Method of evaluating geometric properties in measurement cells in an analytical ultracentrifugation apparatus

Abstract

A method of evaluating geometric properties in measurement cells in an analytical ultracentrifugation (AUC) apparatus may include applying light to a plurality of measurement cells arranged in the AUC apparatus, while centrifuging the plurality of measurement cells. The light may have a wavelength ranging from 190 nm to 800 nm, preferably from 320 nm to 800 nm, and more preferably 500 nm. The method may further include detecting a radial position of a top wall and / or a bottom wall of one or more channels within one or more of the plurality of measurement cells, and then generating data comprising data pertaining to the radial position of the top wall and / or the bottom wall of the one or more channels within the plurality of measurement cell(s). Based on the data or readout, geometric properties of the measurement cells can be determined, including whether the measurement cells are aligned or misaligned.

Description

[0001] METHOD OF EVALUATING GEOMETRIC PROPERTIES IN MEASUREMENT CELLS IN AN ANALYTICAL ULTRACENTRIFUGATION APPARATUS

[0002] FIELD OF THE DISCLOSURE

[0003] [1] Various exemplary embodiments relate to a method of evaluating the geometric properties of one or more channels within one or more of a plurality of measurement cells in an analytical ultracentrifugation (AUC) apparatus, and more specifically relate to evaluating the alignment of measurement cells in an AUC apparatus, by applying light to one or more channels within one or more of a plurality of measurement cells in the AUC apparatus to detect certain radial positions in the one or more channels within one or more of the plurality of measurement cells to determine whether one or more of the plurality of measurement cells are aligned or misaligned.

[0004] BACKGROUND

[0005] [2] In an analytical centrifuge, an optical detection system is used to determine and monitor a sedimentation rate or profile of a sample to determine various metrics for the sample, such as, but not limited to, size, weight, density, shape, and heterogeneity of the analytes in solution. The sample is typically analyzed using light by the light source and optical detectors within the AUC apparatus, i.e. the light source and optical detectors are typically integrated into the AUC apparatus. The wavelength of the light used within an AUC apparatus is within the UV-VIS absorbance range.

[0006] [3] Measurement cells are required within an AUC apparatus to allow light to pass through the analyte during the run time of the AUC apparatus. Each measurement cell includes a housing, two windows, and a centerpiece with one or more channels (also known by the skilled person as sectors) and filling holes for the channels, closed with a screw plug in the housing. The measurement cells are placed in the rotor slots of a rotor of the AUC.

[0007] [4] Sedimentation-velocity analytical ultracentrifugation (SV-AUC) is a biophysical method to characterize analytes in solution. Analytes will sediment when subjected to the gravitational forces applied by the centrifuge based on size, weight, density, shape, and heterogeneity of the analytes in solution. During SV-AUC, analytes sediment rapidly due to the high speeds employed. In all cases, the applied centrifugal force causes movement of the solutes / species to the outside rotor position, more particularly to the bottom of the cell. The speed of this movement is determined and used to obtain the sedimentation coefficient, which is the primary output parameter of the SV-AUC experiment. Further data processing and fitting of statistical models yields the content of the species in solution, as well as the diffusion coefficients and molecular weight (MW) of the components with good accuracy.

[0008] [5] Depending on the selected speed of rotation, SV-AUC can be applied for the analysis of solutes, ranging from small peptides to viruses, and aggregates thereof. Typical pharmaceutical applications for SV-AUC are the quantification of aggregates in protein formulations, and the characterization of virus preparations, where, e.g., the presence of empty and filled viral particles can be evaluated. SV-AUC is furthermore applied to determine the aggregation properties or different assembly states of nanoparticles such as lipid-nanoparticles containing nucleic acids.

[0009] [6] Accuracy depends on the ability of the analytes to move freely in the solution column, which is guaranteed by the sector-shaped cell geometry and alignment of the cell with the direction of the centrifugal force. When the cell’s central symmetry line goes through the center of the rotor, the analytes will not collide with the cell walls and will move towards the bottom of the cell without any disturbances to the sedimentation process. Under these conditions, artificial concentration gradients can also be produced. However, recent studies found that even one degree of misalignment may impede the sedimentation process and affect the results, for example leading to the detection of an apparently higher quantity of protein aggregates than in perfectly aligned cells. This observation came along with the development of different types of alignment tools.

[0010] [7] The correct alignment of the measurement cells within each rotor slot is key for the precision and accuracy of SV-AUC analysis. Whereas alignment can be done by aligning the calibration marks on the cell and the rotor housing based on naked eye observation, alignment tools further contribute to correct alignment. Mechanical alignment tools have been developed as separate instruments, as well as tools which evaluate the alignment by optical means. A simple mechanical tool is available that can be used to align the cell in the rotor based on the calibration marks on the cell and the rotor. Optical alignment tools have been developed where cell alignment is achieved by adjusting the position of the centerpiece’s middle wall to optically match the calibration line of the alignment tool, independent of the calibration marks on the cell and the rotor.

[0011] [8] A need exists (i) to confirm the alignment of the measurement cells within the rotor of the centrifuge, (ii) to confirm the comparability and / or quality of different measurement cells, (iii) to verify the consistency of rotor positions, (iv) to establish acceptance limits for the alignment, and (v) combinations thereof. These different aspects are useful in assessing the validity of the obtained SV-AUC results. For example, it is particularly desirable to be able to generate reliable data that can be documented to elevate the setup of a Good Manufacturing Practice (GMP) compliant analysis approach for analytical ultracentrifugation.

[0012] [9] Disadvantages of the prior art alignment tools are, for example, the need to obtain an additional alignment tool, an overlooking of internal misalignment (e.g., if the calibration mark on the rotor housing is misaligned), and a lack of documentation of the alignment. Although alignment with optical alignment tools is independent of internal misalignment and works well with empty cells, such tools may have the disadvantage that the alignment with the tool’s calibration line becomes difficult when cells are filled with a sample due to reduced visibility of the calibration line.

