ON DEMAND: ANTHONY GRABSKI, Ph.D Director of Research and Development Semba Biosciences Inc.

    Octave™ Applications

Applications: Proteins

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2014 Bench top continuous chromatography: an enabling platform for highly productive mAb purification Grabski, A., Zilberman, A., Van Oosbree, T., Mierendorf, R. BioProcess International Conference, Boston USA

ABSTRACT: For this study we purified a humanized IgG1a mAb from CHO culture fluid using the Octave System in a SMB-PAC process and an AKTA System in a standard batch PAC process. Previously, we have established that mAb produced from a Step-SMB PAC process performed on the Octave platform was equivalent or superior in quality to that produced by the analogous batch process. In this study, we demonstrated that this finding was consistent between three Protein A adsorbents: an agarose-based MabSelect SuRe (GE Life Sciences), and two polymethacrylate-based resins: TOYOPEARL AF-rProtein A-650F and TOYOPEARL AF-rProtein A HC-650F (Tosoh Bioscience). Importantly, TOYOPEARL AF-rProtein A HC-650F demonstrated the best performance in Step-SMB at a mAb titer of 7.5 g/L: productivity was 97 g mAb/L resin/h with 97% yield.

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2014 Continuous separation of protein loaded nanoparticles by simulated moving bed chromatography Satzer, P., Wellhoefer, M. and Jungbauer, A. J. Chromatogr. A 1349  44-49

ABSTRACT: For scale up and efficient production of protein loaded nanoparticles continuous separation by size exclusion chromatography in simulated moving bed (SMB) mode helps do reduce unbound protein concentration and increase yields for perfectly covered particles. Silica nanoparticles were loaded with an excess of beta casein or bovine serum albumin (BSA) and the loaded particles purified by size exclusion chromatography using Sephacryl 300 as stationary phase in a four zone SMB. We determined our working points for the SMB from batch separations and the triangle theory described by Mazzotti et al. with an SMB setup of one Sephacryl 300 26/70mm column per zone with switch times of 5 min for BSA and 7 min for beta casein. In the case of BSA the Raffinate contained loaded nanoparticles of 63% purity with 98% recovery and the extract was essentially particle free (95% purity). We showed that the low purity of the Raffinate was only due to BSA multimers present in the used protein solution. In the case of beta casein where no multimers are present we achieved 89% purity and 90% recovery of loaded nanoparticles in the Raffinate and an extract free of particles (92% purity). Using a tangential flow filtration unit with 5 kDa cutoff membrane we proved that the extract can be concentrated for recycling of protein and buffer. The calculated space-time-yield for loaded nanoparticles was 0.25 g of loaded nanoparticles per hour and liter of used resin. This proves that the presented process is suitable for large scale production for industrial purposes.

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2013 Bench top continuous chromatography: an enabling platform for bioprocess development Mierendorf, R., Zilberman, A., Thalley, B., Grabski, A. Integrated Continuous Biomanufacturing Conference, Barcelona, Spain

SUMMARY: For this study we purified a humanized IgG1a monoclonal antibody from CHO culture fluid using the Octave System in a step mode continuous Protein A capture (PAC) process and an AKTA System in a standard batch PAC process. We compared the performance of three commercial Protein A adsorbents in continuous and single-column modes, as determined by measuring host cell protein (HCP), residual DNA, and monomer content in the purified fractions. In all cases the purity achieved by SMBC was equivalent or slightly better than the single-column process.

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2013 Continuous processing of recombinant proteins: Integration of inclusion body solubilization and refolding using simulated moving bed size exclusion chromatography with buffer recycling Wellhoefer, M., Sprinzl, W., Hahn, R. and Jungbauer, A. J. Chromatogr. A 1319  107-117

