US20150329892A1

US20150329892A1 - Apparatus and Method for Optical Sampling in Miniature Bioprocessing Vessels

Info

Publication number: US20150329892A1
Application number: US14/709,685
Authority: US
Inventors: Edwin John Koerperick; Jonathon Todd Olesberg; Christine Esther Evans; Mark Allen Arnold; Gary Wray Small
Original assignee: ASL ANALYTICAL Inc
Current assignee: ASL ANALYTICAL Inc
Priority date: 2014-05-13
Filing date: 2015-05-12
Publication date: 2015-11-19
Also published as: US20150330903A1; US9404072B2; US9360422B2; US20150330894A1

Abstract

An optical sampling apparatus for miniature-scale bioprocessing vessels includes features for optical interrogation of the bioprocessing vessel contents by means of transmission or transflection spectroscopy. This optical interrogation allows for the determination of quantities and parameters of substances in fluids contained within the bioprocessing vessels during bioprocesses. Multiple such bioprocessing vessels with the optical interrogation features may be mounted in a receiver for conducting multiple bioprocesses simultaneously. A translatable probe may be used to interact with each of the bioprocessing vessels in the receiver.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patent application No. 61/992,735, filed May 13, 2014, for “Optical Interfaces for Bioprocessing Vessels.” Such application is incorporated herein by reference in its entirety.

BACKGROUND

The present invention relates to optical sampling means for providing optical communication between an optical instrument and a disposable vessel of polymeric construction for applications including, but not limited to, pharmaceutical, food processing, and chemical manufacturing as well as other laboratory and industrial processes.
The use of optical and electronic instrumentation to monitor and control the contents of vessels and changes taking place therein is well known in the art. Processing and storage of, for example, food, beverage, chemical, agricultural, fuel, and pharmaceutical products have historically taken place primarily in multiple-use vessels comprised of stainless steel and/or glass. Numerous hardware approaches enabling interrogation and analysis of the contents of such vessels by, for example, optical, electronic, and electrochemical techniques have been described in the art. Dissolved oxygen may be measured by, for example, electrochemical probes with oxygen-permeable membranes, as well as fluorescent sensor techniques. Measurement of pH is possible by electrochemical techniques as well as fluorescent methods. Probes for measurement of optical characteristics of materials in rigid vessels by transmission, reflection, and attenuated total reflection (ATR) are also known in the art. Such probes are often of tubular form and primarily metal construction, protruding through a head plate or side wall of a vessel and into the fluid under process. Probes and sensors of this general description are commonly designed for robustness and longevity—tolerating use, cleaning, and often sterilization for many process cycles. Such multiple-use probes and sensors typically have form factors that are not accommodating to interfacing with single-use bioreactors, particularly flexible bioreactors and those with small working volumes. Flexible bioreactors, also known as bag bioreactors, lack rigidity—surfaces commonly distort during operation, making attachment and positioning of typical multiple-use probes difficult and unstable. Bioreactors with small working volumes simply do not have the surface area or volume to support many of the sensors and probes that are common in the art. Moreover, such prior art sensors and probes do not commonly fit within the model of single-use technology as they are not disposable and must be in contact with the process fluid, thereby requiring the cleaning, sterilization, and aseptic insertion steps that single-use technology seeks to avoid.
Regular cleaning and maintenance of multiple-use vessels is required to maintain process integrity, and sterile conditions are often necessary, demanding yet more laborious and/or costly cleaning and sterilization procedures. The maintenance, cleaning, and disinfection of multiple-use process vessels coupled with the high initial cost of the equipment has led to accelerating adoption of single-use, disposable vessels in multiple industries. These single-use vessels are most commonly constructed of polymers and are often purchased pre-sterilized such that the user may immediately put them to use. As such, sensors that will come into contact with the fluid are commonly integrated into the vessel before sterilization and sterilized with the vessel. Any sensors or connections to the vessel that are not integrated and sterilized with the vessel may be externally sterilized and installed via aseptic ports. While use of sensors or probes that are not installed into the vessel prior to sterilization of the vessel is feasible, it is typically undesirable due to the additional labor required of the end user as well as the increased probability of contamination. Such single-use vessels offer several additional benefits over conventional multiple-use bioreactors: ease of use; reduced setup labor for end users; significantly reduced cleanup time; and lower equipment costs. Single-use disposable bioreactors are available in a variety of sizes and form factors—working volumes range from sub-milliliter to thousands of liters.
A key aspect in bioprocessing is being able to transition processes from small-scale experiments in the research lab to a large-scale production environment. The research and effort to transition from small-scale experiments to production is known as scale-up, and this process is commonly challenging and time consuming. Scale-up often comprises three major phases—the research phase where initial studies are performed and processes are selected and verified; the pilot plant phase where processes are further studied, refined, and verified in higher volume processes; and the production phase where large-scale manufacturing is performed. The conditions present in small-volume research bioreactors may be markedly different from those present in the larger bioreactors in the pilot plant and on the production floor. Indeed, processes can vary considerably even between different bioreactors in the research lab. In order to execute the scale-up process in the most efficient manner possible, it is desirable to have the ability to optimize a plurality of process parameters and constituent concentrations, and often to be able to control such parameters and constituent concentrations. Ideally such monitoring and control capabilities will be uniform throughout the various stages of scale-up. Bioreactors having working volumes of microliters to few milliliters are commonly known as micro-bioreactors, and are often configured such that multiple micro-bioreactors are used to perform experiments in parallel. Such multiplexed experiments with cell culture or fermentation processes enable evaluation of process conditions, cell lines, or other variables in an efficient manner. So-called miniature-bioreactors commonly have working volumes of tens to few hundreds of milliliters, and may offer another step in the scale-up process. Similarly to micro-bioreactors, mini-bioreactors are often configured in groups for parallel experimentation, though with a working volume that better represents more standard process conditions. While reliable monitoring of constituent concentrations of fluids in bioprocesses such as nutrient analyte concentrations remains challenging even in large-volume bioreactors, the challenge is amplified with micro- and mini-bioreactors given the space constraints and form factors. Sensor technologies capable of providing such fluid constituent concentration information, and ideally control of such concentrations, in bioreactors used across the product development arc from research lab to production plant are desired in the biotechnology and pharmaceutical industries.
Sensors for measurement of a variety of parameters within single-use vessels have been demonstrated. For example, analysis of physical and chemical conditions such as pH and dissolved oxygen (DO) is possible by means of sensors comprising fluorescent dots within the bioreactor fluid. Single-use and disposable temperature and pressure sensors have been demonstrated. Optical interfaces for vessels of polymeric construction, which may be single-use and/or flexible vessels, are also known in the art, though to a far lesser extent than similar interfaces for multiple-use vessels. Interfaces for transmission, reflection, and ATR optical measurements have been disclosed; however these interfaces and ports are generally not optimized for near-infrared spectroscopic applications. Numerous polymers are available that are at least partially transparent to visible and short-wave infrared (SWIR), though these polymers are often substantially opaque or exhibit significant absorption structure at wavelengths longer than 1.5 μm.
Bioreactors commonly require frequent monitoring and strict control in order to ensure optimal environmental and nutritional conditions for fermentation, cell cultures, or similar processes contained therein. While sensors are available to continuously measure parameters such as DO and pH as is hardware and software to control these parameters, sensors and systems to monitor nutrients and chemical constituents in an automated fashion and control the levels thereof have historically been largely absent in the art. This is the case for both multiple- and single-use bioreactors, however sensor solutions to interface with single-use bioreactors have been particularly lacking.
Measurement of chemical constituents by spectroscopic methods, particularly infrared spectroscopic methods, presents a robust means to monitor said chemical constituents and control levels thereof within bioreactors and process vessels in general. In order to optically interface with polymeric vessels and their contents, integrated and robust optical interface solutions are desired. These solutions may be substantially transparent in the wavelength range of interest, thereby enabling high measurement stability and optical throughput. The requirement of material transparency is particularly challenging for infrared spectroscopy, principally near- and mid-infrared spectroscopy, where optical absorption by many commonly used polymers is unacceptably high when polymer thicknesses are within the satisfactory range to maintain mechanical integrity. When in an optical spectroscopic configuration, embodiments of optical sensors where the path or sample length through the vessel contents is selectable and/or controlled may be desirable for some applications. Embodiments where any optical elements that are to come in contact with the vessel contents are fused to the vessel and sterilized with the vessel are often preferable to solutions where optical monitoring components are inserted aseptically subsequent to sterilization.

