WO2016116947A1

WO2016116947A1 - A coiled flow inverter reactor for continuous refolding of denatured recombinant proteins and other mixing operations

Info

Publication number: WO2016116947A1
Application number: PCT/IN2016/000022
Authority: WO
Inventors: Anurag Rathore; Krishna Deo Prasad Nigam; Mili Pathak; Harshit Agarwal; Abhishek Kumar Sharma; Nikhil Kateja; Vishwanath Hebbi
Original assignee: Indian Institutes of Technology
Current assignee: Indian Institutes of Technology
Priority date: 2015-01-21
Filing date: 2016-01-20
Publication date: 2016-07-28
Anticipated expiration: 2017-07-21

Abstract

The present invention provides an innovative reactor design based on coiled flow inverter geometry for carrying out continuous protein refolding reactions in-vitro by dilution method. The reactor consists of an inline mixer followed by a design to provide cross sectional mixing of diluted denatured proteins through flow inversion thereby changing the direction of centrifugal force in helically-coiled tube. The unique feature of Coiled Flow Inverter Reactor (CFIR) is that it improves contact amongst the various entities such as chaotropes, stabilizers, buffer ions, reducing and oxidizing agents and the protein molecules, within the cross section in a low shear environment, thus providing a sharp residence time distribution and stabilizing the refolding reaction by reducing the side reactions that otherwise result in formation of aggregates and oxidized impurities and result in reduced refolding yield. The reactor is also exemplified for its versatility of being applicable to other biotech unit operations by demonstration of continuous precipitation of impurities from cell culture harvest of a monoclonal antibody therapeutic. The proposed invention can be used for achieving homogeneity in concentrations of protein and other reactants throughout the process so as to achieve the desirable outcome of the resulting reaction for protein refolding, precipitation, viral inactivation etc.

Description

A COILED FLOW INVERTER REACTOR FOR CONTINUOUS REFOLDING OF DENATURED RECOMBINANT PROTEINS AND OTHER MIXING

OPERATIONS

CROSS-REFERENCE TO RELATED APPLICATION(S)

5 This patent application claims the priority of the Indian patent application numbered 185/DEL/2015 filed on January 21 , 2015.

FIELD OF INVENTION

The present invention relates with the designing of a novel reactor. More 10 particularly, the present invention involves the use of a Coiled Flow Inverter Reactor (CFIR) for achieving homogeneity in concentrations of protein and other reactants throughout the process so as to achieve the desirable outcome of the resulting reaction. The reaction could be refolding, precipitation, viral inactivation etc. The reactor consists of an inline mixer 15 followed by a coiled flow inverter that is designed to provide cross sectional mixing of held up protein solution.

BACKGROUND OF THE INVENTION

20 Recombinant therapeutic proteins are traditionally processed in batch mode. However, increase in market demand and simultaneous cost pressures have motivated the industry to translate various unit operations into continuous operations so as to create a streamlined continuous bioprocessing train. One of the most important concerns in this translation is

25 to ensure that the process conditions and attributes are successfully replicated. As for protein solutions being processed, ensuring homogeneity and stability is of utmost importance, with the failure in doing so likely to result in poor quality attributes in the product. This invention relates to ensuring successful translation of biotech unit operations that require concentrations of protein and other reactants to be kept homogenous throughout the process so as to achieve the desirable outcome of the resulting reaction. The invention does so by employing a modified design of a coiled flow inverter (CFI) first introduced in U.S. Patent No. 7,337,835, which causes cross sectional mixing of the holdup via flow inversion that is achieved by changing the direction of centrifugal force in helically-coiled tubes. The effectiveness of these devices has been studied with respect to Dean Number for their applications as heat exchangers. CFIR is a configuration of equally spaced helical coils in each bank that are substantially in one common plane, the axis of each helical coil is at an angle of 90° bent to the adjacent helical coil. These bends at right angles cause flow inversion of liquids, thus enhancing cross sectional mixing in the tube. The configuration can be appropriately utilized to provide a sharper residence time distribution along with good cross-sectional mixing.

In order to demonstrate the application of the invention for various biotech unit operations, we exemplify it with its implementation for two major operations - protein refolding of denatured proteins and impurity precipitation in clarified cell culture harvest of a monoclonal antibody therapeutic.

