Process for production and purification of lethal toxin neutralizing factor (LTNF) from <i>E. coli</i> and its economic analysis

Hebbi, Vishwanath; Pandi, Kathiresan; Kumar, Devendra; Komives, Claire; Rathore, Anurag S.

doi:10.1002/jctb.5537

Cited by 6 publications

(17 citation statements)

References 43 publications

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“…The production of lethal toxin‐neutralizing factor (LTNF) by Escherichia coli ( E. coli ) was chosen for implementation. LTNF is a peptide purified from opossum ( Didelphis virginiana ) serum, which neutralizes snake venom and acts against toxins from insects, plant, and bacteria; it is used primarily as antivenom for snakebites . The air supply and substrate feeding pump were shut down to mimic the utility failure (accidental shutdown) of actuators often encountered in process operation to determine how the controller responds to these disruptions and how well the process recovers based on control action.…”

Section: Introductionmentioning

confidence: 99%

“…LTNF is a peptide purified from opossum (Didelphis virginiana) serum, which neutralizes snake venom and acts against toxins from insects, plant, and bacteria; it is used primarily as antivenom for snakebites. [25][26][27] The air supply and substrate feeding pump were shut down to mimic the utility failure (accidental shutdown) of actuators often encountered in process operation to determine how the controller responds to these disruptions and how well the process recovers based on control action. Through this study, we investigated the window of opportunity that is created by employing an advanced controller when disruptions caused by utility failure occur during process operation.…”

Section: Introductionmentioning

confidence: 99%

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Implementing Process Analytical Technology for the Production of Recombinant Proteins in Escherichia coli Using an Advanced Controller Scheme

Priyanka

Kumar

Gomes

et al. 2019

Biotechnology Journal

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The performance of a bioreactor in meeting process goals is affected by the microorganism used, medium composition, and operating conditions. A typical bioreactor uses a supervisory control and data acquisition (SCADA) system for control, and a combination of software and hardware tools for real‐time data analysis. However, when the process is disrupted by utility or instrumentation failure, typical process controllers may be unable to reinstate normal operating conditions before the cells in the reactor shift to unfavorable metabolic regimes. The objective of this study is to examine how the response of a controller affects process recovery when disruptive incidences occur under a process analytical technology (PAT) framework. The process used for this investigation is the production of lethal toxin‐neutralizing factor (LTNF) by Escherichia coli (E. coli), which is controlled by a decoupled input–output‐linearizing controller (DIOLC). The performance of the DIOLC is compared to a proportional integral derivative (PID) controller subjected to the same conditions. The disruptions are introduced manually and the effect of controller action on process recovery and LTNF synthesis is measured in terms of peak purity and concentration. It is observed that DIOLC performs better after reinstating operating conditions and results in a meaningful improvement in performance.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Implementing Process Analytical Technology for the Production of Recombinant Proteins in Escherichia coli Using an Advanced Controller Scheme

Priyanka

Kumar

Gomes

et al. 2019

Biotechnology Journal

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“…Interestingly, the first 15 amino acids of the N-terminal sequence of the full protein have been found to contain the activity of the complete protein . From a digest of the LTNF protein, it has been reported that the N-terminal portion (10–15 amino acids) of the protein maintained the potency of the full protein when administered to mice. , The short peptide LKAMDPTPPLW with 11 amino acid residues from the N-terminal of the native LTNF protein has been produced recombinantly by our group and is henceforth referred to as rLTNF . We have previously demonstrated the activity of rLTNF against rattlesnake venom and proposed a manufacturing process with economic analysis. , …”

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confidence: 99%

“…The need for fast and reliable methods for monitoring cleavage reactions is prevalent in many areas of biologics manufacturing. An RP-HPLC method for monitoring the cleavage of rLTNF concatemer into monomer has been published previously . Cation exchange elute of rLTNF concatemer was treated with α-chymotrypsin to induce cleavage at the C-terminal of tryptophan to yield the monomer.…”

mentioning

confidence: 99%

“…2,3 The short peptide LKAMDPTPPLW with 11 amino acid residues from the N-terminal of the native LTNF protein has been produced recombinantly by our group and is henceforth referred to as rLTNF. 4 We have previously demonstrated the activity of rLTNF against rattlesnake venom and proposed a manufacturing process with economic analysis. 2,3 Peptides can be manufactured either by chemical synthesis or via recombinant DNA technologies.…”

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Process Analytical Technology Implementation for Peptide Manufacturing: Cleavage Reaction of Recombinant Lethal Toxin Neutralizing Factor Concatemer as a Case Study

Hebbi¹,

Kumar²,

Rathore³

2020

Anal. Chem.