BRIEF SUMMARY

As used herein, the terms “optical” and “light” refer to electromagnetic radiation having vacuum wavelengths between 300-20,000 nm.
As used herein, “near infrared”, “near-infrared”, and “NIR” mean the region of the electromagnetic spectrum generally spanning wavenumbers between 3300 cm⁻¹and 14,000 cm⁻¹(corresponding to wavelengths of approximately 0.7 μm to 3.0 μm).
As used herein, “interrogation” and “sampling” mean illuminating a sample with optical radiation and collecting at least a portion of the radiation having interacted with said sample for optical analysis.
As used herein, “working volume” refers to the typical volume of fluid contained within a vessel or container during a process and is most commonly less than the total volume of fluid that the vessel could retain.
As used herein, “miniature,” and “mini,” when used in reference to bioprocessing vessels means bioprocessing vessels having working volumes less than or equal to 0.25 liters.
As used herein, “constituent” means a chemical analyte, protein, DNA, component in a fluid, cell, or solid suspended in a fluid.
The present invention relates to miniature-bioprocessing vessels comprising features for optical interrogation of fluids contained within such bioprocessing vessels. Embodiments of receiver assemblies for receiving, housing, and positioning embodiments of such bioprocessing vessels are also provided. Embodiments of such receivers may also provide optical elements, sensors, and means for optical communication with optical instruments, and may be configured to receive a plurality of bioprocessing vessels. An optical instrument may be used in conjunction with embodiments of the present invention to determine and/or control quantities of substances in fluids contained within bioprocessing vessels. The invention pertains to optical transmission and transflection measurements in general and particularly to near-infrared spectroscopic analytical techniques.
A plurality of embodiments of bioprocessing vessels comprising features for optical interrogation is described herein. Embodiments of bioprocessing vessels provided by the present invention typically comprise at least one rigid polymer sidewall and may be comprised entirely of rigid polymer materials. In such embodiments, at least a portion of the polymeric vessel may be substantially transparent to the wavelengths of electromagnetic radiation being utilized either due to inherent lack of absorption or to use of a suitably thin section of polymer. In one embodiment of the present invention, an optical sampling region is provided whereby features extending outward from the primary volume of a bioprocessing vessel at least partially define the optical sampling region by providing a defined length of optical path through a fluid contained within the bioprocessing vessel. In another embodiment, an optical sampling region is provided whereby features extending into the primary volume of a bioprocessing vessel at least partially define the optical sampling region.
Embodiments are also described whereby bioprocessing vessels comprise integral optical probes extending into the vessel. In some embodiments, optical waveguides and/or optical elements within an optical probe communicate light into the fluid within a bioprocessing vessel where it may interact with the fluid, and a portion of the light having interacted with the fluid may be communicated by additional optical waveguides and/or elements to a sensor or optical instrument. In one embodiment, integral probes are provided where the input and output optical communication is provided on a single surface integrated with the probe. In another embodiment, input and output optical communication are provided on different sides of the optical probe coupled to different sides of a vessel. In yet another embodiment, only input optical waveguides and/or elements are provided, and light is sensed by a sensor in a receiver for the bioprocessing vessel.
The detailed description and drawings provided herein will offer additional scope to certain implementations of the present invention. It should be understood that the described implementations are provided as examples only. Those skilled in the art will recognize that numerous variations and modifications of the described implementations are within the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an isometric view of a disposable bioprocessing vessel having features for optical sampling that extend outward from the primary volume of the vessel on the side of the vessel.

FIG. 2 shows a top sectional view of the bioprocessing vessel in FIG. 1 and an associated optical reader.

