.
The Agilent Bravo with AssayMAP technology provides a robust system for hIgG titer in support of biopharmaceutical discovery and development. The 96AM head is designed to maintain syringe dead volumes at less-than 500 nl, which is important in eliminating air bubbles and allows direct displacement of fl uid to maintain constant fl ow rate regardless of column resistance. The direct displacement mode can be used with either AssayMAP cartridges or bare probes. By controlling whether cartridges are mounted during aspirate and dispense cycles, the system can dictate uni-directional fl ow through the cartridges. The head can also use pipette tips in airdisplacement mode, delivering 2 µl at ±5% CV, which is identical to the Agilent Bravo 96LT head. While the normal velocity range of 1-500 µl/s is maintained for use with pipette tips, in direct-displacement mode fl ow rates can be controlled to as low as 1 µl/ minute to address challenging separations. The combination of fl ow control with direct displacement fl uid handling in a multichannel system enables high throughput microchromatography.
Two critical factors for developing a successful chromatography method are quantitative binding of desired targets, and effi cient removal of non-specifi c material. Flow rate control is of key importance for both factors to optimize interactions between the sample and column bed material. This is especially important for assays which rely on enzymatic reactions. Because the 96 AM system combines a wide range of fl ow rates with high-quality microscale media cartridges it will be useful for developing additional affi nity purifi cation methods as well as enzyme-based applications such as immunoassays and glycan analyses.
AssayMAP® is technology patented by BioSystem Development. AssayMAP® is a registered trademark of BioSystem Development.
Learn more about Advances in Liquid Handling on our Whitepapers Web Portal at www.bioscienceworld.ca
