Method and system for determining viscosity of a liquid sample

Abstract

A method and system for determining the viscosity of a liquid sample is provided. The method and system utilize an acoustic liquid processor (101). The acoustic liquid processor (101) has a first position (105) adapted to receive a liquid sample. The acoustic liquid processor (101) is configured to apply one or more acoustic signals (217) to the liquid sample at the first position (105) until a specified volume of the liquid sample is transferred from the first position (105) to a second position (109) of the acoustic liquid processor (101). The acoustic liquid processor (101) has a controller (202) configured to determine the viscosity of the liquid sample based on the number of acoustic signals (217) required to transfer the specified volume of the liquid sample from the first position (105) to the second position (109).

Description

[Technical Field]

[0001] The present disclosure is directed to systems and methods for determining the viscosity of small liquid samples, such as proteins. [Background technology]

[0002] In the life sciences, it is sometimes necessary to measure the viscosity of liquid samples containing proteins. Reliable measurements of these viscosities are often essential for ongoing research and the development of protein therapeutic products. The cone and plate method is commonly used to measure viscosity in these situations. The cone and plate method is a dynamic viscometric method that measures the viscosity of a sample based on applied rotational shear stress and changes in shear rate. The cone and plate method is generally recognized by those skilled in the art for its high precision and accuracy. Other viscometric methods, such as the Rheosense Initium and Malvern Viscosizer, can also be used.

[0003] The process of moving liquids while applying an acoustic signal is known as acoustic droplet ejection (ADE). ADE is discussed in detail in B. Hadimioglu, R. Stearns, and R. Ellson, “Moving Liquids with Sound: The Physics of Acoustic Droplet Ejection for Robust Laboratory Automation in Life Sciences,” J Lab Autom., vol. 21, no. 1, pp. 4-18, February 2016, doi:10.1177 / 2211068215615096, which is incorporated herein by reference. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] B. Hadimioglu, R. Stearns, and R. Ellson, “Moving Liquids with Sound: The Physics of Acoustic Droplet Ejection for Robust Laboratory Automation in Life Sciences” J Lab Autom., vol. 21, no. 1, pp. 4-18, Feb. 2016, doi:10.1177 / 2211068215615096 Summary of the Invention [Means for solving the problem]

[0005] One aspect of the present disclosure provides a method including: (a) placing a protein sample at a first position of an acoustic liquid processor; (b) applying one or more first acoustic signals using the acoustic liquid processor until a specified amount of sample is transferred from the first position to a second position of the acoustic liquid processor; and (c) determining the viscosity of the sample based on the number of one or more first acoustic signals required to transfer the specified amount of sample from the first position to the second position. Additionally or alternatively, the viscosity can be determined based on any of: (i) the SubEject power (dB) required to transfer the specified amount of sample to the second position; (ii) the SubEject amplitude (volts) required to transfer the specified amount of sample to the second position; (iii) the NewEject amplitude (volts) required to transfer the specified amount of sample to the second position; (iv) the NewEject amplitude + Threshold (volts) required to transfer the specified amount of sample to the second position; and / or (v) the power difference (volts) required to transfer the specified amount of sample to the second position.

[0006] Another aspect of the present disclosure provides a method that includes (a) placing a protein sample at a first location of an acoustic liquid processor, (b) repeatedly applying one or more acoustic signals to the sample at the first location using the acoustic liquid processor until it is determined that a specified amount of sample has been transferred from the first location to a second location, and (c) determining the viscosity of the sample based on the number of repetitions required to transfer the specified amount of sample to the second location. Additionally or alternatively, the viscosity can be determined based on the SubEject output (dB), SubEject amplitude (Volts), NewEject amplitude (Volts), the sum of NewEject amplitude + Threshold B (Volts), or the output difference (Volts) required to transfer the specified amount of sample to the second location.

[0007] Another aspect of the present disclosure is an acoustic liquid processor. The acoustic liquid processor has a first location with one or more wells adapted to receive a sample. The acoustic liquid processor is configured to apply one or more first acoustic signals to a sample at the first location until a specified amount of sample is transferred from the first location to a second location of the acoustic liquid processor. The acoustic liquid processor has a controller configured to determine a viscosity of the sample based on the number of first acoustic signals required to transfer the specified amount of sample from the first location to the second location. Additionally or alternatively, the viscosity can be determined based on the SubEject power (dB) required to transfer the specified amount of sample to the second location, the SubEject amplitude (volts) required to transfer the specified amount of sample to the second location, the NewEject amplitude (volts) required to transfer the specified amount of sample to the second location, the NewEject amplitude + Threshold (volts) required to transfer the specified amount of sample to the second location, or the power difference (volts) required to transfer the specified amount of sample to the second location.