FIG. 3 shows an isometric view of a disposable bioprocessing vessel having features for optical sampling that extend outward from the primary volume of the vessel on the bottom of the vessel.

FIG. 4 shows a side sectional view of the bioprocessing vessel in FIG. 3.

FIG. 5 depicts a polymer laminate comprising multiple polymer materials.

FIG. 6 shows a top sectional view of a disposable bioprocessing vessel having features for optical sampling that extend outward from the primary volume of the vessel on the side of the vessel and include a reflector.

FIG. 7 shows a side sectional view of a bioprocessing vessel having features for optical sampling that protrude into the primary volume of the vessel and form a single optical path length.

FIG. 8 shows a side sectional view of a bioprocessing vessel having features for optical sampling that protrude into the primary volume of the vessel and form two optical path lengths.

FIG. 9 shows a side view of a disposable bioprocessing vessel comprising a second polymer in the optical sampling region.

FIG. 10 shows a top sectional view of the bioprocessing vessel in FIG. 9.

FIG. 11 shows an isometric view of a disposable bioprocessing vessel with an integral optical probe.

FIG. 12 shows a side sectional view of the bioprocessing vessel in FIG. 11.

FIG. 13 shows a side sectional view of a disposable bioprocessing vessel with an integral optical probe having an optical sampling region transverse to the axis of the probe.

FIG. 14 shows a side sectional view of a disposable bioprocessing vessel with an integral optical probe providing an optical sampling region between the end of the optical probe and the bottom of the bioprocessing vessel.

FIG. 15 shows an isometric view of a receiver for a plurality of disposable bioprocessing vessels with several disposable bioprocessing vessels installed therein.

FIG. 16 shows a detailed view of the isometric view of the receiver base assembly in FIG. 15.

FIG. 17 shows a sectional view of the receiver base assembly of FIG. 15 with three embodiments of disposable bioprocessing vessels installed.