[0013]

[0010] An unmet need therefore exists to determine the measurement cell’s alignment without requiring an additional alignment tool. Also, the method could allow misaligned cells to be adjusted before starting the actual experiment (to improve data quality), and / or to document the alignment status of cells during the experiments (to improve data integrity). A further unmet need exists to compare the rotor positions and the differences between measurement cells, so that a full picture of the system setup can be obtained and documented with respect to cell alignment.

[0014] SUMMARY OF THE DISCLOSURE

[0015]

[0011] In various embodiments, geometric properties of one or more channels within one or more of a plurality of measurement cells in an AUC apparatus may be evaluated according to the method disclosed herein. The method may include applying light to the one or more channels, while operating the AUC apparatus. The method may include detecting an intensity of light transmitted by the one or more channels and then generating data pertaining to the intensity of light transmitted by the one or more channels. The method may include determining when one or more transitions of the intensity of transmitted light occurred based on the data. The method may include determining one or more specific distances in the one or more channels to the center of the rotor of the AUC apparatus (herein also referred to as “radial positions” or “radius”) at the one or more positions when the one or more transitions of the intensity of transmitted light occurred. The method may further include determining one or more geometric properties of the one or more channels based on the one or more specific distances to the center of the rotor of the AUC apparatus.

[0016]

[0012] In various embodiments, the alignment of measurement cells in an AUC apparatus may be evaluated according to the method disclosed herein. The method may include applying light to one or more channels within one or more of a plurality of measurement cells arranged in the AUC apparatus, while centrifuging the measurement cells. The light may have a wavelength ranging from 190 nm to 800 nm, preferably ranging from 320 nm to 800 nm, and more preferably of 500 nm. The method may further include detecting a radial position of a wall facing the inside (i.e. center) of the rotor (herein also referred to as “top wall”) and a wall facing the outside of the rotor (herein also referred to as “bottom wall”) of one or more channels within one or more measurement cells and then generating data pertaining to the radial position of the top wall and the bottom wall of the one or more channels within the measurement cell(s). Lastly, the method may include determining whether one or more of the plurality of measurement cells is aligned or misaligned based on the data.

[0017]

[0013] In various embodiments, a computer-readable medium may have a program stored thereon where said program includes instructions that, when executed by an AUC apparatus, may perform the method(s) described above.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019]

[0014] In the drawings:

[0015] FIGs. 1A to 1C depict various alignments of two channels within one measurement cell of the AUC apparatus, with FIG 1A showing a correctly aligned cell, and FIGs IB and 1C showing misaligned cells;

[0020]

[0016] FIGs. 2A and 2B depict a graph (full line: sample channel, dotted line: reference channel) corresponding to scans of empty measurement cells that were scanned by the UV / VlS-optical system of the AUC apparatus at a wavelength of light of 280 nm and 500 nm, respectively, with the inlets showing the scans zoomed into the top wall region and bottom wall region of the cell, and with the full and dotted lines showing the scan of the sample and reference channel, respectively;

[0021]

[0017] FIGs. 3A and 3B depict a graph (full line: sample channel, dotted line: reference channel) corresponding to scans of measurement cells with an antibody solution that were scanned by the UV / VlS-optical system of the AUC apparatus at a wavelength of light of 280 nm and 500 nm, respectively, with the inlets showing the scans zoomed into the top wall region and bottom wall region of the cell

[0022]

[0018] FIG. 4 depicts the radial distance shift at the bottom walls of seven measurement cells in different misalignment experiments with empty measurement cells;

[0023]

[0019] FIGs. 5A and 5B depict graphs (full line: sample channel, dotted line: reference channel) corresponding to scans of the top and bottom walls, respectively, of the channels in the measurement cell that is misaligned as shown in the image of FIG. 5C, and of which the radial distance shift at the bottom wall is shown in the filled bar of misalignment experiment 1 in FIG. 4;

[0024]

[0020] FIGs. 6A and 6B depict graphs (full line: sample channel, dotted line: reference channel) corresponding to scans at the top and bottom walls, respectively, of the channels in the measurement cell that is misaligned as shown in the image of FIG. 6C, and of which the radial distance shift at the bottom wall is shown in the filled bar of misalignment experiment 7 in FIG. 4;

[0025]

[0021] FIG. 7 depicts the correlation between the misalignment angle derived from images and the corresponding radial distance shift in pm of the top walls of the reference and sample channel (“Inside”) and of the bottom walls of the reference and sample channel channels (“Outside”), determined using the method described herein with empty measurement cells;

[0026]

[0022] FIGs. 8A to 8D are graphs depicting the results of an evaluation of the accuracy of measurement cell geometric properties and assembly, and of the accuracy of the rotor calibration marks when using the method described herein with empty measurement cells;

[0027]

[0023] FIGs. 9A to 9D are graphs depicting the results of an evaluation of the accuracy of measurement cell geometric properties and assembly, and of the accuracy of the rotor calibration marks when using the method described herein with measurement cells containing an antibody solution;

[0028]

[0024] FIGs. 10A to 10D are graphs depicting the results of an evaluation of the accuracy of measurement cell geometric properties and assembly, and of the accuracy of the rotor calibration marks when using the method described herein after using a Spin Analytical optical alignment tool for adjusting alignment;

[0029]

[0025] FIGs. 11A to 11D are graphs that depict the results of an evaluation of the geometric cell properties and the alignment profile for a different rotor when using the method described herein;

[0030]

[0026] FIGs. 12A and 12B are graphs that depict the results of an evaluation of varying speeds when using the method described herein;

[0031]

[0027] FIG. 13 shows schematic drawings and photos of measurement cells in an AUC apparatus illustrating the position of the cell channels and the alignment or misalignment of the calibration marks on the measurement cell and the rotor housing in measurements 1-5 of the case study with a monoclonal antibody described in Example 7; and

[0032]

[0028] FIGs. 14A to 14F are graphs that depict the results of the case study with monoclonal antibody described in Example 7. DETAILED DESCRIPTION

[0033]

[0029] In the following detailed description, reference is made to the accompanying drawings which form part thereof and in which specific embodiments are shown for illustrative purposes in which the invention may be practiced. Since components of embodiments may be positioned in a number of different orientations, the directional terminology is for illustrative purposes and is not limiting in any way. It is to be understood that other embodiments may be used and structural or logical changes may be made without departing from the scope of protection of the present invention. It is to be understood that the features of the various exemplary embodiments described herein may be combined with each other, unless specifically stated otherwise. The following detailed description is therefore not to be understood in a limiting sense, and the scope of protection of the present invention is defined by the appended claims.

