阅读说明：本技术 在连续流动体系中保持窄停留时间分布的机械方法 (Mechanical method for maintaining narrow residence time distribution in continuous flow systems ) 是由 R.图切利 J.考尔马雷 L.梅西尔 M.荷尔斯泰因 C.吉莱斯皮 R.希尔 于 2018-04-18 设计创作，主要内容包括：在连续流动体系中维持窄停留时间分布的方法,其特别适用于病毒灭活,如在蛋白纯化过程期间。将流体样品引入轴向流动通道,并使其以离散包或区域在其中流动以使停留时间分布和轴向分散最小化。本文所述的实施方案避免或最小化了在蛋白纯化过程期间使用大罐或储器进行病毒灭活的需要；减少了病毒灭活所需的总时间,和/或减少了在蛋白纯化过程期间进行病毒灭活操作所需的总物理空间,这继而减少了纯化过程的总占地面积。(A method for maintaining a narrow residence time distribution in a continuous flow system is particularly useful for viral inactivation, such as during a protein purification process. A fluid sample is introduced into the axial flow channel and caused to flow therein in discrete packets or zones to minimize residence time distribution and axial dispersion. The embodiments described herein avoid or minimize the need for large tanks or reservoirs for virus inactivation during the protein purification process; the overall time required for virus inactivation is reduced, and/or the overall physical space required for performing virus inactivation operations during the protein purification process is reduced, which in turn reduces the overall footprint of the purification process.)

1. A method for maintaining a narrow residence time distribution of a fluid sample flowing in a fluidic channel having an axial length, comprising flowing the fluid sample in discrete packets along the axial length within the fluidic channel.

2. The method of claim 1, wherein the fluid sample is flowed in discrete packets by applying a compressive force to the fluidic channel.

3. The method of claim 1, wherein the fluid sample has a nominal residence time of one to two minutes in the fluidic channel.

4. The method of claim 1, wherein the fluid sample has a nominal residence time of two to four minutes in the fluidic channel.

5. The method of claim 1, wherein the fluid sample has a nominal residence time of four to six minutes in the fluidic channel.

6. The method of claim 1, wherein the fluid sample has a nominal residence time of six to eight minutes in the fluidic channel.

7. The method of claim 1, wherein the fluid sample has a nominal residence time of eight to ten minutes in the fluidic channel.

8. The method of claim 1, wherein the fluid sample has a nominal residence time of ten to fifteen minutes in the fluidic channel.

9. The method of claim 1, wherein the fluid sample has a nominal residence time of fifteen to thirty minutes in the fluidic channel.

10. A method for inactivating one or more viruses in a sample comprising a target molecule, wherein the method comprises flowing the sample in discrete packets in a flow channel during a process of purifying the target molecule while continuously exposing the sample to inactivation conditions.

11. A method for inactivating one or more viruses in a fluid sample comprising a target molecule, comprising subjecting the fluid sample to a protein a affinity chromatography process, thereby obtaining an eluate; continuously transferring the eluate to an axial flow channel to mix one or more viral inactivating agents with the eluate; and flowing the eluate in the axial channel in discrete packets for a time sufficient to inactivate the virus.

12. A method for inactivating one or more viruses in a fluid sample comprising a target molecule, comprising subjecting the fluid sample to an ion exchange chromatography process, thereby obtaining an eluate; continuously transferring the eluate to an axial flow channel to mix one or more viral inactivating agents with the eluate; and flowing the eluate in the axial channel in discrete packets for a time sufficient to inactivate the virus.

13. The method of claim 1, wherein the fluid sample has a nominal residence time of one to two minutes in the fluidic channel.

14. The method of claim 1, wherein the fluid sample has a nominal residence time of two to four minutes in the fluidic channel.

15. The method of claim 1, wherein the fluid sample has a nominal residence time of four to six minutes in the fluidic channel.

16. The method of claim 1, wherein the fluid sample has a nominal residence time of six to eight minutes in the fluidic channel.

17. The method of claim 1, wherein the fluid sample has a nominal residence time of eight to ten minutes in the fluidic channel.

18. The method of claim 1, wherein the fluid sample has a nominal residence time of ten to fifteen minutes in the fluidic channel.

