Monitoring ceramic hydroxyapatite media degradation using dynamic image analysis and uniaxial confined bulk compression.

Ceramic hydroxyapatite (CHT) is a multimodal chromatographic medium widely used in the pharmaceutical industry for the purification of biomolecules. CHT is a sintered form of hydroxyapatite crystals with moderate stability at acidic conditions. This moderate stability may lead to underperformance of CHT packed bed lifetime, especially under acidic conditions, which should be monitored by diagnostic tools to design optimal buffer systems for the step. This study presents the application of dynamic image analysis (DIA) and uniaxial confined bulk compression (UCBC) to monitor CHT particle degradation as a function of buffer composition. DIA was used to evaluate changes in solidity and morphology, while UCBC was used to evaluate changes in resistance to uniaxial compression. All properties were studied as a function of bed position and operational parameters. Results show that when CHT is exposed to acidic pH, adding phosphate and/or calcium at concentrations of 1 mM minimizes changes in particle solidity and mechanical strength. Changes in CHT morphological properties (i.e., convexity, aspect ratio) are also affected by the presence of calcium and/or phosphate in the inlet buffers. Furthermore, calcium and phosphate have a positive effect on the mechanical behavior of CHT, which is related to changes in the CHT particle solidity.

Techno-economic evaluation of an inclusion body solubilization and recombinant protein refolding process.

Expression of recombinant proteins in Escherichia coli is normally accompanied by the formation of inclusion bodies (IBs). To obtain the protein product in an active (native) soluble form, the IBs must be first solubilized, and thereafter, the soluble, often denatured and reduced protein must be refolded. Several technically feasible alternatives to conduct IBs solubilization and on-column refolding have been proposed in recent years. However, rarely these on-column refolding alternatives have been evaluated from an economical point of view, questioning the feasibility of their implementation at a preparative scale. The presented study assesses the economic performance of four distinct process alternatives that include pH induced IBs solubilization and protein refolding (pH_IndSR); IBs solubilization using urea, dithiothreitol (DTT), and alkaline pH followed by batch size-exclusion protein refolding; inclusion bodies (IBs) solubilization using urea, DTT, and alkaline pH followed by simulated moving bed (SMB) size-exclusion protein refolding, and IBs solubilization using urea, DTT and alkaline pH followed by batch dilution protein refolding. The economic performance was judged on the basis of the direct fixed capital, and the production cost per unit of product (P(C)). This work shows that (1) pH_IndSR system is a relatively economical process, because of the low IBs solubilization cost; (2) substituting ß-mercaptoethanol for dithiothreithol is an attractive alternative, as it significantly decreases the product cost contribution from the IBs solubilization; and (3) protein refolding by size-exclusion chromatography becomes economically attractive by changing the mode of operation of the chromatographic reactor from batch to continuous using SMB technology.

Size-exclusion chromatographic protein refolding: fundamentals, modeling and operation.

Size-exclusion chromatography (SEC) has proven its capability to refold a variety of proteins using a range of gel filtration column materials, demonstrated in the growing body of experimental evidence. However, little effort has been allocated to the development of mechanistic models describing size-exclusion chromatographic refolding reactors (SECRR). Mechanistic models are important since they provide a link between process variables like denatured and reduced protein feed concentration (C(f,D&R)), flow rate, column length, etc., and performance indicators like refolding yield (Y(N)), thereby opening the possibility for in silico design of SECRRs. A critical step, in the formulation of such models, is the selection of an adequate reaction mechanism, which provides the direct link between the separation and the refolding yield. Therefore, in this work we present a methodology using a SEC refolding reactor model, supported by a library of reaction mechanisms, to estimate a suitable reaction scheme using experimental SEC refolding data. SEC refolding data is used since it provides information about the mass distribution of monomers and aggregates after refolding, information not readily available from batch dilution refolding data alone. Additionally, this work presents (1) a systematic analysis of the reaction mechanisms considered using characteristic time analysis and Damköhler maps, revealing (a) the direct effect of a given reaction mechanism on the shape of the SEC refolding chromatogram (number of peaks and resolution) and (b) the effect that the competition between convection, refolding and aggregation is likely to have on the SEC refolding yield; (2) a comparison between the SECR reactor and the batch dilution refolding reactor based on mechanistic modeling, quantitatively showing the advantages of the former over the latter; and (3) the successful application of the modeling based strategy to study the SEC refolding data of an industrially relevant protein. In principle, the presented modeling strategy can be applied to any protein refolded using any gel filtration material, providing the proper mass balances and activity measurements are available.

Ion-exchange chromatographic protein refolding.

The application of ion-exchange (IEX) chromatography to protein refolding (IExR) has been successfully proven, as supported by various studies using different model proteins, ion-exchange media and flow configurations. Ion-exchange refolding offers a relatively high degree of process intensification, represented by the possibility of performing protein refolding, product purification and product concentration, in one unit operation. Besides its high degree of process intensification, IExR offers an additional set of key advantages including: spatial isolation of the bound protein molecules and the controllable change in chemical composition using gradients. Despite of the acknowledgement of the former advantages, the lack of mechanistic understanding on how they influence the process performance of the ion-exchange refolding reactor, limits the ability to exploit them in order to optimize the performance of the unit. This paper presents a quantitative analysis that assesses the effect that the spatial isolation and the urea gradient, have on the IExR performance, judged on the basis of the refolding yield (Y(N)) and the fractional mass recovery (f(Prot,Rec)). Additionally, this work discusses the effect of the protein load, the protein loading state (i.e., native, denatured, denatured and reduced (D&R)) and the adsorbent type on f(Prot,Rec). The presented work shows: (1) that the protein load has a direct effect on f(Prot,Rec), and the magnitude of this effect depends on the loading state of the protein solution and the adsorbent type; (2) that irrespectively of the type of adsorbent used, the saturation capacity of a denatured protein is less than the native protein and that this difference can be linked to differences in accessible binding surface area; (3) that there is a clear correlation between fractional surface coverage (θ) and f(Prot,Rec), indicating that the former could serve as a good descriptor to assess spatial isolation, and (4) that the urea gradient has a direct link with the variations on the refolding yield, and this link can be quantitatively estimated using as descriptor the urea gradient slope (ξ). Overall, the information provided in this paper aims at the eventual development of rational design or selection strategies of ion-exchange media for the satisfactory and successful refolding of a target protein.

Efficient solubilization of inclusion bodies.

The overexpression of recombinant proteins in Escherichia coli leads in most cases to their accumulation in the form of insoluble aggregates referred to as inclusion bodies (IBs). To obtain an active product, the IBs must be solubilized and thereafter the soluble monomeric protein needs to be refolded. In this work we studied the solubilization behavior of a model-protein expressed as IBs at high protein concentrations, using a statistically designed experiment to determine which of the process parameters, or their interaction, have the greatest impact on the amount of soluble protein and the fraction of soluble monomer. The experimental methodology employed pointed out an optimum balance between maximum protein solubility and minimum fraction of soluble aggregates. The optimized conditions solubilized the IBs without the formation of insoluble aggregates; moreover, the fraction of soluble monomer was approximately 75% while the fraction of soluble aggregates was approximately 5%. Overall this approach guarantees a better use of the solubilization reagents, which brings an economical and technical benefit, at both large and lab scale and may be broadly applicable for the production of recombinant proteins.