[0034]

[0030] The inventors have surprisingly discovered a method of applying the UV / VIS optical detection system of an AUC apparatus to one or more of a plurality of measurement cells to assess one or more geometric properties of one or more channels within the one or more measurement cells, and / or of applying the UV / VIS optical detection system of an AUC apparatus to one or more of a plurality of measurement cells to determine an alignment of the measurement cells within the AUC apparatus. Such a UV / VIS optical detection system may be the UV / VIS optical detection system integrated into the AUC apparatus in various embodiments.

[0035]

[0031] The UV / VIS optical detection system may be used to confirm the alignment of the measurement cells within the AUC apparatus, to confirm the comparability and / or quality of different measurement cells, to verify the consistency of rotor positions, to establish acceptance limits for the alignment, and combinations thereof. The method may be performed based on a UV / VIS optical scanning procedure, while running the AUC experiment. The method may, mutatis mutandis, be used to evaluate the alignment of the calibration centerpiece. The approach disclosed herein would improve the centrifuge workflow to generate reliable data and, since the method also allows documentation of the geometric properties of the measurement cells, would elevate the setup of a GMP- compliant analysis approach for analytical centrifugation.

[0032] The terms, “cells” and “measurement cells” are used herein interchangeably. A measurement cell may be a single measurement cell that fits into a single slot of a rotor. Each measurement cell includes a housing, two windows, and a centerpiece with one or more channels (also known by the skilled person as sectors) and filling holes for the channels, closed with a screw plug in the housing. A measurement cell may have two channels therein to enable a reference to be alongside the sample within a single slot of the rotor.

[0036]

[0033] The inventors have further found that a difference between certain radial equivalent positions in two or more channels of a measurement cell or a deviation from a known aligned position of one or more of the channels of a measurement cell (herein referred to as a “radial distance shift”), such as, for example, the radial distance shift of the top wall positions or the bottom wall positions of the reference and sample channel, can be measured during scanning of the measurement cells in an AUC apparatus, where the certain equivalent positions may be defined on the basis of a transition of the intensity of transmitted light during the scan. Such radial distance shift can assist in determining whether the measurement cells are aligned or misaligned without requiring a mechanical or optical alignment tool. The data generated from the UV / VlS-scans, such as in a readout, from the AUC apparatus can be used to re-adjust misaligned cells before starting the actual experiment to improve data quality and / or allow documentation of correct alignment after the experiment has been completed, thereby improving data integrity. Further, this approach can be used to compare the rotor positions and geometric differences between measurement cells to obtain a full picture of the system setup with respect to measurement cell alignment. Also, the method can be used as a quality control tool in the manufacturing of measurement cells. In general, the alignment method can be included in the workflow prior to starting an AUC experiment. This allows the skilled person to, for example, confirm that a certain measurement cell was properly aligned, or to readjust misaligned cells before restarting the run with correctly aligned measurement cells. Various uses of the method disclosed described herein can be summarized as described in Table 1:

[0034] Table 1.

[0037]

[0035] In embodiments, a method of evaluating the geometric properties of one or more channels within one or more of a plurality of measurement cells in an AUC apparatus has been discovered. The method may include applying light to the one or more channels, while operating the AUC apparatus. The method may include detecting an intensity of light transmitted by the one or more channels and then generating data pertaining to the intensity of light transmitted by the one or more channels. The method may include determining when one or more transitions of the intensity of transmitted light occurred based on the data. The method may include determining one or more specific distances in the one or more channels to the center of the rotor of the AUC apparatus at the one or more positions when the one or more transitions of the intensity of transmitted light occurred. The method may include determining one or more geometric properties of the one or more channels based on the one or more specific distances to the center of the rotor of the analytical ultracentrifugation apparatus.

[0038]

[0036] In some embodiments, the one or more measurement cells comprise a centerpiece, wherein the one or more channels are located within the centerpiece. In embodiments, the method is carried out with standard measurement cells. In other words, the measurement cells do not include an external component introduced or inserted into the measurement cell beyond the usual AUC measurement cell components.

[0037] The method may include storing the one or more geometric properties in a computer-readable format. The one or more positions when the one or more transitions of the intensity of transmitted light occur may correspond to the top wall of the one or more channels and / or the bottom wall of the one or more channels.

[0039]

[0038] The one or more geometric properties may be or may include the orientation of the one or more channels towards the center of the rotor within one or more of the plurality of measurement cells. Specific examples of geometric properties may be or include the alignment of the channels, the accuracy of rotor calibration marks, the accuracy of a cell assembly, the cell length, the counterbalance alignment, and combinations thereof. Being able to evaluate the accuracy of rotor calibration marks is relevant since it was found that often reassembled measurement cells may show misaligned marks on the bottom, possibly through wear and tear, and that manufacturing tolerance limits for the calibration marks on the rotors may in certain cases lead to consistent misalignment of the cells, even when the marks on the rotor and the cell housing match exactly. Being able to evaluate the alignment status of the calibration counterbalance is relevant for confirming accuracy of the AUC results, and the method disclosed herein can, mutatis mutandis, be applied to evaluate the alignment of the counterbalance centerpiece, when scanning the sample and reference slit-assembly of the calibration centerpiece in analogy to scanning the sample and reference channel of a measurement cell.

