Cascade Tangential Flow Filtration Systems for Perfusion of Cell Culture

Abstract

A multi-stage TFF apparatus, comprising a bioreactor capable of holding cell culture media; a first stage TFF device in fluid communication with the bioreactor; a storage tank in fluid communication with the first stage TFF device; a first recirculating loop in fluid communication with the first stage TFF device and the bioreactor; a second stage TFF device, in fluid communication with and downstream from the storage tank; and a second recirculating loop in fluid communication with the second stage TFF device and the storage tank.

Claims

1. A multi-stage TFF apparatus, comprising: a bioreactor capable of holding cell culture media; a first stage TFF device in fluid communication with the bioreactor; a storage tank in fluid communication with the first stage TFF device; a first recirculating loop in fluid communication with the first stage TFF device and the bioreactor; a second stage TFF device, in fluid communication with and downstream from the storage tank; and a second recirculating loop in fluid communication with the second stage TFF device and the storage tank.

2. The multi-stage TFF apparatus of claim 1, further comprising a first feed pump disposed between the bioreactor and the first stage TFF device.

3. The multi-stage TFF apparatus of claim 1, further comprising a second feed pump disposed between the storage tank and the second stage TFF device.

4. The multi-stage TFF apparatus of claim 1, further comprising a first permeate pump disposed between the first stage TFF device and the storage tank.

5. The multi-stage TFF apparatus of claim 1, further comprising a second permeate pump disposed downstream of the second stage TFF device.

6. The multi-stage TFF apparatus of claim 1, wherein the first stage TFF device is a cell retention device.

7. A method for processing biological fluids, comprising: providing biological fluids within a cell culture media to a bioreactor and growing biological cells therein; delivering the biological fluid to a first stage TFF device, wherein cells are retained within the first stage TFF device and producing a permeate; perfusing the biological fluid by returning the biological fluid to the bioreactor via first recirculating loop; delivering the permeate to a storage tank; feeding the permeate from the storage tank to a second stage TFF device, wherein the permeate is clarified, wherein some of the permeate is recirculated via a second recirculating loop to the storage tank and a second permeate is delivered to final fill container for storage as a biological product.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0009] FIG. 1 depicts a cell-culturing system comprising a bioreactor, a storage tank, and a multi-stage TFF apparatus according to embodiments of the disclosure;

[0010] FIG. 2 depicts a graph showing a cell culture performance, viable cell density and viability as a function of time within a first TFF device, embodiments according to the disclosure;

[0011] FIG. 3 depicts a graph of product sieving by percent, according to embodiments of the disclosure;

[0012] FIG. 4 depicts a graph of clarification performance of a second TFF device, according to embodiments of the disclosure;

[0013] FIG. 5 depicts a graph of turbidity of a product concentration in a permeate and a product concentration of a feed, according to embodiments of the disclosure; and

[0014] FIG. 6 depicts a graph of a performance of a prior art 0.2 μm PES membrane ATF device with the same cell culture.

DETAILED DESCRIPTION OF SOME EMBODIMENTS

[0015] So the manner in which the features disclosed herein can be understood in detail, a more particular description of the embodiments of the disclosure, briefly summarized above, may be had by reference to the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this disclosure and are therefore not to be considered limiting of its scope, for the embodiments described and shown may admit to other equally effective embodiments. It is also to be understood that elements and features of one embodiment may be found in other embodiments without further recitation and that identical reference numerals are sometimes used to indicate comparable elements that are common to the figures.

[0016] The terms “bioreactor,” “bag,” and “container” are generally used interchangeably within this disclosure. A flexible bioreactor, bag, or container connotes a flexible vessel that can be folded, collapsed, and expanded and/or the like, capable of containing, for example, a biological fluid. A single use bioreactor, bag, or container, typically also flexible, is a vessel that is used once and discarded.

[0017] The term “sterile” is defined as a condition of being free from contaminants and, particularly within the bioprocessing industry, free from undesirable viruses, bacteria, germs, and other microorganisms.

[0018] The term “upstream” is defined as first step processes in the processing of biological materials, such as microbes/cells, mAbs, proteins, including therapeutic proteins, viral vectors, etc., are grown or inoculated in bioreactors within cell culture media, under controlled conditions, to manufacture certain types of biological products.