19. The method of claim 11, wherein the fluid sample has a nominal residence time of fifteen to thirty minutes in the fluidic channel.

20. The method of claim 11, wherein the elution fluid is flowed in discrete packets by applying a compressive force to the fluid channel.

21. The method of claim 11, wherein the target is an antibody or an Fc region-containing protein.

22. A fluid channel having an axial length, wherein the fluid channel comprises a plurality of discrete packets of a fluid sample comprising a target molecule and one or more viral inactivating agents.

23. The fluidic channel of claim 22, wherein the fluid sample comprises a virus.

Background

The large scale production and economic surrounding purification of therapeutic proteins, particularly monoclonal antibodies, is an increasingly important issue for the biopharmaceutical industry. Therapeutic proteins are typically produced in mammalian cells or bacterial cells that have been engineered to produce a protein of interest. However, once produced, the protein of interest needs to be separated from various impurities such as Host Cell Proteins (HCPs), endotoxins, viruses, DNA, etc.

In a typical purification process, the cell culture harvest is subjected to a clarification step to remove cell debris. The clarified cell culture harvest comprising the protein of interest is then subjected to one or more chromatography steps, which may include an affinity chromatography step or a cation exchange chromatography step. To ensure viral safety of the therapeutic candidate and compliance with regulatory requirements, viral clearance unit operations are performed during the purification process. Such steps include protein a and ion exchange chromatography, filtration and low pH/chemical inactivation. Viral inactivation is typically performed after a chromatography step (e.g., after affinity chromatography or after cation exchange chromatography). In a typical large scale purification process, a chromatographic elution pool containing a protein of interest is collected in a large tank or reservoir and subjected to a virus inactivation step/process under mixing for an extended period of time, which may take hours to a day or more, to achieve complete inactivation of any virus that may be present in the elution pool.

In monoclonal antibody (mAb) processing, for example, a series of individual unit operations are performed in batch mode, wherein storage tanks are used to store materials between unit operations and to facilitate any necessary solution conditioning between steps. Typically, the material is collected in a tank where it is conditioned to achieve target inactivation conditions. This can be achieved by adding an acid to reach the low pH target level or by adding a detergent during detergent based inactivation. Next, the material is transferred to a second tank where it is maintained under inactivation conditions for a prescribed incubation time. The purpose of the transfer is to eliminate the risk of droplets on the wall of the first tank, which may not have reached the target inactivation conditions and which may contain viral particles. By transferring the material to a different tank, this risk is reduced.

Several virus inactivation techniques are known in the art, including exposure of the protein solution to certain temperatures, pH or radiation, and exposure to certain chemical agents such as detergents and/or salts. One virus inactivation method involves large storage tanks where the material is maintained under inactivation conditions such as low pH and/or exposure to detergent for 60 minutes. This static hold step is a bottleneck towards continuous processing.

However, virus killing kinetics indicate that inactivation times can be significantly shorter than 60 minutes, which indicates that treatment times can be significantly reduced, that static storage tanks for virus inactivation can be eliminated, and that the method can be more suitable for continuous treatment and/or flow inactivation.

Recently, it has been desired to have a continuous process in which unit operations are linked together and manual solution conditioning is minimized. To facilitate this, efforts are underway to develop on-line treatment methods to enable on-line virus inactivation and other on-line solution conditioning. The challenge in continuous processing is the efficient movement of fluid from point a to point B. One example is plug flow movement of fluid through a length of tubing. The flows involved in mAb treatment typically fall into laminar flow regime (reynolds numbers less than 2100). In this state, the molecules are dispersed by radial diffusion, with the result that the solute pulse expands axially along the flow direction. This is known as taylor dispersion and is shown schematically in fig. 1. Laminar Poiseuille flow results in a parabolic velocity profile. The leading and trailing ends of the pulse start with a sharp interface but become parabolic in shape due to laminar flow of the fluid. Axial expansion continues over time and the molecules become more dispersed over the tube length. The effect of axial dispersion was observed in the resulting concentration profile obtained by pulsed injection of the marker species at the tube outlet, as shown in figure 2. This concentration profile reflects a broad distribution of the residence time of the marker species. Such varying residence times may lead to uncertainty as to whether all fluids have sufficient residence time in the virus-inactivation environment, or to system sizes that are too large to ensure that no molecules exit faster than expected. Due to the sufficiently long residence time to ensure virus inactivation, the proteins (products) are exposed to inactivation conditions for too long a residence time, which has undesirable consequences such as potential degradation and aggregation.