Recombinant proteins are often over expressed in microbial systems such as E.coli, resulting in formation of insoluble inclusion bodies. This necessitates denaturation of the product in these inclusion bodies and requires refolding in order to attain the native structure. The traditional and most widely used industrial process is in batch reactor, wherein denatured protein is diluted in refolding buffer placed in stirred tank (Bade et al, 2012). This often ends in very dilute solutions with large handling of process volumes and is time consuming. This poses the need for designing a continuous refolding reactor that meets the process requirements. In an attempt to solve the problem, various researchers have attempted to design and use continuous stirred tank reactor (CSTR), CSTR with recycling, and tubular reactor to make the refolding process continuous. The subject matter of U.S. Patent No. 4,999,422 shows that the use of CSTR for continuous refolding provides broad residence time distribution that has been known to lead to misfolded and degraded proteins along with native protein. Further, shear and foaming can cause aggregation and product loss. The other U.S. Patent No. 8,067,201 describes an approach involving dialysis based refolding wherein yield of refolded solution is 90% with 32X dilution. This approach in addition to above mentioned drawbacks increases the volume and restricts process operation from happening in real continuous mode.

Schlegl et al (2005) have proposed use of a CSTR along with a diafiltration circuit to lower the denaturant levels. The folded protein is removed selectively and unfolded protein is recycled back. This, however, poses a problem of selective removal of refolded protein.

Pan et al (2014) have designed a tubular reactor for continuous refolding of proteins with dilution of 10X and residence time of 5 to 21 hours for different proteins. This tubular reactor works as a PFR with a long run time. Theoretically, a CSTR provides better mixing but is bound to have a broad residence time distribution, whereas a plug flow reactor has a narrower residence time distribution but may lack the necessary mixing required for refolding reaction.

In recent decades, although upstream process development has reduced the overall cost of monoclonal antibodies significantly by increasing titer values several fold, this has increased the pressure on downstream processing to develop the potential for making biologies production cost effective. For example, one of the most cost intensive downstream processes is Protein A (ProA) affinity capture (Warikoo et al., 2015). Precipitation in general is a low cost, high-yielding capture step and combines ease of scalability with cost effectiveness. Precipitation reactions can be performed in both batch and continuous mode. In addition to economic benefits, continuous manufacturing offers several decisive advantages, such as a higher degree of automation, a reduction in manual work and therefore less chance of human error (Hammerschmidt et al., 2015).

Continuous precipitation has been performed in a variety of configurations. Tavare and Patwardhan (1992) have performed crystallization of copper sulphate, nickel ammonium sulfate, and soy protein in continuous mixed suspension and mixed product removal reactors (MSMPR). The product crystal size distributions at steady state was measured and correlated in terms of power law kinetics to understand the effect of various observable variables. An MSMPR precipitator with a volume of 273ml was used by Raphael and Rohani in 1996 for isoelectric precipitation of sunflower protein.

Another configuration most widely explored by researchers is the straight tube configuration. A 20 m long, 6 mm diameter glass tubular precipitator was used for isoelectric precipitation of sunflower protein by Raphael and Rohani (1999).

Pan et al (2014) have used an integrated continuous tubular reactor system for processing an autoprotease expressed as inclusion bodies. The inclusion bodies were suspended and fed into the tubular reactor system for continuous dissolving, refolding and precipitation. Refolding and precipitation yield in tubular reactor were similar to batch reactor and process was stable for at least 20h. Productivity (in mg/l/h) was found to be twice in tubular reactor compared to batch reactor.

Hammerschmidt et al. (2014) propounded the theory that inexpensive precipitants such as CaC and ethanol in continuous mode can be a cost- effective replacement for ProA capture [7]. The authors proposed that mAb purification processes using continuous precipitation have lower cost of goods when compared to ProA affinity capture at all stages of the life cycle of a therapeutic antibody (i.e clinical phase I, II and III, as well as, full commercial production).

Hammerschmidt et al. (2015) have also transferred a two-stage batch precipitation process consisting of a mineral salt (CaCb) and an organic solvent (ethanol) to a continuous mode using continuous tubular reactors. Both reactors were operated for several hours at steady state without manual intervention, delivering antibody at a constant yield and purity. An overall yield excess of 90 percent, with a host cell protein reduction from 42,777 to 9000 ppm and a DNA reduction from 359 ppm to 7 ppm was achieved. The aggregate levels were found to below 1 % under all conditions tested.

