Normal Phase High-Performance Liquid Chromatography of Pneumocandins: In Situ Modification of Silica with L-Proline To Separate Structural Analogues

Nti-Gyabaah, Joseph; Antia, Firoz D.; Dahlgren, Mary Ellen; Göklen, Kent E.

doi:10.1021/bp050319t

Cited by 6 publications

(2 citation statements)

References 26 publications

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“…Order Rate law Integrated rate law (Nti-Gyabaah et al, 2006;Roush et al, 2005). Use of the rigid ceramic beads afforded tolerance of elevated process temperatures as well as being compatible with the hexylene glycol modifier.…”

Section: Redox Reaction Rate Modelingmentioning

confidence: 99%

“…The approach leverages use of a ceramic CEX resin combined with a mobile phase modifier, hexylene glycol. There are limited prior examples of this approach employed for the resolution of impurities of similar biophysical characteristics (Nti‐Gyabaah et al, 2006; Roush et al, 2005). Use of the rigid ceramic beads afforded tolerance of elevated process temperatures as well as being compatible with the hexylene glycol modifier.…”

Section: Case Study #3: In‐situ Mixed Mode (Cex)mentioning

confidence: 99%

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Insulin purification—Innovation continuum via synthesis of fundamentals, technology, and modeling

Roush

Iammarino

Chmielowski

et al. 2023

Biotech & Bioengineering

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Advancement in all disciplines (art, science, education, and engineering) requires a careful balance of disruption and advancement of classical techniques. Often technologies are created with a limited understanding of fundamental principles and are prematurely abandoned. Over time, knowledge improves, new opportunities are identified, and technology is reassessed in a different light leading to a renaissance. Recovery of biological products is currently experiencing such a renaissance. Crystallization is one example of an elegant and ancient technology that has been applied in many fields and was employed to purify insulins from naturally occurring sources. Crystallization can also be utilized to determine protein structures. However, a multitude of parameters can impact protein crystallization and the “hit rate” for identifying protein crystals is relatively low, so much so that the development of a crystallization process is often viewed as a combination of art and science even today. Supplying the worldwide requirement for insulin (and associated variants) requires significant advances in process intensification to support scale of production and to minimize the overall cost to enable broader access. Expanding beyond insulin, the increasing complexity and diversity of biologics agents challenge the current purification methodologies. To harness the full potential of biologics, there is a need to fully explore a broader range of purification technologies, including nonchromatographic approaches. This impetus requires one to challenge and revisit the classical techniques including crystallization, chromatography, and filtration from a different vantage point and with a new set of tools, including molecular modeling. Fortunately, computational biophysics tools now exist to provide insights into mechanisms of protein/ligand interactions and molecular assembly processes (including crystallization) that can be used to support de novo process development. For example, specific regions or motifs of insulins and ligands can be identified and used as targets to support crystallization or purification development. Although the modeling tools have been developed and validated for insulin systems, the same tools can be applied to more complex modalities and to other areas including formulation, where the issue of aggregation and concentration‐dependent oligomerization could be mechanistically modeled. This paper will illustrate a case study juxtaposing historical approaches to insulin downstream processes to a recent production process highlighting the application and evolution of technologies. Insulin production from Escherichia coli via inclusion bodies is an elegant example since it incorporates virtually all the unit operations associated with protein production—recovery of cells, lysis, solubilization, refolding, purification, and crystallization. The case study will include an example of an innovative application of existing membrane technology to combine three‐unit operations into one, significantly reducing solids handling and buffer consumption. Ironically, a new separations technology was developed over the course of the case study that could further simplify and intensify the downstream process, emphasizing and highlighting the ever‐accelerating pace of innovation in downstream processing. Molecular biophysics modeling was also employed to enhance the mechanistic understanding of the crystallization and purification processes.

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Section: Redox Reaction Rate Modelingmentioning

confidence: 99%

Section: Case Study #3: In‐situ Mixed Mode (Cex)mentioning

confidence: 99%