[0040]

[0039] The method may further include determining that a misalignment of the one or more channels has occurred when the data comprises at least a portion of data pertaining to a radial distance shift of the one or more channels. The method may further include stopping the AUC apparatus based on the determination that the one or more channels within one or more of the plurality of measurement cells are misaligned, and adjusting the orientation of the measurement cells such that the measurement cells are aligned before the evaluation of the sedimentation properties of the samples. For example, misaligned measurement cells may be aligned (ij visually to a calibration mark, (ii) by applying an alignment tool to the misaligned measurement cells, (hi) based on an orientation previously determined by the method disclosed herein or a radial distance shift obtained by the method disclosed herein, or both, or (iv) combinations thereof.

[0040] The light may have a wavelength ranging from 190 to 800 nm, preferably from 320 nm to 800 nm, or more preferably of 500 nm. The applied light may have a wavelength that allows for sample-filled channels (e.g. protein-filled samples) to be compared to the buffer- filled channel within the same measurement cell.

[0041]

[0041] In embodiments, a method of aligning measurement cells in an AUC apparatus has been discovered that may allow for determining whether one or more of a plurality of measurement cells is aligned or misaligned. The method may include applying light to one or more of a plurality of measurement cells arranged in the AUC apparatus, while centrifuging the measurement cells. The light may have a wavelength ranging from 190 nm to 800 nm, preferably ranging from 320 nm to 800 nm, and more preferably of 500 nm. The method may further include detecting a radial position of a top wall and a bottom wall of one or more channels within one or more of the plurality of measurement cells, and then generating data, e.g. a readout, that includes data pertaining to at least the radial position of the top wall and the bottom wall of the channel(s) within the measurement cell(s). The method may further include determining whether one or more of the plurality of measurement cells are aligned or misaligned based on the data or readout.

[0042]

[0042] In various embodiments, the method may further include aligning the one or more of plurality of measurement cells visually to a calibration mark, applying an alignment tool to the one or more of plurality of measurement cells, or both, prior to the step of applying light according to the method. In addition, the method may also include adjusting the alignment of measurement cells when one or more of the plurality of measurement cells is determined to be misaligned. The method described herein is particularly useful in combination with an optical alignment tool, to consistently enable high quality data.

[0043]

[0043] The method can be used to determine the alignment status of AUC measurement cells in a rotor under the conditions of an actively running rotor. The centrifuging of the measurement cells according to various embodiments of the method may occur at a speed ranging from 2,000 rpm to 60,000 rpm, preferably from 2,000 rpm to 42,000 rpm, more preferably from 2,000 to 4,000 rpm, or most preferably of 3,000 rpm. In various embodiments, the AUC apparatus is operated at a speed which does not change the sedimentation properties of the sample in the one or more channels, such as at a speed from 2,000 rpm to 42,000 rpm, more preferably from 2,000 to 4,000 rpm, or most preferably of 3,000 rpm. The speed at which sedimentation occurs or does not occur is largely dependent on the samples that are being measured, and the skilled person would understand which speeds would not change the sedimentation properties of a given sample when operating the method. After determining the geometric properties, the method may further include evaluating the sedimentation properties of the sample in the one or more channels. Moreover, the method described herein also works for typical runs of a standard experiment at high speed where the very first scan at 500 nm can be used for the evaluation of the geometric properties, which allows, for example, to confirm and document a correct alignment after the experiment. It is noted that the results of the very first scan is routinely discarded from an AUC sample analysis according to the present standard measurement protocols. However, this scan can, as shown herein, be used for an alignment check at 500 nm without loss in data quality of the sample analysis. Although the latter mode would not allow the skilled person to stop and correct for any misaligned measurement cells, the obtained information from the readout would still allow the skilled person to document the alignment status of the measurement cells. Consequently, the method described herein improves data quality and data integrity, which are both particularly important in regulated QC and / or GMP settings.

[0044]

[0044] In various embodiments, the data or a readout of the data may include the radial position of the top wall(s) of the one or more channels and the radial position of the bottom wall(s) of the one or more channels. The evaluation of the alignment status may result in data or a readout including data pertaining to a radial distance shift that ranges from -800 to 800 microns, such as from -200 to 200 microns. The radial distance shift may, for example, be obtained by calculating the difference between the radial positions of the equivalent positions of the reference channel and sample channel in a measurement cell. A radial distance shift of 0 microns would correspond with perfect alignment. When using the method for evaluating the alignment status of the counterbalance centerpiece, the geometry of the centerpiece is such that perfect alignment is achieved when the radial distance between the top positions of the sample and reference slit on the one hand and the bottom positions of these slits on the other hand is the same.

[0045]

[0045] The method may further include obtaining one or more images for one or more of the plurality of measurement cells where each image includes a rotor calibration mark and a mark on the cell housing for each measurement cell of the plurality of measurement cells. The alignment status may be further confirmed or determined when one or more of the images comprises a misalignment angle ranging from -15 ° to 15 °, such as from -3 ° to 3 °, with an angle of 0 ° corresponding with perfect alignment. A misalignment angle is defined herein to mean the angle between a line connecting both calibration marks on the rotor and a line connecting both calibration marks on the cell housing.

[0046]

[0046] In various embodiments, the measurements may be taken when the measurement cells are empty, or when the measurement cells include a solution arranged therein. If a solution is arranged therein, the solution may include an antibody, a peptide, a protein, a virus, a nanoparticle, a lipid-containing complex, or combinations thereof.

[0047]

[0047] In various embodiments, a computer-readable medium may have a program stored thereon that includes instructions that, when executed by an AUC apparatus, perform the method described herein. An AUC apparatus may include such a computer-readable medium.

[0048] EXAMPLES

[0049]

[0048] An Optima AUC analytical ultracentrifuge (Beckman-Coulter), featuring an 8 hole An-50 Ti analytical rotor was used with 12-mm epon-charcoal double sector centerpiece AUC cells, i.e. AUC measurement cells that have two channels. The measurement cells were either left empty (unfilled) or were filled with 340 pl of a formulation containing an antibody. Each channel was scanned twice. The measurement cells were aligned either by naked eye based on the calibration marks on the cells and the rotor or by using the Spin Analytical optical alignment tool (Nanolytics Instruments GmbH).