DETAILED DESCRIPTION

An embodiment of a disposable bioprocessing vessel comprising features to provide an optical sampling region is shown in FIG. 1 and FIG. 2. The isometric view in FIG. 1 shows a bioprocessing vessel 100 with features that extend outward sideways from the primary volume of the vessel. The features extending outward from the vessel comprise a first surface 110 and a second surface 120 that provide an optical sampling region 130. Additional surfaces may be provided as necessary to provide a particular geometry of the optical sampling region 130. For example, a curved or stepped surface may be provided adjacent to the optical sampling region 130 in order to promote adequate mixing of the fluid within the optical sampling region 130 and within the entire vessel 100. At least one rigid wall 140 is provided, though the entire bioprocessing vessel 100 may comprise rigid polymer materials. The geometry of the optical sampling region 130 may be configured to best suit the chosen application. For measurements in aqueous solutions with near-infrared electromagnetic radiation, the optical path defined by the distance between first 110 and second 120 surfaces (distance through the fluid within the vessel) will preferably be between 0.5 mm and 2.0 mm. This range of optical path length provides acceptably low attenuation due to water absorption and sufficient interaction length with the fluid sample to provide near-infrared measurements of substances within the fluid. The optical path length is taken as the path length that the beam takes through the fluid and does not include the wall thicknesses of the first 110 or second 120 surfaces. A transmission or transflection measurement may be provided by embodiments of the present invention. In a transmission measurement configuration, electromagnetic radiation having interacted with the fluid is communicated through the second surface 120 and detected outside of the disposable bioprocessing vessel. In a transflection measurement, a portion of the electromagnetic radiation having been communicated through the first surface 110 is reflected back towards the first surface 110 by the fluid and substances contained therein, and a portion of the electromagnetic radiation having interacted with the fluid and having reached the second surface is reflected from the second surface 120 (or a reflective element 190 thereon or therein, depicted in FIG. 6) towards the first surface 110. As electromagnetic radiation from the portions having been transmitted through and reflected from the fluid sample in the optical sampling region 130 comprises the resulting radiation incident on the first surface 110 in the direction of arrowed line B, a transflection measurement may be provided.
The polymer materials that comprise the first 110 and second 120 surfaces in the optical sampling region 130 will preferably be materials being sufficiently transparent to the wavelength range of electromagnetic radiation used for optical interrogation. In the near-infrared wavelength range of the electromagnetic radiation spectrum, perfluorinated polymers such as fluorinated ethylene propylene are preferable due to their low optical absorption, chemical compatibility, and classification as USP Class VI compliant materials. Alternatively, if polymers exhibiting substantial absorption in the wavelength range of interest are to be used, at least portions of the first 110 and second 120 surfaces in the optical sampling region 130 may be manufactured to be sufficiently thin to provide sufficient optical throughput in the wavelength range of interest. For example, polycarbonate exhibits strong absorption features in the near-infrared wavelength range, however the transmission of polycarbonate is acceptable if the polycarbonate is sufficiently thin, and preferably less than 0.25 mm thick. Due to the sterility requirements common in bioprocessing applications, polymers chosen for manufacturing of the bioprocessing vessel 100 and optical sampling region 130 will preferably be amenable to sterilization by one or more techniques. Sterilization by irradiating with gamma or beta radiation is a common technique for disposable polymer components in bioprocessing applications. Both sterilant gas such as ethylene oxide or heat sterilization by autoclave are also sterilization options, and embodiments will preferably withstand sterilization by at least one sterilization technique and remain FDA and/or USP Class VI compliant after sterilization.
The sectional view in FIG. 2 shows the bioprocessing vessel of FIG. 1 with an optical reader 150 configured to provide communication of electromagnetic radiation into and out of the fluid within the optical sampling region 130. The optical reader 150 may comprise a housing 160 and optical elements 170 such as waveguides or lenses to communicate light to and from the optical sampling region 130. Electromagnetic radiation from an optical instrument or light source and traveling in the direction of arrowed line A may be communicated through one or more optical elements 170 in the optical reader 150, and through the first surface 110. A portion of the electromagnetic radiation having interacted with the fluid within the disposable bioprocessing vessel 100 may be communicated through the second surface 120 and additional optical elements 170 within the optical reader 150. The optical reader 150 may then communicate a portion of the resulting electromagnetic radiation to the optical instrument or a separate sensor for sensing.
In an embodiment similar to the bioprocessing vessel 100 with features that extend outward sideways from the primary volume of the vessel, an embodiment of a bioprocessing vessel 180 with features extending outward through the bottom of the vessel are also provided. First 110 and second 120 surfaces are provided and form an optical sampling region 130. Said bioprocessing vessel 180 shown in FIG. 3 and FIG. 4 provides similar functionality to the bioprocessing vessel having features extending outward sideways 100. The positioning of the optical sampling region 130 at the side or bottom of the bioprocessing vessel allows flexibility in the configuration of the receiving and optical sampling components that interface with the vessel. The first 110 and second 120 surfaces need not extend out from the primary volume of the disposable bioprocessing vessel in the shape depicted in FIG. 3 and FIG. 4, and indeed it may be preferable to minimize the extent of the protrusion to ensure adequate mixing and fluid homogeneity. Baffles or other directional surfaces (not shown) may be provided to encourage fluid mixing within the sampling region 130. In one embodiment, the first 110 and second 120 surfaces may comprise step-variable features to provide more than one optical path length through the fluid. The ability to select from a plurality of optical path lengths is particularly advantageous in bioprocessing applications where the turbidity of the fluid may change substantially throughout the process. For example, in a first part of a cell culture or fermentation process where the turbidity is low, a longer optical path length may be chosen to increase the optical interaction length with the fluid and substances contained therein. In a subsequent part of the process when the turbidity is high due to cell growth, a shorter optical path length may be chosen to reduce the attenuation from the cells and thereby increase the optical signal.
Embodiments comprising disposable bioprocessing vessels with polymer regions for optical wavelength reference operations are provided by the present invention. Polymers for optical wavelength referencing may be the same polymer as the primary polymer comprising the bioprocessing vessel, or may be a different polymer having more desirable properties for wavelength reference operations. The disposable bioprocessing vessel 180 embodiment shown in FIG. 4 comprises an additional polymer element 185 configured for optical wavelength reference operations. Said additional polymer element 185 is configured such that an optical beam incident in the direction of arrowed line E may pass through said second polymer element 185 without traversing the fluid contained within the bioprocessing vessel 180. Such a configuration where an optical beam used for wavelength reference operations traverses only the reference polymer and not any fluid within the bioprocessing vessel is preferable so that no optical signature from the fluid (which may change over time) is included in the wavelength reference operation.
Embodiments of the present invention comprising composite polymer laminates are also provided. A disposable bioprocessing vessel or portion thereof may comprise a plurality of polymer layers adjacent to one another as shown in FIG. 5. Such a composite polymer 200 may comprise for example a first 210, second 220, and third 230 polymer layer. The composite polymer 200 may be formed by any number of methods including layers that are co-extruded, fusibly bonded, adhesively bonded, thermally bonded, ultrasonically bonded, or connected at seams. Such composite polymer laminates may find application where various properties are required of the bioprocessing vessel that cannot be easily accomplished with a single polymer. For example, a first polymer being FDA or USP Class VI compliant may be used as a liner in contact with the fluid contents of the vessel while a second polymer may comprise an outer structural layer of the vessel, and need not be in compliance with FDA or USP guidelines as no surfaces of the second polymer are wetted. Use of a transition polymer between an inner and an outer polymer may be used to improve adhesion between the inner and outer polymers. Additional polymers beyond the inner and outer polymers may also be used for example to affect oxygen permeability of the composite polymer structure.
The embodiment shown in FIG. 6 provides a transflection measurement configuration. Such a configuration enables collection of electromagnetic radiation having portions both having been reflected from the fluid and materials contained therein as well as having been transmitted through said fluid and materials contained therein. The embodiment shown in FIG. 6 provides an extension of the disposable bioprocessing vessel 100 of FIG. 1 and FIG. 2 wherein the second surface 120 further comprises a reflector 190. The reflector 190 may comprise for example an optical element such as a mirror, a dielectric coating, or a metallic coating, and may or may not be attached to the second surface 120.