[0019] The term “downstream” indicates those processes in which biological products are harvested, purified, formulated and packaged.

[0020] The term “clarification” is defined as a downstream process, wherein large insoluble contaminants, usually whole cells and cell debris are separated from the feedstock or harvest.

[0021] The term “purification” is defined as a downstream process, wherein bulk contaminants and impurities, including host cell proteins, DNA and process residuals are removed from the product stream.

[0022] The term “polishing” is defined as a downstream process, wherein trace contaminants or impurities that resemble the product closely in physical and chemical properties are eliminated from the purified product stream.

[0023] The term “chromatography” is defined as a downstream process suitable for biological chromatographic techniques, comprising, but not limited to, protein A chromatography, affinity chromatography, hydrophobic interaction chromatography, column chromatography, and ion exchange chromatography, e.g., anion exchange chromatography, and cation exchange chromatography.

[0024] FIG. 1 depicts a cell-culturing system comprising a bioreactor, a storage tank, and a multi-stage TFF apparatus; according to embodiments of the disclosure. FIG. 1 depicts a cell-culturing system 100 comprising a bioreactor 102, a storage tank 104. and, collectively, a multi-stage TFF apparatus comprising a first stage TFF device 106a, and a second stage TFF device 106b; according to embodiments of the disclosure. The bioreactor 102 is capable of culturing cells that produce antibodies, viruses, and/or other biologics. The bioreactor 102 is capable of being operated under perfusion conditions. For example, cell culture media is supplied periodically or continuously as cell cultures to the first stage TFF device 106a. In some embodiments, the downstream processing is a first stage TFF device 106a. In some embodiments, the first stage TFF device 106a is a cell retention device externally connected to the bioreactor to retain the viable cells within the bioreactor while continuously removing the spent media and product.

[0025] The cell-culturing system 100 further comprises a storage tank 104. The storage tank 104 is disposed in fluid communication and between the first stage TFF device 106a, which is, for example, a cell retention device and a second stage TFF device 106b. A first feed pump 108a is capable of delivering, for e.g., a product stream directly to downstream processing apparatus without further clarifying the product stream. A permeate is delivered from the first stage TFF device 106a to the storage tank 104 via a first permeate pump 110a. The first stage TFF device 106a also returns fluid to the bioreactor 102 via a first recirculating loop 112a. A feed from the storage tank 104 is delivered, via a feed pump 108b, to the second stage TFF device 106b for clarification. A loop returns some fluid to the storage tank 104 using a second permeate pump 108b and a second recirculating loop 112b.

[0026] According to embodiments of the disclosure, the first stage TFF 106a device is a cell retention device externally connected to the bioreactor capable of retaining the viable cells within the bioreactor while removing, optionally continuously, the spent media and product. A large pore size membrane (5-10 μm) is used to mitigate membrane fouling and prevent loss of product sieving over a long period of time during perfusion cell culture. The permeate from the first stage TFF 106a is stored in an intermediate storage tank, from which the feed is introduced continuously to the second stage TFF 106b, and flow rate in and out each TFF stage is similar to maintain the mass balance. The second stage TFF device 106b separates cell debris and other impurities from the product molecule using a small pore size microfiltration membrane (for example, a ≤0.2 μm). The processed product can be fed into the next unit operation (for e.g., Protein A chromatography for monoclonal antibodies (mAbs) and/or other downstream biological processes known to those in the art) without further clarification.

[0027] 2 depicts a graph showing a cell culture performance, viable cell density and viability as a function of time within a first TFF device, embodiments according to the disclosure. As can be seen, the viability of the cells remains nearly constant at approximately 95% during a 15-day duration. Also, a viable cell density reaches 100×10.sup.6 cells/mL after 6 days and maintains this level, or increases, over a subsequent 9 days.

[0028] FIG. 3 depicts a graph of product sieving by percent, according to embodiments of the disclosure. As can be seen, a product sieving percent % (defined as product concentration in the first permeate divided by the product concentration in the bioreactor), maintains at around 100% over 15 days, which indicates that nearly all products passed through the membrane during the entire cell culture process.