In continuous or semi-continuous flow systems, it is desirable to provide a method for maintaining a narrow residence time distribution.

It would be desirable to provide a continuous or semi-continuous flow system for biomolecule purification, in particular for protein purification.

SUMMARY

Embodiments disclosed herein provide methods of maintaining narrow residence time distributions in continuous flow systems that are particularly useful for viral inactivation, such as during protein purification processes.

Embodiments described herein eliminate or minimize the need for large tanks or reservoirs for virus inactivation during the protein purification process, reduce the total time required for virus inactivation, and/or reduce the total physical space required for virus inactivation operations during the protein purification process, which in turn reduces the total footprint of the purification process. Furthermore, this increases the certainty that all molecules have undergone the shortest residence time, thereby providing some safety factor for ensuring inactivation while minimizing prolonged retention.

In some embodiments, a method for inactivating one or more viruses that may be present in a sample during purification is provided, wherein the method comprises maintaining a narrow residence distribution in a continuous flow system by separating a fluid into discrete regions or packets (packets) as the fluid flows in the axial direction of a flow channel (e.g., a tube). The flow channel may be used as a incubation chamber. This allows for sufficient residence time of all species of the fluid in the flow channel, which in turn allows for inactivation of viruses as the fluid flows in the flow channel and mixes with the one or more viral inactivating agents. The flow channel may be made of a variety of materials and shapes, including circular plastic tubing and "Smart FLEXWARE" formed by welding two pieces of plastic together in a pattern to form the channel^®"macroscopic fluid flow path assembly, commercially available from millipore sigma (U.S. patent No. 9,181,941B 2, U.S. patent No. 9,051,929B 2).

In certain embodiments, the incubation chamber, flow channel, or tube is configured to provide effective radial mixing and minimal axial mixing, which results in a narrow or reduced residence time distribution, and wherein the volume of the chamber or tube does not experience changes due to pressure and temperature. In some embodiments, the incubation chamber, flow channel, or tube is a single-use chamber or tube and is sterilizable.

In certain embodiments, methods are provided for inactivating one or more viruses that may be present in a fluid sample comprising a target molecule (e.g., an antibody or Fc region-containing protein), comprising subjecting the fluid sample to a protein a affinity chromatography process or an ion exchange chromatography process to obtain an eluate; continuously introducing the eluate into an axial flow channel to mix one or more viral inactivating agents with the eluate in the flow channel; and flowing the eluate in the axial flow channel in discrete packets for a time sufficient to inactivate the virus. In certain embodiments, the chromatographic process is performed in a continuous mode. The eluate from the affinity chromatography process may be a real-time eluate from the column that enters the system in all of its gradients in pH, conductivity, concentration, etc., or may be a pool of elutions that are then subjected to inactivation after homogenization.

In some embodiments, the in-line incubation chamber, flow channel, or tube may be implemented in a process in which the storage pool or tank is located directly upstream, downstream, or both of the chamber, channel, or tube. In some embodiments, the in-line incubation chamber, flow channel, or tube may be implemented in a process in which the chamber, channel, or tube directly connects two unit operations, such as an upstream protein a chromatography operation and a downstream cation exchange operation.

Brief Description of Drawings

FIG. 1 is a schematic illustration of fluid flow in a channel;

FIG. 2 is a graph of a concentration profile produced by pulsed input of a marker species in a channel (a finite volume of marker species injected into a trunk stream at a fast rate so as to produce a uniform plug of marker species in which the concentration profile at each end of the plug approaches a step change) according to the prior art;

FIG. 3 is a graph of UV exposure versus time for various flow channels;

FIG. 4 is a graph of UV exposure versus volume for various flow channels;

fig. 5 is a perspective view of a device adapted to generate a compressive force on a flow channel to form a fluid packet according to certain embodiments;

FIG. 6 is a perspective view of the flow passage wrapped around the mandrel shown in FIG. 5;

FIG. 7 is a perspective view of a flow channel wrapped around a mandrel placed in the device of FIG. 5;