The present invention intends to provide a system for achieving homogeneity in concentrations of protein and other reactants throughout the process so as to achieve the desirable outcome of the resulting reaction. The reaction could be refolding, precipitation, viral inactivation etc. The proposed invention has been successfully used for performing refolding of GCSF. Further, the CFIR has also been successfully used for translating batch precipitation process into a continuous one by continuous mixing of a clarified harvest stream getting homogenized with a stream of acid. Both applications demonstrate the realization of the benefits expected from continuous processing. The above mentioned shortcomings, disadvantages and problems are addressed herein, which will be understood by reading the following specification. REFERENCES

1. Bade, P. D., Kotu, S. P., & Rathore, A. S. (2012). Optimization of a refolding step for a therapeutic fusion protein in the quality by design (QbD) paradigm. Journal of Separation Science, 35(22), 3160-3169.

2. Galliher, P. M. (1991). U.S. Patent No. 4,999,422. Washington, DC: U.S.

Patent and Trademark Office.

3. Lin, Z., & Morin, P. (201 1). U.S. Patent No. 8,067,201. Washington, DC:

U.S. Patent and Trademark Office.

4. Schlegl, R., Tscheliessnig, A., Necina, R., Wandl, R., & Jungbauer, A.

(2005). Refolding of proteins in a CSTR. Chemical Engineering Science, 60(21), 5770-5780.

5. Pan, S., Zelger, M., Hahn, R., & Jungbauer, A. (2014). Continuous protein refolding in a tubular reactor. Chemical Engineering Science, 1 16, 763-772.

6. Saxena, A. K., & Nigam, K. D. P. (1984). Coiled configuration for flow inversion and its effect on residence time distribution. AIChE Journal,

30(3), 363-368.

7. Nigam, K. D. P. (2008). U.S. Patent No. 7,337,835. Washington, DC: U.S. Patent and Trademark Office.

8. Warikoo, V., & Godawat, R. (2015). A new use for existing technology- continuous precipitation for purification of recombination proteins.

Biotechnology Journal.

9. Hammerschmidt, N., Hintersteiner, B., Lingg, N., & Jungbauer, A. (2015). Continuous precipitation of IgG from CHO cell culture supernatant in a tubular reactor. Biotechnology Journal.

10. Tavare, N. S., & Patwardhan, V. (1992). Agglomeration in a continuous MSMPR crystallizer. AIChE Journal, 38(3), 377-384. 1. Raphael, M., & Rohani, S. (1996). Isoelectric precipitation of sunflower protein in an MSMPR precipitator: Modelling of PSD with aggregation. Chemical Engineering Science, 51(19), 4379-4384.

2. Raphael, M., & Rohani, S. (1999). Sunflower protein precipitation in a tubular precipitator. The Canadian Journal of Chemical Engineering,

77(3), 540-554.

3. Pan, S., Zelger, M., Jungbauer, A., & Hahn, R. (2014). Integrated continuous dissolution, refolding and tag removal of fusion proteins from inclusion bodies in a tubular reactor. Journal of Biotechnology, 185(XX), 39-50. Hammerschmidt, N., Tscheliessnig, A., Sommer, R., Helk, B., & Jungbauer, A. (2014). Economics of recombinant antibody production processes at various scales: IndustryDstandard compared to continuous precipitation. Biotechnology journal, 9(6), 766-775

4. Mannall, G. J., Titchener Hooker, N. J., Chase, H. A., & Dalby, P. A.

(2006). A critical assessment of the impact of mixing on dilution refolding.

Biotechnology and Bioengineering, 93(5), 955-963.

OBJECTS OF THE INVENTION The main object of the present invention is to provide an effective reactor design, enabling continuous processing of protein dispersions in biotech unit operations.

The other object of the present invention is to provide a reactor which enables effective mixing required for biotech unit operations in order to maintain homogenous physicochemical environment for the proteins and other biomolecules to refold to improve contacting in case of a reaction and avoid protein aggregation. Another object of the present invention is to provide an effective reactor design based on coiled flow inverter geometry. Another object of the present invention is to construct a coiled flow inverter reactor in a Single Use Technology paradigm where the entire configuration can be made out of autoclavable materials. Yet another object of the present invention is to provide a reactor designed for refolding which can be configured to work for various proteins by configuring well defined design parameters such as the residence time, flow rates, buffer composition etc. Another object of the present invention is to provide a reactor designed for precipitation of impurities in a cell culture harvest which can be configured to work for various proteins by configuring well defined design parameters such as the residence time, flow rates, buffer composition etc. Another object of the present invention is to provide a versatile reactor configuration for protein processing for a variety of biotech unit operations such asviral inactivation.