[0050] Example 1: Determination of the alignment status at different wavelengths with empty and filled cells

[0051]

[0049] According to the method described herein, the alignment status of the measurement cells was experimentally evaluated by using the UV / V1S- optical system of the Optima AUC. In this example, the AUC’s UV / V1S detector is used to assess the radial positions of the top and bottom walls of the two channels within the measurement cell. For this purpose, empty measurement cells as well as measurement cells filled with an antibody solution were scanned at 280 nm or 500 nm, while the Optima AUC was operated at a centrifugation speed of 3,000 rpm. The radial positions of the top wall of the cell channels (facing inside towards the rotor center) and bottom wall of the cell channels (facing outside away from the rotor center) were obtained from the scans. The radial positions of the top and bottom walls correspond with a transition of the intensity of transmitted light. To obtain the radial distance shift between the top or bottom walls of the two channels, the difference between the radial positions of the equivalent positions of the left and the right channel (also known as the reference channel and sample channel) is calculated.

[0052]

[0050] The scans are shown in FIGs. 2 and 3. For the empty measurement cells, the intensity in the two channels of each measurement cell were indistinguishable (FIGs. 2A and 2B). For the measurement cells filled with an antibody solution, the intensity transitions at the radial positions between 6.1 and 6.3 cm in FIGs 3A and 3B arise from the solutions in the channels. The 280 nm scan of the sample channel is influenced by the antibody absorbance (FIG. 3A), but the scan at 500 nm is only minimally influenced by the presence of a sample solution (FIG. 3B). Thus, the 500 nm scan can be used to compare the channels with better precision, especially if any of the measurement cells are filled with a solution.

[0053] Example 2: Determination of the correlation between the alignment assay and image evaluation of the alignment

[0054]

[0051] Empty measurement cells were loaded into the rotor positions. The rotor was placed on a stative, and a camera was used to document the alignment marks on the bottom of the rotor. The image was evaluated with the image processing software Image] to define the angle of misalignment based on the angle between a line connecting both calibration marks on the rotor and a line connecting both calibration marks on the cell housing.

[0055]

[0052] For this example, seven two-channel epoxy AUC measurement cells were used. The seven measurement cells were aligned and misaligned within the rotor. The values of the radial distance shifts obtained for the misalignment in pm with the method described in Example 1 (with scanning at 500 nm and a centrifugation speed of 3,000 rpm) were then plotted for each of the (mis-)aligned cells in the different misalignment experiments (FIG 4). Scanning at 500 nm detected clear differences in the bottom positions (FIGs. 5B and 6B) with also a substantial shift between the scans at the top position (FIGs. 5A and 6A).

[0053] Next, the angle of misalignment was determined from images taken for each of the measurement cells (as for example shown in FIGs. 5C and 6C) in such a way that the rotor calibration marks and the marks on the cell housings can be seen and evaluated with the Image] software as described above. This misalignment angle was plotted against the values of the radial distance shifts obtained for the misalignment in pm (FIG. 7). A clear correlation between the misalignment angle and the determined radial distance shift of the top walls (“inside”-values) and bottom walls (“outside”-values) of the channels scanned at 500 nm is observed (FIG. 7) with the correlation being better for the bottom position than for the top position, noting the deviation of the correlation for the top positions in case of larger positive misalignment angles.

[0056] Example 3: Systematic rotation of measurement cells to different rotor slots to evaluate cell- and rotor-specific geometric properties

[0057]

[0054] Without the use of the method described herein, the operator must rely on the correctness of the calibration marks at the rotor backside and the correctness of the assembly status of the measurement cells for the alignment quality of the measurement cells in the AUC apparatus. During disassembly and reassembly of the measurement cells, it may be possible that a correct and fully aligned assembly is not achieved, such that the centerpiece is slightly twisted against the alignment axis of the cell housing. This then results in a systematically inaccurate alignment when aligning the calibration marks at the cell housing and the rotor.

[0058]

[0055] To distinguish rotor-based misalignment from measurement cell-based misalignment, seven empty AUC-cells were aligned by using the calibration marks in a Ti- 50 rotor (S / N 14U707). The top and bottom positions of the cells were evaluated as described in Example 1, with scanning at 500 nm and a centrifugation speed of 3,000 rpm. Subsequently, the cells were systematically rotated by moving them to the next rotor slot position until all slot positions were analyzed twice. This dataset was then evaluated to see whether systematic differences were obtained. Two geometric properties could be derived from these alignment evaluations. Firstly, the channel length delta, which can be defined from the difference between the radial distance shift of the channel top walls and the radial distance shift of the channel bottom walls, was determined using equation Eq. 3 below. The channel length delta is considered as a cell specific property originating from channel geometries. This channel length delta may reveal slight differences in channel length for the sample and reference channel, which is independent of the alignment status. The obtained channel length delta results are organized in FIG. 8A per cell housing, and in FIG. 8B per rotor slot. Secondly, the average misalignment was calculated, using the radial distance shifts of channel top walls and bottom walls, as shown in equation Eq. 4 below. This average misalignment is a measure of the misalignment of the cell in the rotor. The average misalignment results are organized in FIG. 8C per cell housing, and in FIG. 8D per rotor slot.

[0059]

[0056] For both parameters, either cell specific deviations or rotor slot position specific deviations were found. For the channel length delta, one particular measurement cell (Cell 2) of all 14 measurements gave a negative value, while the other six measurement cells gave a slightly positive value (FIG. 8A]. This parameter was independent of the slot position in the rotor (FIG. 8B]. When considering the average misalignment, a positive misalignment value was obtained for the measurement cells 1 to 4, whereas a negative misalignment value was obtained for the measurement cells 5 to 7 (FIG. 8C].