FIG. 8 is a graph showing the time of virus inactivation for various Phi6 titers;

FIG. 9 is a graph showing the time of virus inactivation for various XMuLV retroviral titers;

FIG. 10 is a graph showing detergent-based virus inactivation kinetics for Phi6 titers;

FIG. 11 is a graph of the residence time distribution of Phi6 virus in the coil and conventional flow channels;

fig. 12 is a schematic of an on-line continuous inactivation process according to certain embodiments;

FIG. 13A is a schematic illustration of a windmill design for compressing flow channels to form a bale, according to certain embodiments;

fig. 13B shows top and perspective views of a conveyor design for compressing a flow channel to form a bale, according to certain embodiments; and

fig. 13C is a schematic illustration of a roller design for compressing a flow channel to form a packet, according to certain embodiments.

Detailed description of the invention

The term "in-line" or "in-line operation" refers to the process of moving a liquid sample through a tube or some other conduit or flow channel without storage in a container. The term "viral inactivation" or "inactivation of a virus" refers to the treatment of a sample comprising one or more viruses in such a way that the one or more viruses are no longer able to replicate or become inactive. In the methods described herein, the terms "virus" and "viral" are used interchangeably. Viral inactivation may be achieved by physical means, such as heat, ultraviolet light, ultrasonic vibration, or using chemical means, such as changing the pH or adding chemicals (e.g., detergents). Viral inactivation is typically a process step used during most mammalian protein purification processes, especially in the case of purification of therapeutic proteins from mammalian-derived expression systems. In the methods described herein, virus inactivation is performed in a fluid flow channel, where the sample is made to travel in discrete regions or packets. It is understood that failure to detect one or more viruses in a sample using standard assays known in the art and those described herein indicates complete inactivation of the one or more viruses upon treatment of the sample with one or more virus-inactivating agents.

The term "discrete region" or "packet" refers to a separately defined volume separated from an adjoining volume by an intervening barrier.

The term "virus inactivating agent" or "virus scavenger" refers to any physical or chemical means capable of inactivating or disabling replication of one or more viruses. A virus-inactivating agent as used in the methods described herein may include a change in solution conditions (e.g., pH, conductivity, temperature, etc.) or the addition of detergents, salts, acids (e.g., acetic acid, which is present in a molar concentration to a pH of 3.6 or 3.7), polymers, solvents, small molecules, drug molecules, or any other suitable entity, or the like, or any combination thereof, that interacts with one or more viruses in the sample, or physical means (e.g., exposure to UV light, vibration, etc.) such that exposure to the virus-inactivating agent inactivates or disables replication of the one or more viruses. In a particular embodiment, the viral inactivating agent is a change in pH, wherein the viral inactivating agent is mixed with a sample comprising the target molecule (e.g., an eluate from a protein a binding and elution chromatography step) in a flow channel, where the sample is flowed in discrete regions or packets.

The term "continuous process" as used herein includes a method of purifying a target molecule that includes two or more process steps (or unit operations) such that the output from one process step flows directly into the next process step in the method without interruption, and wherein two or more process steps may be performed simultaneously for at least a portion of their duration. In other words, in the case of a continuous process, it is not necessary to complete a process step before the next process step starts, as long as a portion of the sample always moves through the process step.

Similarly, a "semi-continuous method" may include operation in a continuous mode for a set period of time, with one or more unit operations being periodically interrupted. Such as stopping the loading of the feed to allow other speed limiting steps to be completed during the continuous capture operation.

Conventional methods for protein purification typically involve cell culture methods, e.g., using recombinantly engineered mammalian or bacterial cell lines to produce a protein of interest (e.g., a monoclonal antibody), followed by a cell harvesting step to remove cells and cell debris from the cell culture broth. The cell harvesting step is typically followed by a capture step, which is typically followed by one or more chromatography steps, also referred to as polishing steps, which typically comprise one or more of cation exchange chromatography and/or anion exchange chromatography and/or hydrophobic interaction chromatography and/or mixed mode chromatography and/or hydroxyapatite chromatography, size exclusion chromatography, depth filtration or the use of activated carbon. A virus inactivation step may also be included after the capture step. The purification step is usually followed by viral filtration and ultrafiltration/diafiltration, which completes the purification process.