ABSTRACT: An integrated process which combines continuous inclusion body dissolution with NaOH and continuous matrix-assisted refolding based on closed-loop simulated moving bed size exclusion chromatography was designed and experimentally evaluated at laboratory scale. Inclusion bodies from Npro fusion pep6His and Npro fusion MCP1 from high cell density fermentation were continuously dissolved with NaOH, filtered and mixed with concentrated refolding buffer prior to refolding by size exclusion chromatography (SEC). This process enabled an isocratic operation of the simulated moving bed (SMB) system with a closed-loop set-up with refolding buffer as the desorbent buffer and buffer recycling by concentrating the raffinate using tangential flow filtration. With this continuous refolding process, we increased the refolding and cleavage yield of both model proteins by 10% compared to batch dilution refolding. Furthermore, more than 99% of the refolding buffer of the raffinate could be recycled which reduced the buffer consumption significantly. Based on the actual refolding data, we compared throughput, productivity, and buffer consumption between two batch dilution refolding processes – one using urea for IB dissolution, the other one using NaOH for IB dissolution – and our continuous refolding process. The higher complexity of the continuous refolding process was rewarded with higher throughput and productivity as well as significantly lower buffer consumption compared to the batch dilution refolding processes.

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 2011 Comparison of purification of recombinant n-demthylase B (NdmB) using simulated moving bed affinity chromatography (SMB) and FPLC Moreno-Cid Mora, J.A., Xu, J., Gopishetty, S.R. and Subramanian, M. Current Topics in Industrial Biotechnology Conference, Iowa City USA

SUMMARY: The Octave 100 System was used in Step SMBC mode to purify a histidine-tagged recombinant protein (NdmB) expressed in E. coli by immobilized metal affinity chromatography (IMAC). For comparison the same cell lysate was used with the same adsorbent in a single column batch process (FPLC). The SMBC process used eight 5-ml columns and FPLC used one 40-mL (XK26) column. Equal amount of protein was loaded on to both systems. The recoveries of NdmB from SMBC and FPLC were 30% and 23% respectively. Based on band intensity on SDS-PAGE of NdmB, it was assessed that SMBC achieved 95 % purity whereas from FPLC it was 91%. Measuring the overall productivity in terms of (a) time taken for purification, 150 minutes vs. 350 minutes (SMBC vs FPLC), (ii) buffer use, 1 vs 1.5 times (SMBC vs FPLC), and (iii) column regeneration (no additional unit operation for SMB), SMBC method was better than the FPLC (batch chromatography).

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2010 Protein purification by SMB: Taking the chromatography platform to the next level Zilberman, A., Grabski, A. and Mierendorf, R. World Biopharm Forum, Cambridge, UK

SUMMARY: The poster demonstrates use of the Octave System in isocratic mode to separate proteins by continuous size exclusion chromatography (SEC), and in step mode for Protein A affinity purification of a monoclonal antibody. Comparisons with single column purification revealed that the SMBC process resulted in improved efficiency in chromatography media and buffer utilization.

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2009 Simulated moving bed chromatography Grabski, A. and Mierendorf, R. Gen. Eng. Biotechnol. News 10  54–55

SUMMARY: The article describes the two basic modes of operation of the Octave System and their application to protein purification. An example shows Protein A affinity purification of a monoclonal antibody from concentrated tissue culture fluid using POROS® MabCapture™ A resin. Antibody recovery was 95%, purity was greater than 99%, and productivity of 200 mg/h was achieved with 8 x 1 mL columns at loading flow velocity of 900 cm/h.

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2009 Continuous highly efficient protein purification using simulated moving bed chromatography Grabski, A., Zilberman, A., and Mierendorf, R. Protein Engineering Summit, Boston USA

SUMMARY: The poster describes the Octave System and demonstrates continuous IMAC purification of several human histidine-tagged fusion proteins expressed in E. coli. A comparison with single column purification revealed that the SMBC process resulted in significant gains in purity.

Applications: Small Molecules

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2015 Size-exclusion simulated moving bed for separatingorganophosphorus flame retardants from a polymer George S. Weeden Jr., Lei Ling, Nicholas H. Soepriatna, Nien-Hwa Linda Wang J. Chromatogr. A 1422 99-116

ABSTRACT: Over 500,000 t of flame retardants in electronic wastes are consigned to landfills each year. A room-temperature, size-exclusion simulated moving bed (SEC-SMB) was developed to recover high purity(>99%) flame retardants with high yield (>99%). The SSWD method for ternary mixtures was developed for SEC-SMB. Fourteen decision variables were optimized to obtain the lowest separation cost within1 min. The estimated cost is less than 10% of the purchase cost of the flame retardants. The estimated cost of the optimized SEC-SMB is less than 3% of that of a conventional batch SEC processes. Fast start-up methods were developed to reduce the SMB start-up time by more than 18-fold. SEC-SMB can be an economical method for separating small molecules from polymers.