Still another object of the present invention is that the reactor designed should seamlessly connect as a module with other unit operations before and after within an integrated/ continuous bioprocessing platform.

These and other objects and advantages of the present invention will become readily apparent from the following detailed description.

SUMMARY OF THE INVENTION

The present invention relates to an innovative reactor (CFIR) system based on coiled flow inverter geometry which consists of a number of banks of tubes, each formed by four discretely wound helical coils which are appreciably coplanar, with each coil having equal number of turns and such that axes of adjacent coils is separated by a right angle before and after 90° bend as shown in Fig. 1.

In yet another embodiment of the invention, the reactor continuously processes a stream protein dispersion, possibly mixed with additives during the process keeping it homogenous and stable. The coiled flow inverter geometry imparts secondary flow in the cross-section of the tube that changes direction after each bend, thus causing mixing in a sustained fashion. The mixing flattens the velocity profile such that the residence time distribution and hence, if applicable, the degree of conversion in the process at a given point along the length is narrow and thus the process can be configured to achieve high biologically active protein purity at the outlet for a given protein. This maintains the homogeneity and stability of the protein solution, preventing aggregation and precipitation of the protein, respectively.

In the particular case of refolding, the reactor processes a continuous stream of denatured protein by diluting it with a buffer that creates an environment conducive for proper refolding in order to provide a stable stream of biologically active protein at the outlet.

In yet another embodiment of the invention, the reactor is also capable of continuously precipitating out impurities in clarified harvest from a mammalian cell culture based bioreactor by providing a homogeneous precipitation environment to the harvest for narrow residence time distributions.

These and other aspects of the embodiments herein will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however that the following description while indicating preferred embodiments and numerous specific details thereof are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications.

DESCRIPTION OF ACCOMPANYING DRAWINGS

The other objects, features and advantages will occur to those skilled in the art from the following description of the preferred embodiment and the accompanying drawings

Figure 1 shows the schematic of a 90° bend of a Coiled Flow Inverter (CFI) of one of the embodiment of the invention wherein 5 turns are used in each of the arm. The curvature ratio is 8.4 and the Deans number is 3.27.

Figure 2 is a process flow diagram of refolding in CFIR.

Figure 3 shows the refolding protocol of recombinant human granulocyte colony stimulating factor(GCSF). It can be seen that the unfolded protein refolds to give rise to a saturated equilibrium level of the percentage native (correctly folded) protein.

Figure 4 shows comparison of continuous (a) and batch (b) refolding process for recombinant human granulocyte colony stimulating factor (rhGCSF).

Figure 5 presents the schematics for the experimental set-up of precipitation process performed using coiled flow inverter reactor. Although specific features of the present invention are shown in some drawings and not in others. This is done for convenience only as each feature may be combined with any or all of the other features in accordance with the present invention.

DETAILED DESCRIPTION OF INVENTION

The invention will now be described in detail with reference to a few preferred embodiments, as illustrated in accompanying drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be apparent to one skilled in the art that the invention may be practiced without some or all of these specific details. In other instances, well- known features and/or process steps have not been described in detail in order to not unnecessarily obscure the invention. The features and advantages of the invention may be better understood with reference to the drawings and discussions that follow.

The various embodiments of the present invention provides a reactor design configured for processing of protein dispersions continuously while maintaining the stability of the dispersion.

In particular, continuous refolding of denatured proteins is accomplished by processing of continuously flowing streams of refolding buffer and reduced recombinant proteins to obtain high productivity of refolded protein at the outlet continuously.

The old traditional batch process involves dilution of a protein suspension and requires initial mixing of there folding buffer and the reduced inclusion bodies, followed by sustained mixing of the resulting solution until the native conformation is attained. Thus, a refolding process has two critical aspects: initial mixing and process mixing. Initial mixing is critical for process performance (Mannall et al, 2006), as poor mixing can lead to regions of higher intrinsic intermediate concentration that can cause aggregation of proteins. The sustained process mixing is generally required to homogenize the protein solution during the course of refolding to prevent intermolecular interactions among proteins and hence prevent protein aggregation.