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The α-chymotrypsin-based cleavage reaction is necessary for manufacturing peptides using rDNA technology with tandem repeats. The current work showcases application of process analytical technology (PAT) tools for monitoring and control of this reaction, using recombinant Lethal Toxin Neutralizing Factor (rLTNF) as a case study. At-line Fourier Transform infrared spectroscopy (ATR-FTIR) combined with attenuated total internal reflectance sampling accessory was exploited to monitor the reaction. PLS spectral calibration models were created for real-time quantification of concentrations of rLTNF concatemer and urea in the reaction mixture. An end-toend PAT monitoring and control strategy was developed to address potential deviations and ensure that targets for yield, purity, and impurity profile are met for each batch. The impact of various deviations of process parameters outside the operating space, such as deviations in the reaction buffer, concentration of concatemer in the IBs, enzyme loading relative to protein concentration, and reaction time with late quenching were investigated. Variation in impurity profile over time in the case of late reaction quenching was determined through HPLC and mass spectrometry. It has been demonstrated how process signatures from the PAT tools across various batches and campaigns can be analyzed to facilitate real-time process monitoring and control.

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Status and future developments for downstream processing of biological products

Jungbauer,

Ferreira,

Butler

et al. 2024

Biotech & Bioengineering

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Governments and biopharmaceutical organizations aggressively leveraged expeditious communication capabilities, decision models, and global strategies to make a COVID‐19 vaccine happen within a period of 12 months. This was an unusual effort and cannot be transferred to normal times. However, this focus on a single vaccine has also led to other treatments and drug developments being sidelined. Society expects the pharmaceutical industry to provide an uninterrupted supply of medicines. However, it is often overlooked how complex the manufacture of these compounds is and what logistics are required, not to mention the time needed to develop new drugs. The overarching theme, therefore, is patient access and how we can help ensure access and extend it to low‐ and middle‐income countries. Despite unceasing efforts to make medications available to all patient populations, this must never be done at the expense of patient safety. A major fraction of the costs in biopharmaceutical manufacturing are for drug discovery, process development, and clinical studies. Infrastructure costs are very difficult to quantify because they often depend on whether a greenfield facility or an existing, depreciated facility is used or adapted for a new product. To accelerate process development concepts of platform process and prior knowledge are increasingly leveraged. While more traditional protein therapeutics continue to dominate the field, we are also experiencing the exciting emergence and evolution of other therapeutic formats (bispecifics, tetravalent mAbs, antibody‐drug conjugates, enzymes, peptides, etc.) that offer unique treatment options for patients. Protein modalities are still dominant, but new modalities are being developed that can be learned from including advanced therapeutics‐like cell and gene therapies. The industry must develop a model‐based strategy for process development and technologies such as continuous integrated biomanufacturing must be adopted. The overall conclusion is that the pandemic pace was unsustainable, focused on vaccine delivery at the expense of other modalities/disease targets, and had implications for professional and personal life (work‐life balance). Routinely reducing development time from 10 years to 1 year is nearly impossible to achieve. Environmental aspects of sustainable downstream processing are also described.

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Process for production and purification of lethal toxin neutralizing factor (LTNF) from E. coli and its economic analysis

Abstract: The developed process is able to produce purified rLTNF with 78±2% recovery. The study shows that recombinant technology can be used to produce rLTNF cost effectively and shows potential as a substitute for currently available antivenoms against snakebite.

Cited by 6 publications

References 43 publications

Implementing Process Analytical Technology for the Production of Recombinant Proteins in Escherichia coli Using an Advanced Controller Scheme

Implementing Process Analytical Technology for the Production of Recombinant Proteins in Escherichia coli Using an Advanced Controller Scheme

Process Analytical Technology Implementation for Peptide Manufacturing: Cleavage Reaction of Recombinant Lethal Toxin Neutralizing Factor Concatemer as a Case Study

Status and future developments for downstream processing of biological products

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