In another embodiment, features that extend inward into the disposable bioprocessing vessel are provided for optical interrogation of the fluid within the vessel. An embodiment shown in FIG. 7 provides a disposable bioprocessing vessel 240 comprising two optical access features 260 extending inward into the interior of the vessel to form an optical sampling region 250 having a fixed optical path length through the fluid. Optical elements such as mirrors or prisms mounted in a receiver or optical reader may be used to provide optical communication to the fluid within the bioprocessing vessel 240. In another embodiment shown in FIG. 8, a disposable bioprocessing vessel 270 comprising optical access features 280 providing two or more optical path lengths is provided. In this embodiment, the optical sampling region 290 comprises two sampling regions providing distinct optical path lengths, a first optical sampling region 300 and a second optical sampling region 310 providing a longer optical path length than the first optical sampling region 300. An optical beam traversing the first optical sampling region 300 providing the shorter optical path length through the fluid is represented by dashed arrowed line C, and an optical beam traversing the second optical sampling region 310 providing the longer optical path length through the fluid within the bioprocessing vessel is represented by arrowed line D. As noted previously, provision of more than one optical path length provides flexibility in sampling for example bioprocesses where cell growth occurs during a process and results in increasing turbidity and hence increasing attenuation of optical beams traversing the fluid.
Due to the fact that many polymers exhibit strong absorption features in certain wavelength ranges of the electromagnetic spectrum, it may be advantageous to provide a second polymer serving as an optical window in the optical sampling region of a disposable bioprocessing vessel. For example in the near-infrared wavelength range of the electromagnetic spectrum (comprising wavenumbers between 3300 cm⁻¹and 14,000 cm⁻¹), strong absorption features may arise from C—H, C—O, O—H, and N—H chemical bonds. For this reason it may be preferable to use polymers lacking such chemical bonds in the optical sampling regions of disposable bioprocessing vessels designed for optical interrogation by such wavelengths. Perfluorinated polymers such as Teflon® polytetrafluoroethylene (PTFE), ethylene tetrafluoroethylene (ETFE), Teflon® fluorinated ethylene propylene (FEP), Teflon® amorphous fluoroplastics (AF), and Teflon® perfluoroalkoxy copolymer (PFA) lack the listed chemical bonds and thus may be preferable for polymer materials within the optical path. Alternatively, other polymer materials may be used if sufficiently thin to provide adequate optical transmission in the desired wavelength range. An embodiment is provided that comprises a second polymer being more optically transparent in the near-infrared wavelength range than the primary polymer used in the manufacture of disposable bioprocessing vessels. Such an embodiment is shown in FIG. 9 and FIG. 10—a disposable bioprocessing vessel 320 having a second polymer 330 in the optical sampling region 130 is provided for improved optical transmission at near-infrared wavelengths of the electromagnetic spectrum. A side view of such a vessel is shown in FIG. 9 and a sectional view is shown in FIG. 10. The optical path length provided in the optical sampling region 130 may be defined by the first 110 and second 120 surfaces as with the previously described embodiment 100, or the optical path length may be defined by the second polymer 330 window region.
In yet another embodiment of the present invention, the optical path length may be formed by compression of the first 110 and/or second surfaces 120. The embodiment shown in FIG. 1 and FIG. 2 may provide such an optical path length formed by compression. Compression of the first 110 and/or second 120 surfaces may be provided by elements of the optical reader 150 or by a receiver assembly. In this embodiment, at least portions of the first 110 and second 120 surfaces are partially compressible such that they can be mechanically compressed to provide a defined optical path length. Compression of surfaces to provide a path length enables a highly stable and reproducible path length, which is of consequence for high-performance spectroscopic measurements. Establishing an optical path length by compression may also provide the benefit of reducing the tolerances on manufacturing of the disposable bioprocessing vessels. The merits of establishing an optical path length by compression of polymer components have been established in U.S. patent application Ser. No. 14/631,914, the teachings of which are incorporated by this reference. If rigid surfaces in non-disposable components of an optical reader or receiver are used to compress surfaces on a disposable bioprocessing vessel to form a defined optical path length, tighter tolerances may be used in the manufacturing of the non-disposable components as a higher manufacturing cost would be acceptable in non-disposable components whereas manufacturing costs are desirably minimized in production of disposable components.
Embodiments of disposable bioprocessing vessels comprising integral optical probes extending into the fluid within the bioprocessing vessels are also provided by the present invention. Provision of optical probes enables alternative optical interfacing strategies to optical instrumentation and permits sampling the contents of bioprocessing vessels at locations more central to the vessel rather than at the periphery. One embodiment of a disposable vessel 340 comprising an integral optical probe 350 is shown in the isometric view in FIG. 11 and the sectional view in FIG. 12. In this embodiment, optical waveguides 360 such as optical fibers provide optical communication between optical elements 370 situated within the bioprocessing vessel 340 and an optical reader, sensor, or receiver for the bioprocessing vessel 340. An optical sampling region is provided between the optical elements 370, and the optical path length is determined by the distance between the optical elements 370 within the fluid. Additional optical components (not shown) may be provided in the optical probe 350 to enhance optical throughput or otherwise improve performance or alter functionality. For example, lenses may be provided to image the optical beam through the optical sampling region. It is preferable that all wetted surfaces to come in contact with the fluid in the bioprocessing vessel 340 be comprised of polymers or other suitable materials that are FDA and/or USP Class VI compliant for bioprocessing applications. The integral optical probe 350 may be located in alternative locations on the bioprocessing vessel 340 such as through the top 380, bottom 390, or a sidewall 400.
The embodiment shown in FIG. 11 and FIG. 12 may be configured to provide an optical transmission or transflection measurement. The distance through the fluid between the optical elements 370 forming the optical path length will preferably be between 0.5 mm and 2.0 mm (inclusively) for near-infrared spectroscopic measurements. This range of optical path lengths is favorable when electromagnetic radiation having wavenumbers between 3300 cm-1 and 5600 cm-1 is employed in the optical measurement owing to sufficiently high optical interaction length with the fluid and sufficiently low water absorption.
Additional embodiments of disposable bioprocessing vessels having integral optical probes are shown in FIG. 13 and FIG. 14. These embodiments provide simple optical configurations to enable low-cost manufacturing and ease of alignment with supplemental optical instrumentation. In the embodiment shown in FIG. 13, a disposable bioprocessing vessel 410 is spanned by an optical probe 420 having an optical sampling region 430 transverse to the axis of the probe 420. A first optical waveguide 440 within the optical probe 420 provides optical communication between an optical instrument and the optical sampling region 430 of the probe 420 where a defined optical path length through the fluid is provided. Additional optical components may be provided within the probe 420 to improve optical throughput or otherwise tailor the performance to an application. In this embodiment, a sensor may be placed directly below the disposable bioprocessing vessel 410 within a receiver or the collected light having interacted with the fluid may be communicated to an optical instrument. In one preferred embodiment, a second optical waveguide 450 being larger in diameter than the first optical waveguide 440 is provided to collect light having interacted with the fluid in the optical sampling region 430. Said second optical waveguide 450 may then communicate collected light to an optical instrument or sensor. In this embodiment, if the optical path length remains sufficiently short (e.g. 2.0 mm or less) and the second waveguide 450 is sufficiently larger in diameter than the first optical waveguide 440, high optical throughput may be provided without the provision of additional optical elements. This approach enables reduction in the number of optical components required, thereby reducing manufacturing costs and simplifying the optical geometry.
The embodiment shown in the sectional view in FIG. 14 provides a disposable bioprocessing vessel 460 with an optical sampling region 470 formed between the end of the optical probe 490 and a wall of the bioprocessing vessel 460. In the embodiment shown in FIG. 14, the optical sampling region 470 that forms the optical path length is located between the end of the optical probe 490 and the bottom wall 480 of the bioprocessing vessel 460. Similarly the optical probe 490 may be installed in different locations within the bioprocessing vessel 460 if advantageous for the application. In one embodiment, a single optical waveguide 500 within the optical probe 490 may be provided if the optical path length provided by the optical sampling region 470 is sufficiently short to maintain acceptable optical throughput. Optical transmission and transflection measurements may be provided by certain embodiments of the invention. For example, an optical transflection measurement may be provided by including a reflective element on the bottom wall 480 opposite the optical probe 490 of the bioprocessing vessel 460.