[0060]

[0057] Interestingly, a clear slot specific behavior was also recorded, so that each measurement cell gave an average misalignment value that was roughly 100 pm higher in the slot position 7 ofthe used rotor (S / N 14U707] (FIG. 8D]. Thus, the alignment status was different in slot position 7 compared to the other slot positions, which implied that the calibration marks on the rotor backside are possibly slightly off in slot position 7 of this rotor compared to the other slot positions. These results confirm that it is possible to use the disclosed method and the here described procedure (investigating seven cells in all seven rotor positions) to evaluate the accuracy of each cell assembly and the accuracy of the rotor calibration marks. Thus, the disclosed method allows to perform a geometry and quality assessment of the used cells and rotor, as well as documentation thereof, which is valuable for quality control (QC] and / or GMP applications of AUC measurements.

[0061]

[0058] The same procedure was then used to evaluate measurement cells filled with an antibody solution. Fourteen datasets of manually aligned measurement cells with each measurement cell in each rotor position were obtained as described above for the empty cells. Results are shown in FIG. 9. The alignment properties of the rotor and the used cells did not change because of the antibody solution therein, and the analysis resulted in similar patterns as before with the empty cells (see FIG. 8). The channel length delta results show that the measurement cells are characterized by specific geometric channel differences independent of the slot positions (FIGs. 9A and 9B) as was also derived for the empty cells (FIGs 8A and 8B). Also, similar results as obtained with empty cells were obtained when evaluating the alignment status of the cells with cells filled with an antibody solution (FIG. 9C). Furthermore, a similar rotor slot dependency of the alignment results with respect to slot position 7 of the rotor 14U707 was obtained (FIG. 9D). Thus, the features observed for empty cells can be observed in a highly similar manner if cells filled with a sample and reference solution are scanned at 500 nm.

[0062] Example 4: Evaluation of the Spin Analytical optical alignment tool to minimize the extent of cell- and rotor-specific alignment differences

[0063]

[0059] In a further experiment, the method was used to evaluate the effect of using an alignment tool to minimize the extent of cell- and rotor-specific alignment differences. The results obtained from alignment for the measurement cells by using the calibration marks in Example 3 were compared with the results obtained when using the Spin Analytical optical alignment tool. This tool supports alignment of the cells based on the positioning of the centerpiece’s middle wall and not based on the calibration marks on the cell and rotor backside. More particularly, the tool is used to manually adjust the cell alignment until a best possible match between the centerpiece’s middle wall with the calibration line of the alignment tool was reached. This match was clearly visible when empty cells were used, but when the measurement cells were filled with a solution, refractive artefacts from the solution made the match difficult to see. Therefore, this experiment was only performed with empty cells.

[0060] The measurements and result analysis was performed as described in Example 3. Results are shown in FIG. 10. It was found that aligning with the help of the alignment tool does not change the measurement cell specific properties of the aligned cellular geometry (FIGs. 10A and 10B). However, when using the Spin Analytical optical alignment tool, the results of the different measurement cells exhibited greater similarity to each other in comparison to alignment based on only the calibration marks (FIG. 8). Most of the average misalignment values ranged from +100 pm to -100 pm (FIG. 10C), indicating that no systematic differences were observed between the measurement cells when aligning them based on the orientation of the centerpiece’s middle wall separating the two channels. In addition, also the differences regarding the rotor positions are largely abolished by this procedure (FIG. 10D).

[0064]

[0061] The alignment evaluation with the 500 nm scanning procedure of the method described herein was able to show that measurement cell alignment with the Spin Analytical optical alignment tool is improved compared to alignment based on the calibration marks, which can be affected by rotor-specific inaccuracies and measurement cell specific offsets, as may arise during the assembly of the measurement cells.

[0065] Example 5: Evaluation of individual properties of different rotors

[0066]

[0062] The rotor used in the previous examples was exchanged to evaluate a different Ti- 50 rotor. A Ti-50 rotor from the production year 2023 (S / N 23U1196) was used, and 14 measurement sets were performed with the same 7 measurement cells in all 7 slot positions according to the method described in Example 1 with empty cells, a scanning wavelength of 500 nm and a centrifugation speed of 3,000 rpm. Regarding the channel length delta (FIGs. 11A and 11B), the measurement cells in this rotor behaved identical as when placed in the rotor used in the previous examples. Interestingly, the measurement cells in this rotor had similar alignment offsets as recorded for the S / N 14U707 rotor (compare FIG. 11C with FIG. 8C) but showed no dependency on the slot position (FIG. 11D).

[0067]

[0063] The latter implies that differences in the calibration marks on the rotor need to be considered, when evaluating the quality of Ti-50 rotors. These marks can deviate from the actual accurate position, which can however be routinely detected by using the scanning procedure of the method described herein while running the AUC apparatus. The variation of the degree of accuracy of marking the AUC rotors with calibration marks during production of the AUC rotors, although being within the tolerance associated with rotor and cell specific internal misalignments, is large enough to be detected by the method described herein. This detected variation suggests that the skilled person should apply the scanning procedure of the method described herein to determine whether the measurement cells are all properly aligned, advantageously in combination with using alignment tools that allow adjusting the cell channel positions independent from the calibration marks.

[0068] Example 6: Evaluation of effect of the centrifugation speed on the UV / VIS-based alignment test

[0069]

[0064] Further tests were performed to determine whether consistent results could be obtained with the method described herein using higher rotational speeds. For this, the speed of the centrifuge was increased in steps of 1,000 rpm from 3,000 rpm to 42,000 rpm. At each of the speed increments, the method was performed with a scan at 500 nm to evaluate the channel length delta and the alignment status of the empty measurement cells. The results for the seven cells were found to be very consistent at each speed, and the method was shown to work equally well at centrifugation speeds generally used in AUC experiments with the Ti-50 rotors and the epoxy centerpieces (FIGs. 12A and 12B).

[0070]

[0065] This example confirms that different modes of operation are possible: (i) perform an alignment scan at low rotational speed before the actual run to stop the AUC apparatus and re-adjust misaligned cells, or (ii) evaluate alignment from the data of a standard experiment at intended (high) speed by using the very first scan at 500 nm for the alignment evaluation.

