Development of an integrated continuous PEGylation and purification Process for granulocyte colony stimulating factor

Kateja, Nikhil; Nitika, .; Dureja, Samit; Rathore, Anurag S.

doi:10.1016/j.jbiotec.2020.07.008

Cited by 17 publications

(8 citation statements)

References 30 publications

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“…This technique relies on addition of salt such as citrate which serve both as reaction stopper (thereby avoiding the use of hydroxylamine) as well as phase separator [ 23 ]. This approach of combining reaction and separation is referred to as process integration, and this can be done in many different ways [ 24 , 25 , 26 , 27 , 28 ].…”

Section: Introductionmentioning

confidence: 99%

Membrane-Based Hybrid Method for Purifying PEGylated Proteins

2023

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PEGylated proteins are usually purified using chromatographic methods, which are limited in terms of both speed and scalability. In this paper, we describe a microfiltration membrane-based hybrid method for purifying PEGylated proteins. Polyethylene glycol (or PEG) is a lower critical solution temperature polymer which undergoes phase transition in the presence of a lyotropic salt and forms micelle-like structures which are several microns in size. In the proposed hybrid method, the PEGylated proteins are first converted to their micellar form by the addition of a lyotropic salt (1.65 M ammonium sulfate). While the micelles are retained using a microfiltration membrane, soluble impurities such as the unmodified protein are washed out through the membrane. The PEGylated proteins thus retained by the membrane are recovered by solubilizing them by removing the lyotropic salt. Further, by precisely controlling the salt removal, the different PEGylated forms of the protein, i.e., mono-PEGylated and di-PEGylated forms, are fractionated from each other. Hybrid separation using two different types of microfiltration membrane devices, i.e., a stirred cell and a tangential flow filtration device, are examined in this paper. The membrane-based hybrid method for purifying PEGylated proteins is both fast and scalable.

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Section: Introductionmentioning

confidence: 99%

Membrane-Based Hybrid Method for Purifying PEGylated Proteins

2023

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“…Kinetic models, − which provide broader insights to reaction mechanisms, laboratory-, and industrial-scale process development, have the potential to help automate the bioconjugation process through rapid online parameter optimization in a design of experiment (DOE)-like manner. − For lysine reactions, the multiplicity of conjugation sites has made kinetic modeling challenging. Early PEGylation kinetic models were able to simulate the degree of PEGylation but not distinguish positional PEG isomers.…”

Section: Introductionmentioning

confidence: 99%

Moving Protein PEGylation from an Art to a Data Science

2022

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PEGylation is a well-established and clinically proven half-life extension strategy for protein delivery. Protein modification with aminereactive poly(ethylene glycol) (PEG) generates heterogeneous and complex bioconjugate mixtures, often composed of several PEG positional isomers with varied therapeutic efficacy. Laborious and costly experiments for reaction optimization and purification are needed to generate a therapeutically useful PEG conjugate. Kinetic models which accurately predict the outcome of socalled "random" PEGylation reactions provide an opportunity to bypass extensive wet lab experimentation and streamline the bioconjugation process. In this study, we propose a protein tertiary structure-dependent reactivity model that describes the rate of protein-amine PEGylation and introduces "PEG chain coverage" as a tangible metric to assess the shielding effect of PEG chains. This structure-dependent reactivity model was implemented into three models (linear, structure-based, and machine-learned) to gain insight into how protein-specific molecular descriptors (exposed surface areas, pK a , and surface charge) impacted amine reactivity at each site. Linear and machine-learned models demonstrated over 75% prediction accuracy with butylcholinesterase. Model validation with Somavert, PEGASYS, and phenylalanine ammonia lyase showed good correlation between predicted and experimentally determined degrees of modification. Our structure-dependent reactivity model was also able to simulate PEGylation progress curves and estimate "PEGmer" distribution with accurate predictions across different proteins, PEG linker chemistry, and PEG molecular weights. Moreover, in-depth analysis of these simulated reaction curves highlighted possible PEG conformational transitions (from dumbbell to brush) on the surface of lysozyme, as a function of PEG molecular weight.

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“…The undoubted demonstration of clinical efficacy and safety is a mandatory requisite for a formulation to be released in the pharmaceutical market. For PEGylated proteins, this implies a careful purification of the target product, to be achieved through the recognition and separation of molecules with the wrong number of PEGs as well as the different positional isoforms. ,, For industrial production, this has increased the demand for robust chromatographic methods to exploit its unique purification efficiency and reach optimal yield. − One of the most common techniques used is ion exchange chromatography, as PEGylation leads to the alteration of the protein isoelectric point (pI) and, in turn, different electrostatic interactions with the stationary phase. Positional isomers of PEGylated proteins may have different strengths of electrostatic interactions, while an increased degree of PEGylation leads to more neutral pI, causing the elution at lower ionic strength in ion exchange chromatography. ,, Because of the high similarity of the chemical properties of the isoforms, a baseline separation of each species is rarely achieved in batch chromatographic purification. − In this case, overlapping regions of the target product and other impurities impose a tradeoff between purity and yield.…”

Section: Introductionmentioning

confidence: 99%

Experimental Design of the Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) Unit for the Separation of PEGylated Proteins

Kim

Botti

Angelo

et al. 2021

Ind. Eng. Chem. Res.

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The rapid growth and diffusion of biopharmaceuticals, including polyethylene glycol (PEG)−protein conjugates, required to set up stringent regulatory requirements, in terms of purity and clinical safety. In order to produce proteins that meet these requirements, the development of robust purification methods has been one of the main goals in the field of downstream processing in the last years. Most of these methods rely on single or sequential batch chromatographic separations, enabling one to reach the purity specification but often at the expense of low yield. An appealing alternative is the Multicolumn Countercurrent Solvent Gradient Purification (MCSGP). Thanks to the internal recycling of the fractions where product and impurities coelute, this process can provide a significant improvement in the process yield, preserving the purity specification. The drawback lies in the increased number of process parameters, compared to the batch, which complicates the design and optimization of this unit. In this work, we propose an ad hoc design procedure for the optimization of central-cut separations at a target purity. Using PEGylated lysozyme as a model system, we illustrate the use of this procedure to identify the load, the elution gradient as well as the collection intervals for optimal yield and productivity, at fixed purity specifications, for a batch (single) column system. The obtained optimal process parameters are subsequently transferred to the MCSGP unit. The so-designed MCSGP process exhibited superior performances compared to the corresponding optimal batch process by increasing the yield and productivity by 17.0% and 2.1%, respectively, at the purity specification of 80%.

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Development of an integrated continuous PEGylation and purification Process for granulocyte colony stimulating factor

Cited by 17 publications

References 30 publications

Membrane-Based Hybrid Method for Purifying PEGylated Proteins

Membrane-Based Hybrid Method for Purifying PEGylated Proteins

Moving Protein PEGylation from an Art to a Data Science

Experimental Design of the Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) Unit for the Separation of PEGylated Proteins

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