Patent Application
20180188180
This invention relates generally to Raman spectroscopy and bioreactors and, in particular, to a Raman probe assembly for single-use bioreactor vessels.
Single-use, disposable bioreactor vessels use a bag as opposed to a more expensive reusable culture vessel. The bag itself is typically made of a flexible plastic, but may be encased in a structure such as a rocker or a cuboid, or cylindrical steel support depending upon the experiment or reaction process under analysis. Commercial, single-use bioreactors have been available for over a decade and are now available from several manufacturers. Such bags commonly include one or more “ports” enabling the contents of the bag to be sampled and monitored.
Raman spectroscopy has become a powerful tool for use in conjunction with in situ process analysis. Sophisticated fiber-optic coupled Raman probes are now routinely used for process sampling in various industries, including bioreactor applications. It would be highly advantageous to couple a Raman probe to a bioreactor bag, through an available port, for example, but a tradeoff exists between the expense and complexity of the probe versus the bag assembly, which is designed to be disposable. Certainly the Raman probe itself cannot be disposable. Nonetheless, if certain components of the probe are not precisely aligned within the port, the required accuracy, repeatability, and reaction model transferability between reaction batches and probes will not be achieved.
There have been attempts to provide for disposable couplings to bioreactor vessel ports, but these attempts do not facilitate precise Raman sampling. U.S. Pat. No. 8,008,065 for example, discloses a port and sensor assembly adapted for use with an optical fiber-based phase fluorometric or Raman measurement system. A fiber or fiber bundle used as the excitation light source is anchored into a disposable insert shell through a ferrule or other suitable retention system. The diverging light from the fiber or fiber bundle is preferably collimated using a lens or lens system so that the collimated light is incident on a fluorescent dye spot. The fluorophore absorbs the excitation light and emits a fluorescent signal that impinges upon a collection system which is focused returned to a photo-diode through the same lens and fiber or fiber bundle. The fluorescent dye spot and fiber are mounted in a disposable shell that is inserted into a port affixed to the disposable bioreactor's lining.
While the system just described provides for a limited degree of fluorescence detection, it does not accommodate the exacting focusing requirements demanded of a state-of-the-art Raman sampling probe. Efficient and repeatable Raman sampling in a variably turbid sample medium, such as a bioreaction, requires the probe to be focused at a very short depth into the turbid sample from a disposable port window. Too deep a focus can negatively impact both the strength and the shape of the measured Raman spectrum.
This invention is directed to systems and methods for coupling a Raman probe to a port in a bioreactor vessel containing a reaction medium. The invention is applicable single-use bioreactor vessels in the form of flexible bags containing cell-culture and/or other biological media in liquid form. Such single-use bags are commonly provided with a hollow, tubular port adapted for attachment to various forms of instrumentation. In accordance with the instant invention, a Raman or other focusing probe head is coupled to the port for in-process monitoring.
A point of novelty resides in a unique combination of re-useable and disposable components to maintain precision while reducing costs. The solution made possible by the invention uses a fairly high-precision-thickness window sealed into a relatively inexpensive disposable barb that is inserted into the port of the reaction vessel. The barb is coupled to an optic component, which contains one or more expensive focusing lenses. To accurately locate the focus of the lens assembly at a precise, predetermined depth outside the window in the reaction medium, inventive end-optic hardware and assembly/alignment tooling are used.
The barb component has a distal end including an integral window with proximal and distal surfaces. The barb component is physically configured to be received by the port in a bioreactor vessel such that the distal surface of the window is exposed to the reaction medium. The system further includes an optic component configured to be received by the barb component. The optic component includes a proximal end adapted for coupling to a Raman probe head, and a distal end including a lens for focusing light to, and collecting light from, a sample focus in the reaction medium for analysis by a Raman analyzer coupled to the probe head. In preferred embodiments, the predetermined distance is on the order of 0.005+/−0.001″, though the invention is not limited in this regard.
Apparatus for setting the focus of the lens at the predetermined distance may include an adjustable spacer component disposed between the lens and the window. More particularly, the lens is retained within a lens mount axially moveable within the optic component and, once the predetermined distance is established, the proximal end of the spacer component is bonded to the lens mount with the distal end of the spacer in intimate contact with the proximal surface of the window. A spring in the hollow optic component may be used to bias the lens mount distally to ensure that the distal end of the spacer component maintains contact with the proximal surface of the window, thereby ensuring that the lens maintains a precise relationship to the window.