[0071]

[0066] Example 7: Case study with AUC experiments with misaligned measurement cells using a monoclonal antibody (mAb)

[0072]

[0067] AUC measurements at 35,000 rpm were performed in the Optima AUC analytical ultracentrifuge (Beckman Coulter) and with AUC measurement cells containing two-sector epon-charcoal centerpieces described above. An aged measurement cell with known misalignment from a worn-out centerpiece (i.e. with measurement cell-based misalignment) and a control measurement cell with known correct centerpiece alignment were filled with 340 pl of a 0.4 mg / ml mAb formulation. The cells were either manually aligned by using the calibration marks of the cell housing and the rotor or aligned using the average misalignment obtained by the method of the present disclosure. Data analysis was performed with UltraScan-in-a-Box. Integration of the sedimentation coefficient distribution was performed with fixed bin sets to obtain contents and sedimentation coefficients for the main low-molecular weight species (being mAb monomer), a first class of high molecular weight species (HMWS1, being mAb dimer) and a second class of high molecular weight species (HMWS2, being mAb oligomers). Root-mean square deviation (RMSD) values were obtained to assess the agreement between the raw data and the simulation results.

[0073]

[0068] Measurement 1 was conducted with the control cell with correctly aligned centerpiece with the cell being manually aligned using the calibration marks (see FIG. 13(1)). Measurement 2 was conducted with the aged cell with known misalignment of the centerpiece with the cell being manually aligned using the calibration marks (see FIG. 13(2)). Measurement 3 was conducted with the aged cell with known misalignment of the centerpiece with the cell being aligned using the average misalignment obtained by the method of the present disclosure (see FIG. 13(3)). Measurement 4 was conducted with the control cell where the cell housing of the control cell was intentionally rotated clockwise (i.e. intentionally misaligned, see FIG. 13(4)) to achieve a misalignment corresponding with an average misalignment of more than +150 pm. Measurement 5 was conducted with the control cell where the cell housing of the control cell was intentionally rotated counterclockwise (i.e. intentionally misaligned, see FIG. 13(5)) to achieve a misalignment corresponding with an average misalignment of more than -150 pm.

[0074]

[0069] The overall average misalignment of the cells in measurements 1-5 was evaluated and calculated to illustrate the extent of misalignment (Table 2, FIG. 14A). The overall average misalignment in Table 2 and FIG. 14A is the mean of four (measurement 1) or two (measurements 2 to 5) runs. In addition, the main species (mAb monomer) content (FIG. 14B), the HMWS1 (mAb dimer) content (FIG. 14C), the HMWS2 (mAb oligomer) content (FIG. 14D), the sedimentation coefficient of the main species (FIG. 14E), and the RMSD value of the data analysis (FIG. 14F) are presented.

[0070] Table 2.

[0075] *data range based on two measurements

[0076]

[0071] Wear and tear of the assembly furrow of a centerpiece, which leads to a less stringent fit into the assembly base in the cell housing over time, can lead to the misalignment as observed for the aged cell. Alignment of the aged cell using the calibration marks and evaluation of the average misalignment using an embodiment of the method of the present disclosure identified an average misalignment of about 140 pm (measurement

[0077] 2; FIG 14A; Table 2). However, aligning the aged cell based on correcting the average misalignment as obtained by using an embodiment of the method of the present disclosure showed low average misalignment values (measurement 3; FIG 14A; Table 2), because the rotation of the centerpiece in the aged cell is compensated by a rotation of the cell housing as depicted in FIG. 13(3). Remarkably, the monomer content of mAb determined from the

[0078] AUC measurements was considerably lower in the case of the aged cell aligned by using the calibration marks. Consequently, the content of mAb dimers and mAb oligomers was higher compared to the measurements with the correctly aligned control cell. This indicates that even though the cell appears to be aligned when considering the calibration marks, the misalignment of the centerpiece in the cell housing has a considerable impact on the AUC results. The considerable difference of the mAb monomer content after aligning the aged cell by relying on the calibration marks or by relying on the radial distance shifts as determined by an embodiment of the method of the present disclosure demonstrates the improved accuracy of AUC results obtainable by applying the disclosed method since it allows reducing the impact of internally misaligned centerpieces.

[0079]

[0072] A similar trend was observed when the cell housing of the control cell was intentionally rotated as in measurements 4 and 5. A trend towards apparently lower monomer content (and higher aggregate content) was observed when the control cell was intentionally rotated (FIGs 14B to 14D). Interestingly, the sedimentation coefficients were not affected (FIG 14E), but RMSD values were slightly higher in the aged cell (FIG 14F), which implies an increase in systematic deviations during the fit of the AUC data.

[0080]

[0073] Various examples of the method are described below that relate to what is described above and shown in the figures.

[0081]

[0074] Example 1 refers to a method of evaluating the geometric properties of one or more channels within one or more of a plurality of measurement cells in an analytical ultracentrifugation (AUC) apparatus. The method includes applying light to the one or more channels, while operating the AUC apparatus. The method includes detecting an intensity of light transmitted by the one or more channels and then generating data pertaining to the intensity of light transmitted by the one or more channels. The method includes determining when one or more transitions of the intensity of transmitted light occurred based on the data. The method includes determining one or more specific distances in the one or more channels to the center of the rotor of the AUC apparatus at the one or more positions when the one or more transitions of the intensity of transmitted light occurred. The method includes determining one or more geometric properties of the one or more channels based on the one or more specific distances to the center of the rotor of the analytical ultracentrifugation apparatus.

[0082]

[0075] Example 2 is set up according to example 1, further comprising storing the one or more geometric properties in a computer-readable format.

[0076] Example 3 is set up according to example 1 or 2, wherein the one or more positions where the one or more transitions of the intensity of transmitted light occurred correspond to the top wall of the one or more channels and / or the bottom wall of the one or more channels.