Embodiments of receivers for disposable bioprocessing vessels are also provided by the present invention. Receivers may accommodate a single bioprocessing vessel, but are commonly configured to receive a plurality of bioprocessing vessels to perform multiple bioprocessing experiments simultaneously. Receivers may perform a plurality of functions such as measurement and/or control of temperature, agitation, aeration, pH, dissolved oxygen, cell density, cell viability, and chemical constituent concentrations. One embodiment of a receiver is shown in the isometric view in FIG. 15. In this embodiment, the receiver 510 is configured to receive a plurality of bioprocessing vessels 520. A base assembly 530 is provided with a plurality of stations 540 configured for receiving bioprocessing vessels 520. Each station 540 within the receiver 510 is configured to receive a bioprocessing vessel 520, and may provide components for near-infrared optical interrogation of the contents of the bioprocessing vessel 520. Optical components such as optical fibers, lenses, mirrors, and sensors may be provided within the receiver base assembly 530 in conjunction with each station 540 to provide optical communication between the contents of the bioprocessing vessels 520 and one or more optical instruments. Optical communication between bioprocessing vessels 520 and optical instrumentation may also be provided by an optical interface 560 configured with a mechanical translator 570. In such an embodiment, one or more motors 580 may be used to translate the optical interface 560 on the mechanical translator 570 to a desired bioprocessing vessel 520 where the optical interface 560 performs optical interrogation on the fluid contents of the bioprocessing vessel 520. The optical interface 560 may comprise optical elements such as fibers, lenses, and windows to provide optical communication with bioprocessing vessels 520 and any associated features or integral optical probes. Similarly, the bioprocessing vessels 520 may be mechanically translated and the optical interface 560 may remain stationary.
The view in FIG. 16 provides additional detail on an embodiment of the receiver base assembly 530 and how interfacing with bioprocessing vessels 520 may be performed. With optical sampling, proper alignment of the bioprocessing vessels 520 within the receiver 510 is preferable to ensure satisfactory alignment of optical components. Stations 540 within the receiver base assembly 530 may comprise specific alignment features 550 such as a notch or groove to couple with a corresponding feature on a bioprocessing vessel 520 to ensure satisfactory alignment. Alternatively, features 590 that correspond to optical sampling features on the bioprocessing vessel 520 may serve as the alignment means.
The sectional view in FIG. 17 offers detail on embodiments for interfacing a receiver 510 with three embodiments of disposable bioprocessing vessels. From left to right in the figure, disposable bioprocessing vessels with: optical sampling features extending outward sideways 100 (from FIG. 1 and FIG. 2); an integral optical probe forming a gap with the bottom wall of the vessel 460 (from FIG. 14); and an integral optical probe having an optical sampling region transverse to the axis of the probe 410 (from FIG. 13) are shown housed within stations 540 in the receiver 510. Embodiments of the present invention may provide optical elements such as optical sensors 600 located within the base assembly 530 of the receiver 510. For example, the optical waveguide 500 provided in the optical probe 490 of the bioprocessing vessel 460 may provide optical communication of near-infrared electromagnetic radiation from an optical source or instrument to the optical sampling region 470 forming an optical path length in the fluid, and resultant electromagnetic radiation may be sensed by the sensor 600.
Methods are also provided by the present invention for determining quantities of substances within fluids contained within disposable bioprocessing vessels. Near-infrared electromagnetic radiation may be used to optically interrogate fluids, and the changes sensed in the collected near-infrared radiation after interaction with a fluid may be used to determine quantities of substances within fluids. Bioprocessing vessels located in a receiver assembly may first be selected for optical interrogation. Selection of a vessel may be performed for example mechanically as by translating an optical interface located on a mechanical translator, or optically as by activation of an optical sensor or switch. Near-infrared electromagnetic radiation is then communicated to a disposable bioprocessing vessel. Communication of near-infrared radiation may be provided by optical waveguides such as optical fibers, free-space optical elements, or a combination thereof. Optical communication elements may be provided in the receiver base assembly, on an optical interface, or both. For example, near-infrared electromagnetic radiation from an optical instrument may be communicated to a bioprocessing vessel via optical waveguides, and radiation having interacted with the fluid within a bioprocessing vessel may be sensed by an adjacent optical sensor. Radiation having interacted with the fluid within a bioprocessing vessel may also be communicated to an optical instrument for analysis. Radiation resulting from optical transmission or transflection measurements through the fluid in the bioprocessing vessel may be used by an optical instrument to determine one or more quantities of substances in a fluid.
Optical spectroscopy with near-infrared electromagnetic radiation offers a plurality of advantages for determining quantities of substances in fluids. Optical absorption features in the 3300 cm-1 to 14,000 cm-1 wavenumber range are often present for substances having C—H, O—H, C—O, N—H, S—H, and P—H chemical bonds, offering the possibility to determine quantities of substances containing such chemical bonds using near-infrared spectroscopy. While water is sufficiently strongly absorbing in several wavelength ranges throughout the infrared electromagnetic spectrum to limit the effectiveness of spectroscopic techniques to determine quantities of substances, the 3300 cm-1 to 5600 cm-1 wavenumber range provides a water transmission window centered at approximately 4600 cm-1. In this wavenumber range the water absorption is sufficiently low to allow adequate optical throughput through fluid samples with a sufficiently short optical path length to determine quantities of substances by spectroscopic techniques. In order to provide sufficient optical throughput through a fluid and also provide a satisfactory optical path length for interaction of electromagnetic radiation with the fluid, optical path lengths through fluids ranging from 0.5 mm to 2.0 mm are preferable for embodiments of the present invention. Measurements with near-infrared spectroscopic techniques may be used to determine quantities of substances in fluids such as alcohols, sugars, lipids, organic acids, peptides, and steroidal molecules as such substances often comprise optical absorption features at near-infrared wavelengths due to their chemical bonds. In addition to measurements of optical absorption by transmission or transflection measurement approaches to determine quantities of substances by their absorption spectra, near-infrared spectroscopic techniques may be used to determine parameters such as cell density, cell viability, or turbidity. Due to the reduction in optical scattering with increasing wavelength, optical path lengths between 0.5 mm and 2.0 mm may be used even when conducting high cell density bioprocesses such as Pichia pastoris fermentations. Use of wavenumbers higher than 5600 cm-1 (shorter wavelength than 1.8 μm) often requires short path lengths or operation with low cell density applications due to the increased optical scattering encountered and resulting optical attenuation.
Embodiments of the present invention including disposable bioprocessing vessels and receivers as well as associated methods provide for a plurality of bioprocessing applications such as a storage stage, a growth stage, a product formation stage, a purification stage, and a product formulation stage. For example, a growth stage may include cell culture, fermentation, or other bioprocesses whereby cell growth and/or product formation is desired. Embodiments of the present invention may be provided for processes such as batch processes as well as continuous processes such as perfusion processes. Downstream processes such as product purification may also utilize embodiments of the present invention for determination of constituents in fluids.
Embodiments of the present invention disposable bioprocessing vessels may also comprise polymer regions to provide an optical wavelength reference. The merits of providing polymer materials for wavelength reference operations have been described in U.S. patent application Ser. No. 14/631,917, the teachings of which are incorporated by this reference. Absorption features of polymers may be used advantageously as optical wavelength references, wherein said absorption features are used to provide a comparison of a measured optical spectrum of the polymer with a known optical spectrum of the polymer to determine the wavelength accuracy of an instrument. Such wavelength reference methods may provide enhanced stability of optical systems and measurements due to the establishment of a calibrated wavelength axis of a measurement. Instrumental drift due to for example drift in performance of instrument components or in environmental conditions may cause undesirable changes to an optical system whereby the wavelength axis of the measurement may deviate from an acceptable condition. Periodic verification and correction of the wavelength properties of an optical system by comparison with a known standard material is desirable to mitigate against such undesirable changes and thereby improve the stability of the optical system and accuracy of measurements made with the optical system. In embodiments of the present invention, a second beam of near-infrared electromagnetic radiation may be provided to optically interrogate a polymer region on a disposable bioprocessing vessel comprising a polymer suitable as an optical wavelength reference. Said polymer region will desirably provide no fluid sample within the second optical beam path such that the optical absorption experienced by the beam is only that of the polymer wavelength reference material. Said polymer used as a wavelength reference material will desirably have multiple optical absorption features within the wavelength range of the optical measurement in order to provide multiple features with which to make a comparison against a known optical spectrum of the polymer. In the near-infrared region of the electromagnetic spectrum, polymer materials such as nylon, polycarbonate, Kapton®, polymethylpentene (TPX), and polyether ether ketone (PEEK) may be provided as wavelength reference materials.
The present invention has been described with reference to the foregoing specific implementations. These implementations are intended to be exemplary only, and not limiting to the full scope of the present invention. Many variations and modifications are possible in view of the above teachings. The invention is limited only as set forth in the appended claims. All references cited herein are hereby incorporated by reference to the extent not inconsistent with the disclosure herein. Unless explicitly stated otherwise, flows depicted herein do not require the particular order shown, or sequential order, to achieve desirable results. In addition, other steps may be provided, or steps may be eliminated, from the described flows, and other components may be added to, or removed from, the described systems. Accordingly, other implementations are within the scope of the following claims. Any disclosure of a range is intended to include a disclosure of all ranges within that range and all individual values within that range.