Biopharmaceutical manufacturing requires inactivation or removal of viruses (components from animal sources, including mammalian cells) to ensure drug safety and to meet standards set forth by regulatory agencies such as the Food and Drug Administration (FDA). Typical methods include a number of virus removal steps that cumulatively provide the necessary protection.

Some methods include titrating a solution comprising the target protein to a low pH to destroy any enveloped viruses and viral components. Typically, samples containing the target protein are maintained under these conditions for extended periods of time because of the time required for viral inactivation and, more importantly, time required to ensure uniform mixing for effective viral inactivation. Thus, in the case of large scale methods, the sample comprising the target protein is incubated at low pH for an extended period of time, typically with mixing, to promote effective virus inactivation. Usually two separate tanks are used, where the first tank is used for pH adjustment and the second tank is used for actual incubation maintenance.

The pH conditions are established as a balance between a low pH sufficient to cause inactivation and a high pH sufficient to avoid denaturation of the target protein or to limit the extent of degradation of the product. In addition, the sample must be exposed for a certain amount of time to cause a significant reduction in the value of viral activity, typically 2-6 LRV (log reduction value).

Parameters considered important for the virus inactivation process are pH, exposure time, characteristics of the background solution conditions (e.g., buffer type, buffer concentration), mAb concentration, and temperature, assuming that there is homogeneous mixing. In the case of large scale processes, mixing poses challenges due to large volumes and additional parameters such as mixing rates and mass transfer.

In the case of Fc region containing proteins (e.g., monoclonal antibodies), virus inactivation is typically performed after elution from the binding and elution chromatographic process steps (e.g., protein a affinity chromatography or cation exchange chromatography) because the pH of the elution pool is closer to the pH required for virus inactivation. For example, in the methods used in today's industry, protein a chromatographic elution pool typically has a pH in the range of 3.5 to 4.0, and cation exchange binding and elution chromatographic elution pool typically has a pH of about 5.0.

In most methods currently used in industry, the elution pool containing the target protein is adjusted to the pH required for virus inactivation and held there for a certain length of time, the combination of pH and time having been shown to result in virus inactivation. Longer times are more effective for virus inactivation, particularly in the case of large scale processes, however, longer times are also known to cause protein destruction and protein denaturation, which can lead to the formation of protein aggregates (immunogenic). Prolonged exposure to low pH can lead to precipitation and formation of aggregates, which is undesirable, and often requires the use of deep filters and/or sterile filters to remove such precipitates and aggregates.

The methods described herein enable viral inactivation to be achieved in a continuous or semi-continuous manner, which can significantly reduce the time associated with viral inactivation, and in turn, the time of the overall purification process, relative to most conventional methods.

In some embodiments, the different process steps are linked to operate in a continuous or semi-continuous manner. In some embodiments, the virus inactivation method as described herein constitutes a process step in a continuous or semi-continuous purification process, wherein the sample flows continuously from, for example, a protein a affinity chromatography step or an ion exchange chromatography step to the virus inactivation step to the next step of the process, which is typically a flow-through purification process step. In-line pH inactivation (e.g., Klutz S et al, Continuous viral inactivation at low pH value in anaerobic fermentation manufacturing, Chemical Engineering and Processing 102(2016) 88-101) has been proposed in the prior art, but developing suitable incubators with narrow residence time distributions means that these chambers are oversized and may be more difficult to validate.

In some embodiments, the virus inactivation process step is performed continuously or semi-continuously, i.e., the eluate from a prior process step, such as a prior binding and elution chromatography step (e.g., protein a affinity chromatography, fig. 12), is continuously flowed into the virus inactivation step using one or more fluidic channels, wherein the eluate is flowed in discrete regions or packets, after which in some embodiments, the virus-inactivated eluate may be collected in a storage vessel until the next process step is performed, or in some embodiments, may be fed directly and continuously to the next downstream process step. For example, referring to fig. 12, in certain embodiments, protein a mAb eluents are rapidly brought to a uniform low pH using an in-line acid addition using a precision syringe pump and static mixer. A robust low pH was maintained at variable protein feed concentrations using 1M acetic acid concentrate. The pH can be verified using sampling and off-line sensors. Robust pH control for extended multi-day operation can be achieved without complex continuous feedback control loops and unreliable pH sensors. The inactivation or incubation chamber provides a reliable retention time for robust LRV and rapidly and consistently quenches to the pH required for subsequent steps (e.g., 5-7.5).