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2014 Systematic optimization and experimental validation of ternary simulated moving bed systems Agrawal, G., Sreedhar, B., Kawajiri, Y. J. Chromatogr. A 1356 82-95

ABSTRACT: Over the past several decades, many modifications have been proposed in SMB chromatography in order to effectively separate a binary mixture. However, the separation of a multi-component mixture using SMB is still one of the major challenges. Recently, a computational study was performed which compared various existing isocratic ternary separation operating schemes (including the JO process) in terms of the maximum throughput attained, and Generalized Full Cycle strategy was proposed based on a systematic design, which was found to have significant improvement over existing strategies [Agrawal and Kawajiri (2012)]. Nevertheless, the operating strategies were not experimentally validated. In this study, we validate both JO and Generalized Full Cycle SMB systems experimentally. A simultaneous optimization and model correction scheme has been implemented to arrive at the optimal operating condition which satisfies the optimal productivity as well as the desired purity and recovery of products experimentally.

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2014 Multi-column chromatographic process development using simulated moving bed superstructure and simultaneous optimization – Model correction framework Sreedhar, B., and Kawajiri, Y. Chemical Engineering Science 116  428-441

ABSTRACT: In this work, we demonstrate an improved framework for simulated moving bed (SMB) chromatographic process development using a superstructure formulation. Various optimal column configurations and operations are obtained by solving a multi-objective optimization problem representing the super-structure by maximizing feed throughput and minimizing Desorbent usage. In order to resolve the model mismatch with experimental data, here we utilize the simultaneous optimization – model correction (SOMC) method in which process optimization is carried out in tandem with model correction using data obtained from experimental evaluations. The potential of the superstructure–SOMC framework has been demonstrated by separating glucose and fructose using columns packed with a cation exchange resin with water as the mobile phase. The optimal operation modes found using the superstructure included standard SMB, three zone (3Z) operation, intermittent SMB (I-SMB) and a newly found outlet stream swing (OSS) – partial feed (PF) hybrid operation. All configurations were experimentally implemented using a Semba Octave™ 100 Chromatography System.

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2013 Simulated Moving Bed Chromatography: Separation and Recovery of Sugars and Ionic Liquid from Biomass Hydrolysates Caes, B.R., Van Oosbree, T. R., Lu, F., Ralph, J., Maravelias, C.T. and Raines, R.T. ChemSusChem 6  2083-2089

SUMMARY: This paper reports the use of ionic liquid [BMIM]Cl in combination with HCl to efficiently hydrolyze cellulose in raw corn stover to glucose and xylose, and the separation and recovery of the [BMIM]Cl and sugars at nearly quantitative levels by SMBC with the Octave System. The recovered hydrolysate sugar products were able to be used as feedstocks for microbes, as demonstrated by using E. coli KO11 to produce ethanol, and the residual lignin appeared to largely retain its native structure.

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2012 Dynamic resolution of chiral 2-hydroxy acids by spinach glycolate oxidase and subsequent purification of the (R)-enantiomers using simulated moving bed chromatography Das, S., Grabski, A., Thalley, B., Zilberman, A., Mierendorf, R. and Subramanian, M. Society for Industrial Microbiology and Biotechnology Annual Meeting, Washington DC, USA