[0083]

[0077] Example 4 is set up according to one of examples 1 to 3, wherein the one or more geometric properties is the orientation of the one or more channels towards the center of the rotor within one or more of the plurality of measurement cells.

[0084]

[0078] Example 5 is set up according to one of examples 1 to 4, further comprising determining that a misalignment of the one or more channels has occurred when the data comprises at least a portion of data pertaining to a radial distance shift.

[0085]

[0079] Example 6 is set up according to example 5, further comprising stopping the analytical ultracentrifugation apparatus based on the determination that the one or more channels within the one or more of the plurality of measurement cells are misaligned, and adjusting the orientation of the one or more of the plurality of the measurement cells such that the one or more of the plurality of measurement cells are aligned.

[0086]

[0080] Example 7 is set up according to any one of examples 1 to 6, wherein one or more of the channels comprises a solution arranged therein.

[0087]

[0081] Example 8 is set up according to one of examples 1 to 7, wherein the light has a wavelength ranging from 190 to 800 nm, preferably from 320 nm to 800 nm, or more preferably of 500 nm.

[0088]

[0082] Example 9 is set up according to any one of examples 1 to 8, wherein the analytical ultracentrifugation apparatus is operated at a speed which does not change the sedimentation properties of the sample in the one or more channels.

[0089]

[0083] Example 10 is set up according to any one of examples 7 to 8, further comprising evaluating the sedimentation properties of the sample in the one or more channels.

[0084] Example 11 is set up according to examples 9 to 10, wherein the analytical ultracentrifugation apparatus is operated at a speed ranging from 2,000 to 60,000 rpm, preferably from 2,000 to 42,000 rpm, and most preferably from 2,000 to 4,000 rpm.

[0090]

[0085] Example 12 is set up according to example 5, further comprising obtaining one or more images for one or more of the plurality of measurement cells, wherein each image comprises a rotor calibration mark and a mark on the cell housing for each measurement cell of the plurality of measurement cells.

[0091]

[0086] Example 13 is set up according to example 5, wherein a misalignment is determined when the radial distance shift deviates from 0 pm.

[0092]

[0087] Example 14 refers to a computer-readable medium storing a program that includes instructions that, when executed by an analytical ultracentrifugation apparatus, perform a method according to any of examples 1 to 13.

[0093]

[0088] Example 15 refers to an AUC apparatus that includes the computer-readable medium of example 14.

[0094]

[0089] Example 16 is set up according to any one of examples 1 to 13, wherein the one or more measurement cells comprise a centerpiece, wherein the one or more channels are located within the centerpiece.

[0095]

[0090] Example 17 is set up according to any one of examples 1 to 13 and 16, wherein the method is carried out with standard measurement cells of the analytical ultracentrifugation apparatus.

Claims

1. Patent Claims1. A method of evaluating the geometric properties of one or more channels within one or more of a plurality of measurement cells in an analytical ultracentrifugation apparatus, wherein the method comprises:(a) applying light to the one or more channels, while operating the analytical ultracentrifugation apparatus;(b) detecting an intensity of light transmitted by the one or more channels;(c) generating data pertaining to the intensity of light transmitted by the one or more channels;(d) determining when one or more transitions of the intensity of transmitted light occurred based on the data;(e) determining one or more specific distances in the one or more channels to the center of the rotor of the analytical ultracentrifugation apparatus at the one or more positions when the one or more transitions of the intensity of transmitted light occurred; and(f) determining one or more geometric properties of the one or more channels based on the one or more specific distances to the center of the rotor of the analytical ultracentrifugation apparatus.

2. The method of claim 1, further comprising storing the one or more geometric properties in a computer-readable format.

3. The method of any preceding claim, wherein the one or more positions where the one or more transitions of the intensity of transmitted light occurred correspond to the top wall of the one or more channels and / or the bottom wall of the one or more channels.

4. The method of any preceding claim, wherein the one or more geometric properties is the orientation of the one or more channels towards the center of the rotor within one or more of the plurality of measurement cells.

5. The method of any preceding claim, further comprising determining that a misalignment of the one or more channels has occurred when the data comprises at least a portion of data pertaining to a radial distance shift.

6. The method of claim 5, further comprising: stopping the analytical ultracentrifugation apparatus based on the determination that the one or more channels within the one or more of the plurality of measurement cells are misaligned; and adjusting the orientation of the one or more of the plurality of measurement cells such that the one or more of the plurality of measurement cells is aligned.

7. The method of any preceding claim, wherein one or more channels comprises a solution arranged therein.

8. The method of any of the preceding claims, wherein the light has a wavelength ranging from 190 to 800 nm, preferably from 320 nm to 800 nm, or more preferably of 500 nm.

9. The method of any of the preceding claims, wherein the analytical ultracentrifugation apparatus is operated at a speed which does not change the sedimentation properties of the sample in the one or more channels.

10. The method of claims 7 or 8, further comprising evaluating the sedimentation properties of the sample in the one or more channels.

11. The method of claims 9 or 10, wherein the analytical ultracentrifugation apparatus is operated at a speed ranging from 2,000 to 60,000 rpm, preferably from 2,000 to 42,000 rpm, and most preferably 2,000 to 4,000 rpm.

12. The method of claim 5, further comprising obtaining one or more images for one or more of the plurality of measurement cells, wherein each image comprises a rotor calibration mark and a mark on the cell housing for each measurement cell of the plurality of measurement cells.

13. The method of claim 5, wherein a misalignment is determined when the radial distance shift deviates from 0 pm.

14. A computer-readable medium storing a program including instructions that, when executed by an analytical ultracentrifugation apparatus, performs a method according to any preceding claim.

15. An analytical ultracentrifugation apparatus comprising the computer-readable medium of claim 14.

16. The method of any one of claims 1 to 13, wherein the one or more measurement cells comprise a centerpiece, wherein the one or more channels are located within the centerpiece.

17. The method of any one of claims 1 to 13 and 16, wherein the method is carried out with standard measurement cells of the analytical ultracentrifugation apparatus.

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