[0029] FIG. 4 depicts a graph of clarification performance of a second TFF device, according to embodiments of the disclosure. As can be seen, the product sieving of a clarification process (defined as product concentration in the second permeate divided by the product concentration in the first permeate) by the second stage TFF device products approximately 85-100% sieving percent from time t=0 and remains nearly constant over 22 days. The overall product sieving across the two-stage TFF is greater than 85% throughout the entire process. In comparison, when operating at similar cell culture conditions (viable cell density and viability), the product sieving of ATF or TFF system using small pore size microfiltration membrane rapidly drops to below 50% within 7 days of perfusion cell culture (See references 1 and 2, below). This demonstrates that the proposed system successfully addresses the challenge of product retention using existing ATF and TFF systems.

[0030] FIG. 5 depicts a graph of turbidity of a product concentration in a permeate and a product concentration of a feed, according to embodiments of the disclosure. As can be seen, a permeate from the second stage TFF device has a turbidity of less than 2 (NUT) from time t=0 over the course of 22 days. This demonstrates that the system is able to operate for more than 3 weeks without turbidity breakthrough and the need to switch filters. Both TFF devices can be pre-sterilized and pre-assembled and the whole system can potentially operate in a fully closed, sterilized environment, minimizing the risk of contamination. The permeate from the cascade TFF system as described herein can load directly onto next purification unit operation(s) without further clarification. In comparison, the turbidity of perfusates from open pore membrane filters (i.e., tangential flow depth filters or hollow fiber filters having an average pore size greater than 5 μm) typically range from 10 to 80 NTU, resulting in the need for an additional depth filtration step to remove particles and cell debris from the perfusion permeate stream (See references 2 and 3, below).

[0031] FIG. 6 depicts a graph of a performance of a 0.2 μm PES membrane prior art ATF device with the same cell culture. As can be seen, the cascade TFF system and methods described herein outperform prior art ATF devices and methods with respect to sieving.

[0032] Embodiments of the disclosure also comprise methods for perfusing cells within a cell culture media. For example, some embodiments of the disclosure include a method for processing biological fluids, comprising providing biological fluids within a cell culture media to a bioreactor and growing biological cells therein; delivering the biological fluid to a first stage TFF device, wherein cells are retained within the first stage TFF device and producing a permeate; perfusing the biological fluid by returning the biological fluid to the bioreactor via first recirculating loop; delivering the permeate to a storage tank; feeding the permeate from the storage tank to a second stage TFF device, wherein the permeate is clarified, wherein some of the permeate is recirculated via a second recirculating loop to the storage tank and a second permeate is delivered to final fill container for storage as a biological product.

[0033] Reference throughout this specification to “one embodiment,” “certain embodiments,” “one or more embodiments,” “some embodiments,” or “an embodiment” indicates that a feature, structure, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Therefore, the appearances of the phrases such as “in one or more embodiments,” “in certain embodiments,” “in one embodiment,” “some embodiments,” or “in an embodiment” throughout this specification are not necessarily referring to the same embodiment.

[0034] Although some embodiments have been discussed above, other implementations and applications are also within the scope of the following claims. Although the specification describes, with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It is therefore to be further understood that numerous modifications may be made to the illustrative embodiments and that other arrangements and patterns may be devised without departing from the spirit and scope of the embodiments according to the disclosure. Furthermore, particular features, structures, materials, or characteristics may be combined in any suitable manner in any one or more of the embodiments.

[0035] Publications of patent applications and patents and other non-patent references, cited in this specification are herein incorporated by reference in their entirety in the entire portion cited as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in the manner described above for publications and references.

[0036] Reference (1): Wang, S. B., Godfrey, S., Radoniqi, F., Lin, H., & Coffman, J. (2019). Larger pore size hollow fiber membranes as a solution to the product retention issue in filtration-based perfusion bioreactors. Biotechnology Journal, 14(2), 1800137, which is incorporated by reference in its entirety.

[0037] Reference (2): Pinto, N. D., Napoli, W. N., & Brower, M. (2020). Impact of micro and macroporous TFF membranes on product sieving and chromatography loading for perfusion cell culture. Biotechnology and Bioengineering, 117(1), 117-124, which is incorporated by reference in its entirety.

[0038] Reference (3): WO 2017/180814 A1 to Coffman, which is incorporated by reference in its entirety.