In the present invention, the use of the coiled flow inverter geometry fulfills the mixing requirement for protein folding reaction. The coiled flow inverter geometry consists of a number of banks, each formed by four discretely wound helical coils which are appreciably coplanar, with each coil having equal number of turns and such that axes of adjacent coils is separated by a right angle as shown in Figure 1. While motion of fluid in a helical path leads to secondary flow in the cross section caused by centrifugal force, bending the tube leads to change in the direction of secondary flow pattern, leading to flow inversion and improved mixing. While mixing, coiled flow inverter geometry homogenizes and stabilizes the protein solution, it also flattens the cross sectional velocity profile, hence better emulating a plug flow due to a narrower residence time distribution.

In the present invention, this effect is utilized to get a narrow distribution of degree of refolding at a given cross-section along the tube length. This implies that one can configure a given refolding process to maximize the purity of a biologically active protein at the final outlet. This also implies that there would be no requirement of additional separation and recycling of unfolded protein which otherwise adds to the complications of validation of reprocessing. Moreover, the outlet protein solution can be readily connected to the next unit operation in a seamless fashion, preventing mix-ups and contamination resulting in improved compliance with the current good manufacturing practices. The present invention is exemplified with reference to recombinant human granulocyte colony stimulating factor (rhGCSF). The experimental setup and process flow has been shown in detail in Figures 2 and 3. A bank is a collection of four branches, each with five turns of helix. With the help of two peristaltic pumps, refolding buffer and reduced IBs are pumped at defined flow rates to achieve the required dilution ratio through the coiled flow inverter. An inline mixer is added before the CFIR to provide the fast mixing required at the start of refolding. At the outlet of CFIR the sample is quenched with acetic acid and stored.

In an embodiment of the invention, two peristaltic pumps were used to pump refolding buffer and reduced inclusion bodies in the desired ratio of 5: 1 . Total 3 banks were used and sampling was done at different time points. A dynamic inline mixture was used at the beginning to provide rapid initial mixing. Quenching of the refolded sample was done using inline dilution by acetic acid as shown in Figure 2

Figure 3 explains the refolding protocol occurring inside the invention. The inclusion bodies are first solubilized using solubilization buffer (50 mM Tris buffer pH 10, containing 6 M Urea) by constant mixing on a stirrer at 180 rpm for 45 min at room temperature. Next, the reducing agent (Dithiothreitol (DTT)) is added to reduce the solubilized inclusion bodies. Finally, the reduced solubilized IBs are diluted in the refolding buffer in the desired ratio of 1 :5 using dynamic inline mixture. This solution is then given the desired residence time in CFIR. The samples are quenched using glacial acetic acid at pH 4 to stop refolding reaction and are subjected for further analysis.

The outcome of this refolding protocol is detailed in Figure 4, which shows the comparison of refolding process in batch using stirred tank and continuous mode using CFIR. Both the processes generate negligible aggregates. The optimum dilution for batch process is at 10X and for continuous is 5X. Batch process requires initial 20 min for drip dilution while continuous process works on flash dilution approach. The RP-HPLC data for batch refolding and continuous refolding process depicts that it takes 150 minutes for batch process to give 83% native protein (sampling point 30min, 60min, 120min, 150min, 180min, 240min, 480min, 720min and 960min) while 84% native protein is achieved in 18min using CFIR (sampling point Omin, 18min, 30min, 60min, 90min, 120min, 150min and 180min). As per our calculations, productivity of continuous process will be17 times higher than batch process for the application under consideration.

Thus, in continuous refolding through the present invention, percentage of native protein with time of refold shows the native percentage approach 84% after 1 h of refold. Percentage of refolded native GCSF in batch process as a function of time is determined by RP-HPLC. It is seen that the refolding is complete after 1 h 30 min and results in 84% purity.

The CFIR enables continuous refolding of proteins with high protein concentration with increased purity and hence provides better productivity as seen in Figure 4. It also easily handles large flow rates and large volumes.

In yet another embodiment of the present invention, the reactor can be easily configured for refolding of different proteins by changing flow rates, number of branches and other process parameters.

In other embodiment of the present invention, the reactor can be modified for refolding various other proteins by configuring process parameters such as residence time, buffer composition, flow rate etc. Certain strategies, which involve introduction of additives or temperature change into the refolding environment after certain residence time, can also be conveniently employed in the present embodiment.