According to certain embodiments, a narrow residence time distribution is maintained in a continuous or semi-continuous flow system. The residence time distribution is sufficiently narrow (and reduced compared to conventional designs) to achieve effective viral inactivation of a fluid sample traveling in the system. A suitable narrow residence time distribution may be quantified based on comparison with results obtained from conventional designs. Pulse data (e.g., UV absorption peaks) from different designs may be compared using statistical quantization metrics. For example, the amount of time required for the middle 80% of the fluid to leave the flow channel may be compared. That is, a spread between 10% and 90% area values (where t is_10%Represents the time, t, at which 10% of the fluid has left the channel_90%Representing the time 90% of the fluid has left the channel). This comparison was made for the peaks shown in FIG. 3 and is listed in Table 1 belowOut, it used a tube with 1/8 "ID and 250 inches length to equal a system volume (hold up volume) of 50 mL. Other methods of analyzing peak characteristics, such as those applied by one skilled in the art, e.g., moment analysis, may also be utilized.

TABLE 1

Method of producing a composite material	t10%(s)	t90%(s)	t10%And t90%Difference(s) therebetween
				Straight pipe	251	1416	1165
Coiled pipe (3/8' bar)	166	403	237
				The flow channel	231	260	29

To compare various designs with different system volumes, these data may also be expressed as normalized volumes (normalized to the system volume). This is shown in fig. 4 and is listed in table 2 below:

TABLE 2

Method of producing a composite material	(V/VSystem of)10%	(V/VSystem of)90%	(V/VSystem of)10%And (V/V)System of)90%The difference between
				Straight pipe	0.84	4.72	3.88
Coiled pipe (3/8' bar)	1.02	2.49	1.47
				The flow channel	0.96	1.08	0.12

In these analysis examples, the 10-90% spread of the embodiments disclosed herein is significantly less than the value of the conventional design and constitutes a narrow residence time distribution according to the embodiments disclosed herein, which is reduced from that of the conventional design. In the ideal case of plug flow, this 10-90% expansion will become zero.

In some embodiments, a narrow residence time distribution is created and maintained by using mechanical methods to divide the fluid into distinct or discrete regions or packets along the axial direction of the fluid flow channel in which the sample travels. A discrete packet is a packet or region separated from another packet or region by an intervening barrier; any degree of separation that forms a boundary between a volume of process fluid and an adjacent volume or volumes of process fluid is a discrete region or packet. The barrier may be formed by mechanically pressing the walls of the fluid channel together (as discussed in more detail below). The length of the packet separation (i.e., the length of the compressed region) is not critical; the packets may be separated by very small lengths or much longer lengths, as long as the size is sufficient to maintain separation between the packets.

In some embodiments, distinct or discrete regions or packets of fluid are achieved by applying compressive forces (substantially simulating a peristaltic pump) to the exterior of the fluid flow channel spaced along the axial length of the channel as fluid flows in the channel. Dividing the fluid into zones or packets minimizes axial dispersion and mixing of the fluid in the channels. The axial dispersion of residence time will affect the range of residence times that the particles may experience. Longer residence times may be more desirable for inactivating virus particles at low pH. However, protein particles in the same solution that have undergone excessive residence times at low pH may degrade, which is undesirable. The degree to which the residence time is minimised will depend on the sensitivity of the product (e.g. protein) being treated, but in any case a lower residence time will be beneficial. Thus, the amount of dispersion that can be tolerated will depend on the particular application. For viral clearance applications, minimizing axial dispersion would provide significant advantages in reducing processing time, as there would be greater confidence in viral inactivation within a short time window.

In certain embodiments, the formation of discrete fluid packets or regions allows the fluid species to translate axially along the length of the flow channel such that the distribution at the channel inlet matches or substantially matches the distribution at the channel outlet, thereby increasing the consistency and certainty of the residence time of the fluid species in the channel.

