Determination of robustness and optimal work conditions for a purification process of a therapeutic recombinant protein using response surface methodology

Amadeo, Ignacio; Mauro, Laura V.; Ortí, Eduardo; Forno, Guillermina

doi:10.1002/btpr.588

Cited by 7 publications

(3 citation statements)

References 25 publications

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“…In this optimization response surface methodology (RSM) was applied through a Box–Behnken design (BBD). Although RSM has already been applied in chromatographic purifications of several therapeutic products and proteins such as recombinant erythropoietin on Blue‐Sepharose, so far the application of RSM for the optimization of the chromatographic purification of PEGylated proteins has not been explored. The experimental points were done with a mixture of the above‐mentioned proteins in a 4:1 ratio.…”

Section: Resultsmentioning

confidence: 99%

Optimized purification of mono‐PEGylated lysozyme by heparin affinity chromatography using response surface methodology

Mejía‐Manzano

Lienqueo

Escalante-Vázquez

et al. 2017

J of Chemical Tech & Biotech

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BACKGROUND: The efficient, controlled and robust purification of conjugates from PEGylation has a growing demand in the biopharmaceutical's market. In general, the yield and purity reached through the conventional chromatographic modes are not particularly high or efficient. Affinity chromatography has so far scarcely been explored. The present work introduces the purification of mono-PEGylated lysozyme from a PEGylation reaction by heparin affinity chromatography (HAC) for the first time in a single step. Response surface methodology (RSM), particularly a Box-Behnken design (BBD) was employed to optimize the separation. RESULTS: Protein adsorption of PEGylated and native lysozyme on Heparin Sepharose 6 Fast Flow resin was described byLangmuir isotherms, showing a relatively low affinity for the PEGylated proteins. From the experimental design, optimal elution conditions in a linear gradient of sodium chloride (NaCl) for the three response variables (yield, purity and productivity) were: gradient length of 13 column volumes (CVs), flow at 0.8 mL min −1 and protein load of 1 mg mL −1 . Based on this optimization, a step gradient procedure was designed that achieved the purification of mono-PEGylated lysozyme with approximately 100% yield and purity in comparison with 92.7% and 99.7% with the linear gradient. Productivity was c. 0.048 ± 0.001 mg mL −1 min −1 using 0.05 mol L −1 NaCl for its elution. CONCLUSIONS: Mono-PEGylated lysozyme was completely separated from a PEGylation mixture with high yield and purity using HAC for first time. Applying response surface methodology (RSM), adequate conditions for more than one requirement were found as well as optimal conditions for a linear gradient of NaCl. Based on this optimization a step gradient procedure was designed that achieved the purification of mono-PEGylated lysozyme in one step with advantages respect to time, resolution, yield and purity compared with other chromatographic modes such as hydrophobic interaction chromatography (HIC) and cation exchange chromatography (CEX).

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Section: Resultsmentioning

confidence: 99%

Optimized purification of mono‐PEGylated lysozyme by heparin affinity chromatography using response surface methodology

Mejía‐Manzano

Lienqueo

Escalante-Vázquez

et al. 2017

J of Chemical Tech & Biotech

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“…This helps to assess the impact of any excursion beyond the usual operating ranges. Many techniques involving experimental design are available, from methodologies mainly suitable for screening experiments such as full and partial factorials [16] and Plackett-Burman designs [17], to optimization techniques such as response surface methodology (RSM) [18] and central composite designs [19]. For an extensive review of the strategies used in the optimization of chromatographic systems, refer to [20].…”

Section: Figmentioning

confidence: 99%

Establishment of a Design Space for Biopharmaceutical Purification Processes Using DoE

Amadeo

Mauro²,

Ortí³

et al. 2014

Methods in Molecular Biology

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Recent trends in the pharmaceutical sector are changing the way protein purification processes are designed and executed, moving from operating the process in a fixed point to allowing a permissible region in the operating space known as design space. This trend is driving product development to design quality into the manufacturing process (Quality by Design) and not to rely exclusively on testing quality in the product. A typical purification step has numerous operating parameters that can impact its performance. Therefore, optimization and robustness analysis in purification processes can be time-consuming since they are mainly grounded on experimental work. A valuable approach consists in the combination of an adequate risk analysis technique for selecting the relevant factors influencing process performance and the design of experiment methodology. The latter allows for many process variables which can be studied at the same time; thus, the number of tests will be reduced in comparison with the conventional approach based on trial and error. These multivariate studies permit a detailed exploration in the experimental range and lay the foundation of Quality by Design principles application. This article outlines a recommended sequence of activities toward the establishment of an expanded design space for a purification process.

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“…For most products, water is the chief impurity and isolation steps are designed to remove most of it, reducing the volume of material to be handled and concentrating the product. Solvent extraction, adsorption, ultra filtration, and precipitation are some of the unit operations involved [23].…”

Section: Product Isolationmentioning

confidence: 99%

Integrated Application of Bioprocess Engineering and Biotechniques for Quality & Bulk Drug Manufacturing

Jena¹,

Mahalakshmi²,

G³

2011

JBB

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One of the major challenges facing the pharmaceutical industry today is finding new ways to increase productivity, decrease costs whilst still ultimately developing new therapies that enhance human health. Both Biotechniques & Bioprocess engineering are integrated for bulk drug manufacturing of biopharmaceutical products to outcome the above problem. Different biotechnological processes are used in industries for large scale production of biological products for optimization of yield and the quality of end product. Various recent developments in biotechniques and its collaborated work with bioprocess engineering helps to maintain the quality in manufacturing process. However in this review article, we systemically describe all the techniques and different bioprocess used for manufacturing of drugs. To the author's knowledge this review represents the most exhaust description of different biotechniques, different bioprocess, process development and design criteria in bioprocess.

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Determination of robustness and optimal work conditions for a purification process of a therapeutic recombinant protein using response surface methodology

Cited by 7 publications

References 25 publications

Optimized purification of mono‐PEGylated lysozyme by heparin affinity chromatography using response surface methodology

Optimized purification of mono‐PEGylated lysozyme by heparin affinity chromatography using response surface methodology

Establishment of a Design Space for Biopharmaceutical Purification Processes Using DoE

Integrated Application of Bioprocess Engineering and Biotechniques for Quality & Bulk Drug Manufacturing

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