SUMMARY: Spray-dried whole cells of Pichia pastoris expressing GO from spinach were used for the dynamic resolution of 2-hydroxy acids. As GO is absolutely specific on (S)-enantiomer, GO catalyzed oxidation of racemic (RS)-2–hydroxy acids produced 2-keto acids, only from (S)-enantiomer, keeping (R)-isomer intact. Non-selective reduction of 2-ketoacids using sodium borohydride produced (RS)-2–hydroxy acids to facilitate a one-pot dynamic process for production of (R)-2–hydroxyl acids with high yield (> 95%). GO could be recycled twice in this dynamic process with > 92% yield of the (R)-acids. The entire “one pot – two step” process was carried out in water and at room temperature in order to make the process economical. Finally, (R)-2-hydroxy acids were purified from the crude post-reaction mixtures by cation exchange using a Semba Octave™ simulated moving bed chromatography (SMBC) system. The initial SMBC conditions were determined using triangle theory based separation parameters, and optimized by adjusting those parameters based on the analysis of the purified fractions. Continuous separation of the hydroxy acids from their 2-keto intermediates and other reaction components was achieved with yields >80% and purities >99%.

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2012 Modeled simulated moving bed purification of 2-hydroxybutyric acid Grabski, A., Das, S., Thalley, B., Yun, J., Zilberman, A., Kim, S-I, Subramanian, M., and Mierendorf, R. Preparative and Process Chromatography International Symposium, Boston, USA

SUMMARY: This poster demonstrates the application of ChromWorks™ SMBC simulation software to separation of organic acids on the Octave™ System. A feed stream derived from recombinant yeast culture fluid containing a mixture of (R)-2-hydroxybutyric acid and 2-ketobutryic acid was separated by ion exchange chromatography with the Octave System in isocratic mode. The modeled separation parameters correlated with the experimental results to produce (R)-2-HBA with >99% purity.

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2009 Performance and benefits of macrocyclic glycopeptide CSP’s in enantiomeric purification using bench-top SMB technology Lee, J.T., Campbell, W. and Mierendorf, R. International Symposium on Chirality, Breckenridge, USA

SUMMARY: This poster demonstrates the use of the Octave™ System for chiral separation using macrocyclic glycopeptide CSPs. Supelco’s Astec CHIROBIOTIC™ V2 and CHIROBIOTIC T media (15 micron particle diameter) were packed in eight 1 x 5 cm columns and run on the Octave 10 System. Racemic mixtures of 5-methyl 5-phenylhydantoin, tolpersione, and ketorolac were successfully separated with high purity and recovery.

Applications: Natural Bioactive Compounds

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2014 Simulated moving bed purification of flaxseed oil orbitides: Unprecedented separation of cyclolinopeptides C and E Okinyo-Owiti, D., Burnett, P., Reaney, M. J. Chromatogr. B 965 231-237

ABSTRACT: The purification and enrichment of most natural products with potential pharmaceutical applications has been performed mainly employing conventional batch-mode chromatographic processes. There is a growing interest in use of simulated moving bed (SMB) chromatography for natural product enrichment as this method enables conservation of mobile phase, while increasing productivity of chromatography medium. SMB increases yield while decreasing cost. Cyclolinopeptides C ([1-9-NaC],[1-MetO]-CLB, 3) and E ([1-8-NaC],[1-MetO]-CLE, 8) were extracted as a mixture from flaxseed oil and then enriched using a three-zone simulated moving bed. The current research extends the SMB technology to enrichment of cyclolinopeptides (CLs), a group of biologically active hydrophobic cyclic peptides that occur in flaxseed oil. Of interest are [1-9-NaC],[1-MetO]-CLB (3) and [1-8-NaC],[1-MetO]-CLE (8) that provide synthetic scaffolds for modified CLs. The influence of flow rate (feed, desorbent, and extract) on the separation of [1-9-NaC],[1-MetO]-CLB (3) and [1-8-NaC],[1-MetO]-CLE (8) was investigated.

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2013 Continuous Solid Phase Extraction with Juice Pigments Aretz, C., Maciolek, J., and Kedrowski, B. University of Wisconsin-Oshkosh Scientific Symposium, Oshkosh, USA

SUMMARY: This poster demonstrates use of the Octave 100 System for continuous solid phase extraction (SPE) to produce large amounts of target pigments from fruit juice. This method utilized four columns and the Octave operating in step mode where each column participates in a different stage of separation and rotates through the stages of equilibration, loading, washing, and elution. This rotation of stages effectively allows for continuous separation of pigments from non-pigment compounds, like sugars, in organic juices.