A sample introduced as a pulse at one end of the flow channel will leave the flow channel at a known time with a sharp, well-defined peak. This has advantages for on-line continuous virus inactivation applications where it is desirable to achieve a target minimum residence time for the species flowing through the system. It can also be carried out in systems where the mobile phase (e.g. buffer) conditions are changed or where buffer dilution occurs. This can be done at various places in a typical mAb purification process. One example is the adjustment typically required between the bind-elute cation exchange step and the flow-through anion exchange step. The elution pool from the cation exchange step is typically at a lower pH and higher conductivity than the target value for the anion exchange step. To effectively condition the cation exchange elution pool to the appropriate conditions in a continuous flow system, discrete packets can be formed that will allow for an effective transition to new conditions. A sharp transition region between different buffer types may be provided, thereby minimizing the time and amount of buffer required and allowing for on-line buffer dilution or on-line conditioning applications.

Turning now to fig. 5-7, a suitable apparatus for applying a compressive force to a fluid channel to create a fluid region or packet is shown, according to embodiments disclosed herein. Many other geometries and configurations can be devised which will produce the same effect, such as those shown in fig. 13A-13C. Fig. 6 and 7 show a mandrel 5 around which a fluid passage 10, such as a tube, may be wrapped to secure the fluid passage 10 in place. One or more rollers 15 rotate about the mandrel 5 and apply a constant compressive force to the exterior of the fluid passageway 10 as they rotate. As the roller 15 rotates, fluid within the channel 10 moves axially through the channel. The pressure from the roller 15 keeps the fluid separated into many different zones or packets along the entire length of the fluid channel 10. Compartmentalization or encapsulation minimizes axial dispersion of the fluid and thus provides desirable flow characteristics for applications involving efficient migration of species, such as in-line viral inactivation or transitions from one fluid to another, such as buffer transitions, or buffer dilutions. The force required to completely close all the flow channels across one quadrant of the "peristaltic spiral" design is a function of the type and size of the tube used as the flow channel. For example, when reinforced silicone tubing (3/8 "OD, 1/8" ID) is used, the force required to compress 17 cycles of tubing across one quadrant of the helix (each quadrant separated by a 2.23 "circumference; i.e., every 2.23" (arc length of tubing between rollers)) is 187 lbsf (11 lbsf per cycle of tubing). The spacing between the rollers determines the axial length of each pack. Those skilled in the art will appreciate that the foregoing is merely exemplary; other designs may provide other packet sizes.

In some embodiments, a "windmill" design may be used that operates in a radial mode with varying cross-sections to maintain a constant fluid velocity driven by a roller of constant angular velocity. One or more rollers may be connected to a rotating hub (hub). As the hub rotates, the rollers compress the flow channel, thereby forming a fluid packet along the flow channel. The flow channels may be made of round plastic tubing or may be formed of plastic sheets that are welded together to form the channels (e.g., SmartFlexWARE)^®). Fig. 13A is one example of a mechanical design in which the tubes are arranged in a radial pattern with varying cross-sections. One or more rollers are used to compress the tube while the hub is rotating. In the example shown in fig. 13A, four rollers are shown on the windmill, but a different number of rollers may be used depending on the desired fluid packet size.

In some embodiments, a "conveyor" design may be used, which includes two rollers (rolls) with a pair of pinch rollers that move across the tube array. The pinch rolls traverse along the gap between the two rolls and then turn around again after separating and returning to the other end. The spacing between the pinch rolls is determined based on the desired bale size. Fig. 13B shows an example of a mechanical design in which the tube is wrapped around two rollers and squeezed by rollers that traverse along the gap between the two rollers.

In some embodiments, the rollers may be positioned on the inside of the coils such that they compress the tube against the fixed outer wall. The spacing between the rollers and the tube size are determined based on the desired bale size. Figure 13C shows an example of a mechanical compression method where rollers are placed on the inside of the tube coil and the rollers compress the tube against a fixed outer wall.

The flow rate and tube length can be selected to target a particular residence time. For virus inactivation applications, the residence time is selected to be a time sufficient to achieve virus inactivation within the flow channel, preferably with a safety factor. For example, in the case where a 30 minute inactivation time is required, a safety factor of 2 may be employed, resulting in a target residence time of 60 minutes in the flow channel. In other embodiments, a safety factor may be employed when viral inactivation occurs in less than 1-2 minutes, and a target residence time in the flow channel of 4 or 5 minutes is used. The minimum residence time may also depend on regulatory guidelines in terms of an acceptable safety factor for virus inactivation.