Claims

1. A disposable bioprocessing vessel for containing a fluid sample, the vessel comprising:

at least one rigid wall;

an optical sampling region integral to the rigid wall and comprising a first and second surface to create an optical path between the first and second surfaces within the bioprocessing vessel, said first and second surfaces comprise a polymer at least partially transparent to near-infrared electromagnetic radiation and wherein the first and second surfaces are sufficiently thin to allow near-infrared electromagnetic radiation to pass therethrough, interact with the fluid sample, and be detected outside of the bioprocessing vessel to provide a transmission or transflection measurement of the fluid sample.

2. The disposable bioprocessing vessel of claim 1, wherein said polymer comprises at least one of polycarbonate or fluorinated ethylene propylene (FEP).

3. The disposable bioprocessing vessel of claim 2, wherein at least portions of the first and second surfaces are less than 0.25 mm thick.

4. The disposable bioprocessing vessel of claim 1, wherein said polymer comprises a composite polymer laminate comprising a first layer and a second layer wherein the second layer comprises a different polymer material than the first layer.

5. The disposable bioprocessing vessel of claim 1, wherein a length of the optical path defined by said first and second surfaces is between 0.5 mm and 2.0 mm inclusively.

6. The disposable bioprocessing vessel of claim 1, wherein said disposable bioprocessing vessel is suitable for sterilization by one or more of gamma irradiation, beta irradiation, ethylene oxide, or autoclave.

7. The disposable bioprocessing vessel of claim 1, wherein said second surface further comprises a reflector, wherein said reflector reflects at least a portion of said near-infrared electromagnetic radiation in the direction of the first surface where it may be sensed by a sensor, thereby providing an optical transflection measurement.

8. The disposable bioprocessing vessel of claim 1, wherein said polymer is at least partially transparent to near-infrared electromagnetic radiation transmitted through a fluid sample comprising wavenumbers between 3300 cm⁻¹and 5600 cm⁻¹.

9. The disposable bioprocessing vessel of claim 1, wherein said disposable bioprocessing vessel has a working volume less than or equal to 0.25 liters.