Moreover, the benefits of the current invention can easily be extended to other biotech unit operations that have a requirement of effective mixing and/or sharp residence time distribution. This aspect of the invention is exemplified with reference to the precipitation of monoclonal antibody product. The experimental setup for precipitation is shown in Figure 5, wherein the peristaltic pumps were used to pump the precipitating agent (acetic acid) and mAb solution at the desired flow rates. Acetic acid was injected in the CFIR at multiple points. CFIRis used for mAb purification from clarified harvest by precipitating impurities through pH reduction using acetic acid as a precipitating agent. Precipitation of HCPs depends largely on the addition time of acids in batch. The same was done in CFIR by adding acetic acid at multiple points in (Omin, 10min and 20 min) to slowly increase the concentration of acetic acid. Sampling was done at multiple time points (Omin, 20min, 60min and 120min) to provide hold time for precipitation.

Recovery, DNA and HCP content of the output solution were measured to determine the quality of purified protein after precipitation in CFIR. The result is shown in the Table 1 below:

It can be seen that the clearance of host cell impurities is nearly same as that from protein A-based purification with a yield 91 %. The HMWP% in each fraction was found to be in range of 4.4±0.2%. The described invention is a compact embodiment of a continuous flow reactor that provides sustained mixing of the held-up volume with minimized back mixing and hence a sharp residence time distribution. Further, the incorporation of the CFIR flow design enables a great degree of customizability to the reactor such that one can vary critical design attributes to optimally customize it to any given process. For example:

• The variation in Dean number can be performed conveniently by varying the helical radius and/or tube radius to manipulate the degree of mixing.

· The residence time distribution can be narrowed down conveniently as understood from the geometrical parameters and flow rate in the coiled flow inverter.

Possible applications of the invention include a variety of reactions such as protein refolding, viral inactivation, precipitation, two phase separation, etc. Further, the invention enables inline addition and/or modification at any point along the reactor length to of process conditions to incorporate various process strategies. For example:

^■ It has been demonstrated in the application of the device as a continuous precipitation unit that pH can be conveniently varied at any stage of the process with addition of suitable streams.

^■ It is also a virtue of the tubular nature of the reactor that in principle any person acquainted with the art can accustom the reactor to provide variable temperature profiles, such as a gradient.

The present invention is amenable to a single-use construction and the sharper residence times make it amenable towards effective control schemes in a continuous bioprocessing train. The reactor can be seen as a modular unit connected seamlessly with an inlet and an outlet in a continuous bioprocessing train. The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the appended claims.

Claims

Claims: We claim:

1. A coiled flow inverter reactor (CFIR) system for continuous refolding of denatured recombinant proteins, precipitation of impurities, viral inactivation and other reaction based unit operations comprising of: at least one coiled flow inverter reactor, wherein the inverter is characterized with the plurality of banks of metallic tubes, wherein each bank comprises of equally spaced plurality of helical coils, with each helical coil having equal number of turns and in such a manner that the adjacent coils is separated by a right angle before and after 90° bend, and the CFIR may have an in-line mixer preceding it and a filter following as and when required.

2. The system of claim 1 , wherein the Coiled Flow Inverter of the Reactor has one or more banks.

The system of claim 1 , wherein the each bank of the Coiled Flow Inverter is formed by multiple discretely wound helical coils. The optimal number is four for the GCSF refolding and mAb precipitation systems under consideration.

The system of claim 1 , wherein the Coiled Flow Inverter Reactor, each arm of the bank is characterised with more than 2 turns. The optimal number is five for the GCSF refolding and mAb precipitation systems under consideration.

The system of claim 1 , wherein the Coiled Flow Inverter Reactor, the Deans number is greater than 1.

6. The system of claim 1 , wherein in the Coiled Flow Inverter Reactor, the axes of the coils are in common plane.

7. The system of claim 1 , wherein in the Coiled Flow Inverter Reactor, each coil is made of stainless steel or any other biocompatible material such as Tygon, Silicone, etc.

8. The system of claim 1 , wherein the Coiled Flow Inverter Reactor, the motion of fluid in a helical path leads to secondary flow in the cross section due to the centrifugal force and bending of the helical tubes leads to change in the direction of secondary flow pattern, leading to flow inversion and improved mixing in a sustained manner.

9. The system of claim 1 , wherein the system is used for protein refolding.

10. The system of claim , wherein the system is used for precipitation of impurities in a biotech operation especially during cell culture harvest.

11.The system of claim , wherein the CFIR is used for viral inactivation in biotech operation.

12. The system of claim 1 , wherein the Coiled Flow Inverter is used for handling of dispersions of proteins.

13. The system of claim 1 , wherein the Coiled Flow Inverter is used for continuous operation.