A separate fixture is preferably used as a focus alignment tool to set the lens at a precise position before placement of the optic component into the barb. The tool includes a simulation window to simulate the window in a barb component, a target with a known Raman spectral signature and a Raman probe head coupled to a Raman analyzer. One or more precision shims are used to position the target at the predetermined distance from the distal surface of the simulation window. With the distal end of the spacer component against the simulation window, the axial position of the lens in the lens mount is adjusted with respect to the spacer component to maximize the Raman spectral signature from the sample, whereupon the spacer component is bonded to the lens mount. Conveniently, the target may be a silicon wafer, which has a strong Raman peak that is maximized when positioned at the focal point of the probe.
Methods of coupling an optical sampling probe head to a port into a bioreactor vessel containing a reaction medium are also disclosed and described.
This invention enables a sophisticated Raman sampling probe to be used with disposable/single-use bioreactor vessels/bags by providing a trade-off in terms of those components that may be retained, and those components that are disposable. This is accomplished at low cost, and without a compromise in terms of sampling accuracy and effectiveness.
The preferred embodiments provide for a multi-part system including a disposable barb assembly with an integrated and sealed window operative to pass wavelengths of interest. The disposable assembly, which is received at one end by a conventional reactor bag port, connects at the other end to a Raman probe. A sanitary clamp between the barb assembly and the probe enables the expensive probe components to be re-used. To ensure that the focusing optic integrated into the disposable barb assembly has the required accuracy, the invention includes a focus assembly tool and associated method to simulate the production assembly to place the focus of the lens at the ideal depth in the sample region opposite the window.
The proximal end of end optic 104 is coupled to a probe head component 110, which, in turn couples to one or more different optical fiber assemblies 112, 113. Component 110, which may comprise the MR Probe available from Kaiser Optical Systems, Ann Arbor, Mich., includes filters, beam splitters and optics to receive laser excitation from one fiber and deliver a Raman signal to a spectrograph by way of a collection fiber. As described in issued U.S. Pat. No. 6,907,149, the entire content of which is incorporated herein by reference, the MR Probe head generates collimated, coaxial images of the excitation and collection fibers for focusing onto or into a variety of sample scenarios using a variety of different end optics. Further details of the MR probe may be found at http://www.kosi.com/na_en/products/raman-spectroscopy/raman-probes-sampling/mr-probe-head.php with the understanding that this invention is not limited in terms of the probe head used. Indeed, the invention is readily applicable to any focusing optical probe, including fluorescence probes, such that as used herein “Raman” should be taken to include these other sampling modalities.
Port 202 is in fact integral to the disposable bioreactor bag, such that the window of the inserted barb is immersed into the liquid contents of the bag, thereby enabling Raman monitoring of the liquid. When the reaction is complete, the end optic component 104 is un-clamped and removed from the barb 102. The entire bag/port/barb pieces may then be safely disposed of—such pieces cannot be reused as they have been in contact with, and contaminated by, the bioreaction materials. The optic component 104, however, can be re-used, as it has not been similarly contaminated.
An important point of novelty of this invention is to relegate the precision and expense in the re-useable components, while minimizing the cost of the disposable components. Toward this end, a challenge is setting the focus of the assembled system to be at a very precise depth outside the window in the reaction medium. A representative focus depth in typical applications might be 0.005″+/−0.001″. Maintaining this precision is necessary to maximize sensitivity and consistency of results from batch to batch and from probe to probe. Even if all of the focal depth tolerance were accommodated by the window thickness, this would still require more precise and more costly window thickness specifications. This is undesirable since, by definition, the window must be disposable, as it comes in contact with the reaction.
The solution made possible by this invention is to use a fairly high-precision-thickness window sealed into a fairly simple disposable barb. While the barb may be machined from stainless steel, for example, in the preferred embodiments the barb is constructed from an injection-moldable material using a process that is certified for bio applications. A remaining challenge, however, is to interface the barb to the optic component 104, which contains the focusing lens, in such a way as to accurately put the focus at the desired depth outside the window. This is accomplished using inventive design of the end optic hardware, disposable barb, and assembly/alignment tooling.
The focus-setting tool is intended to optically simulate the end-use Raman sampling environment as used with a disposable bioreactor bag.
A Raman probe head (not shown), is pre-aligned to collimate and combine the Raman excitation and collection fiber paths (i.e., a standard MR Raman probe head available from Kaiser Optical Systems, Inc.) is located at the same nominal distance from the window as in the final product installation. This distance is not critical, as it is in a nominally collimated space.
During setup using the tool, the probe head is connected to a Raman analyzer, the focus lens is bonded into its mount 302, and the spacer component 306 is loaded against the window at reference 401. The axial position of the mounted focus lens is adjusted with respect to spacer 306 to a position that maximizes the Raman signal of the silicon wafer. The focus lens mount 302 and spacer 306 are then bonded in that position with a UV-cure adhesive.
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