Suitable nominal residence times include, but are not limited to, 1-2 minutes, 2-4 minutes, 4-6 minutes, 6-8 minutes, 8-10 minutes, 10-15 minutes, and 15-30 minutes.

The ability to maintain a narrow residence time distribution is advantageous for viral inactivation methods such as mAb treatment, where the virus must stay for a minimum amount of time under defined inactivation conditions such as low pH, exposure to detergents, etc. By maintaining a narrow residence time distribution, the minimum time requirement for virus inactivation can be met while also minimizing exposure of the product (e.g., protein/mAb) to harsh inactivation conditions.

In the case of low pH inactivation, for example, a viral inactivating agent such as an acid may be added as a side stream to the main feed flow path. In certain embodiments, a syringe pump controlled by a software program may be used to add the desired amount of virus-inactivating agent. In some embodiments, a controller for a pump may be provided, the controller having a processing unit and a memory element. The processing unit may be a general purpose computing device such as a microprocessor. Alternatively, it may be a dedicated processing device, such as a Programmable Logic Controller (PLC). The memory elements may utilize any memory technology such as RAM, DRAM, ROM, Flash ROM, EEROM, NVRAM, magnetic media, or any other medium suitable for storing computer-readable data and instructions. The instructions may be those necessary to operate the pump. The controller may also include an input device such as a touch screen, a keyboard, or other suitable device that allows an operator to input a set of parameters to be used by the controller. The input device may also be referred to as a human machine interface or HMI. The controller may have an output adapted to control the pump. These outputs may be analog or digital in nature and may provide a binary output (i.e., on or off), or may provide a range of possible outputs, such as an analog signal or a multi-bit digital output. After the reagents are added and mixed (e.g., by an in-line static mixer), they then enter the flow channel of the incubation chamber through which they flow for the target inactivation time. The amount of reagent added depends on the type of acid, the strength of the acid and the buffering capacity of the feed solution. The feed solution buffering capacity will depend on many factors, including buffer type, buffer concentration, and mAb concentration. Similar methods can be used for detergent inactivation, rather than low pH inactivation.

According to embodiments disclosed herein, a fluid sample introduced into a fluidic channel will widen away from the fluidic channel with minimal peak broadening. The amount of peak broadening observed when applying the methods disclosed herein is significantly less than the amount of peak broadening observed when employing conventional virus inactivation methods. Fig. 4 illustrates a comparison of peak broadening produced by the methods disclosed herein and conventional methods. Peak broadening is shown in fig. 4, where the sample is injected into straight tubes, coiled tubes (tubes wrapped around a 3 inch diameter rod), and tubes that are introduced into discrete fluid packets according to embodiments disclosed herein. All tubes have the same inner diameter and a length corresponding to a theoretical hold-up volume of 50 mL. 0.5 mL of sample was injected into the tube initially containing water, and after sample injection, water was passed through each tube at 10 mL/min (corresponding to a nominal residence time of 5 minutes). The resulting UV trace was collected at the tube exit. Volumes were normalized to system volumes.

In the literature, coiled tubes have been shown to offer advantages over straight tubes due to secondary flow properties (e.g., Dean vortex, U.S. Pat. No. 5,203,002 "current channel membrane filtration"). Dean vortexes have been used in particular for sequential virus inactivation (WO 2015/135844 a1 "Device and method for continuous virus inactivation"). The peaks obtained using the methods disclosed herein are much narrower than those of both straight and coiled tubes, indicating that the fluid species traveling in discrete packets or regions in the flow channel have a narrower residence time distribution in the system.

The methods described herein also result in a smaller physical footprint for the process, for example, by eliminating the need to use a pool tank for virus inactivation or by minimizing the size of the incubation chamber necessary to inactivate viruses with an appropriate safety factor. In general, there is an increasing demand for more flexible manufacturing processes that improve efficiency by reducing the overall physical footprint (i.e., floor area) of the process. The methods described herein can reduce the overall footprint of the purification process by eliminating the large pool tanks typically used for virus inactivation.

Examples