10. The disposable bioprocessing vessel of claim 1, wherein said first and second surfaces extend outward from a primary volume of said bioprocessing vessel and define said optical path.

11. The disposable bioprocessing vessel of claim 1, wherein said first and second surfaces comprise one or more features extending inward into a primary volume of said bioprocessing vessel and defining said optical path therebetween.

12. The disposable bioprocessing vessel of claim 1, wherein said disposable bioprocessing vessel is configured for a process selected from the group consisting of a storage stage, a growth stage, a product formation stage, a purification stage, and a product formulation stage.

13. The disposable bioprocessing vessel of claim 1, wherein said first and second surfaces comprise regions with step-variable distances therebetween, thereby providing a plurality of optical paths of different optical path lengths.

14. The disposable bioprocessing vessel of claim 1, wherein said first and second surfaces comprise a second polymer, wherein the second polymer is more optically transparent than said polymer to near-infrared electromagnetic radiation, wherein at least a portion of said second polymer is within said optical path.

15. The disposable bioprocessing vessel of claim 14, wherein said second polymer comprises fluorinated ethylene propylene (FEP).

16. The disposable bioprocessing vessel of claim 1, wherein said vessel further comprises a region comprising a second polymer and at least a portion of said region does not surround a fluid sample within the disposable bioprocessing vessel, wherein said second polymer provides optical absorption features to enable an optical wavelength reference.

17. The disposable bioprocessing vessel of claim 1, wherein at least portions of said first and second surfaces are at least partially compressible, and wherein a length of an optical path through the fluid sample is defined by compression of said first and second surfaces.

18. A disposable bioprocessing vessel with features for optically sampling a fluid within said bioprocessing vessel, said bioprocessing vessel comprising:

a top, a bottom, and at least one rigid sidewall, wherein the top, bottom, and rigid sidewall define an interior;

an optical probe integral with at least one of the top, bottom, or rigid sidewall and protruding into the interior of said disposable bioprocessing vessel, said integral optical probe comprising at least one optical waveguide;

wherein a portion of said integral optical probe is within the interior of said disposable bioprocessing vessel and provides optical communication between a fluid sample within said disposable bioprocessing vessel and an optical instrument whereby a near-infrared transmission or transflection measurement is provided.

19. The disposable bioprocessing vessel of claim 18, wherein the probe further comprises two optical elements positioned to provide an optical path length therebetween and within the interior of the bioprocessing vessel, whereby an optical transmission or transflection measurement through the fluid sample contained within the interior of said disposable bioprocessing vessel is provided.

20. The disposable bioprocessing vessel of claim 18, wherein said integral optical probe intersects two surfaces defining the interior of said disposable bioprocessing vessel, whereby near-infrared electromagnetic radiation is communicated through one of said surfaces, interacts with a fluid sample within said disposable bioprocessing vessel, and at least a portion of near-infrared electromagnetic radiation having interacted with said fluid sample is communicated through the other of said surfaces thereby providing an optical transmission measurement.

21. The disposable bioprocessing vessel of claim 18, wherein the length of optical path through said fluid is between 0.5 mm and 2.0 mm inclusively.

22. The disposable bioprocessing vessel of claim 18, wherein said disposable bioprocessing vessel has a working volume less than or equal to 0.25 liters.

23. A receiver assembly for receiving one or more disposable bioprocessing vessels and optically interrogating said one or more disposable bioprocessing vessels with near-infrared electromagnetic radiation, said receiver assembly comprising:

a base assembly comprising one or more stations each configured to receive a disposable bioprocessing vessel comprising components for near-infrared optical interrogation, each of said one or more stations comprising features for alignment of said disposable bioprocessing vessels within said stations such that said bioprocessing vessels may be installed in only one orientation; one or more optical assemblies for optical communication of near-infrared electromagnetic radiation between said disposable bioprocessing vessels positioned within said stations and an optical instrument;

wherein said optical assemblies for optical communication comprise one or both of an optical interface not integrated within said base assembly to provide optical communication with said disposable bioprocessing vessels or at least one optical assembly integrated within said base assembly.

24. The receiver assembly of claim 23, further comprising a mechanical translator configured to provide mechanical translation between said base assembly and said optical interface.

25. The receiver assembly of claim 23, wherein said receiver assembly is configured to receive a plurality of disposable bioprocessing vessels.

26. The receiver assembly of claim 23, wherein said stations are configured to receive disposable bioprocessing vessels, and wherein the disposable bioprocessing vessels comprise working volumes less than or equal to 0.25 liters.

27. The receiver assembly of claim 23, wherein said optical interface comprises one or more optical waveguides to communicate near-infrared electromagnetic radiation between an optical instrument and the disposable bioprocessing vessel.

28. The receiver assembly of claim 23, wherein said receiver assembly further comprises one or more optical sensors.

29. A method of determining the quantities of one or more substances in a fluid sample contained within a disposable bioprocessing vessel that is located in a receiver assembly, said method comprising the steps of:

selecting a disposable bioprocessing vessel;

communicating near-infrared electromagnetic radiation from an optical instrument to said disposable bioprocessing vessel;

collecting near-infrared electromagnetic radiation having interacted with a fluid content of said disposable bioprocessing vessel and communicating said electromagnetic radiation to said optical instrument for analysis;

determining the one or more quantities of substances in the fluid sample in said disposable bioprocessing vessel by utilizing the optical instrument to perform an optical transmission or transflection measurement.

30. The method of claim 29, wherein said optical instrument is configured to measure one or more of alcohols, sugars, lipids, organic acids, peptides, steroidal molecules, or proteins.

31. The method of claim 29, wherein said optical instrument is configured to measure one or more of cell density, cell viability, or turbidity.

32. The method of claim 29, wherein the step of determining the one or more quantities of substances is performed in a plurality of bioprocessing vessels.

33. The method of claim 29, wherein said method further comprises the step of communicating a second beam of near-infrared electromagnetic radiation through a portion of said disposable bioprocessing vessel and not interacting with a fluid sample, wherein the optical absorption features of said polymer provide an optical wavelength reference.