[0008] In further accordance with any one or more of the above aspects, the method and / or acoustic liquid processor may further include any one or more of the following suitable forms:

[0009] In some formats, the designated amount of sample to be transferred is all or substantially all of the sample.

[0010] In some formats, the specified amount of sample to be transferred is the minimum amount of sample required to cause a displacement along one or more axes on the meniscus of the portion of the sample remaining in the first position.

[0011] In some formats, the controller is configured to measure the amount of sample transferred using a fluid measurement technique.

[0012] In some forms, the controller is configured to determine the viscosity of the sample based on a set of parameters of the first acoustic signal.

[0013] In some forms, the parameters include at least two of frequency, power, amplitude, wavelength, bandwidth, and period.

[0014] In some forms, the one or more first acoustic signals each have a frequency in the range of 1 mHz to 5 mHz, such as 1 to 4 mHz or 1.5 to 3 mHz. In some forms, the one or more first acoustic signals each have a power in the range of 0.5 dB to 2.5 dB, such as 1 to 2 dB or 1 to 1.5 dB. In some forms, the controller is configured to vary a set of parameters of the acoustic liquid processor until a specified volume of sample is transferred.

[0015] In some forms, the controller is configured to repeatedly increase the frequency of the first acoustic signal.

[0016] In some forms, the controller is configured to iteratively increase the frequency of the first acoustic signal by no more than 0.1 Hz or no more than 0.05 Hz per iteration.

[0017] In some formats, the controller determines whether a specified amount of sample has been transferred.

[0018] In some forms, the controller determines whether a specified amount of sample has been transferred by applying one or more second acoustic signals to a first portion of the sample that was not transferred from the first location, determining the amount of the first portion of the sample that was not transferred based on the application of the one or more second acoustic signals, determining the amount of the second portion of the sample that was transferred based on determining the amount of the first portion of the sample that was not transferred, and comparing the amount of the second and / or first portion of the sample to a specified amount of the sample to be transferred.

[0019] In some formats, the amount of the untransferred first portion of the sample is determined based on an imprint formed on the meniscus of the untransferred first portion of the sample in response to application of the one or more second acoustic signals.

[0020] In some formats, the sample has a volume of at least 12 μL and no more than 80 μL.

[0021] In some formats, the sample has a volume of about 30 μL.

[0022] In some formats, the sample has a volume of about 20 μL.

[0023] In some formats, the first location is a first well of a source plate removably positioned within the acoustic liquid processor, and the second location is a first well of a destination plate removably positioned within the acoustic liquid processor, the first well of the destination plate being inverted relative to the first well of the source plate.

[0024] In some forms, the controller determines the viscosity of the sample by comparing the number of one or more first acoustic signals to a predetermined relationship between the number of one or more first acoustic signals and the viscosity of a set of parameters of the one or more first acoustic signals. For example, in some forms, the controller is calibrated based on a predetermined relationship between the viscosity of a standard or known substance (e.g., cP from a cone-plate) and the number of first acoustic signals (e.g., acoustic repetitions) required to transfer a specified amount of the standard or known substance. In some forms, the controller can be calibrated based on a predetermined relationship between the viscosity of a standard or known substance (e.g., cP from a cone-plate) and the SubEject power (dB) required to transfer a specified amount of the standard or known substance, the SubEject amplitude (volts) required to transfer a specified amount of the standard or known substance, the NewEject amplitude (volts) required to transfer a specified amount of the standard or known substance, the NewEject amplitude + ThreshdB (volts) required to transfer a specified amount of the standard or known substance, and / or the power difference (volts) required to transfer a specified amount of the standard or known substance. The calibrated controller can be used to determine the viscosity of one or more samples, including samples of unknown composition and / or unknown viscosity. The one or more samples can be different from each other. Determining the viscosity of the one or more samples can be done without further calibration of the controller.

[0025] In some forms, the controller updates the predetermined relationship for the set of parameters based on the number of the one or more first acoustic signals and the determined viscosity of the sample.

[0026] In some forms, in a mass analyzer configured to generate mass spectrometry data from a sample, optionally, one or more first acoustic signals are configured to transport the sample or a portion thereof to the mass analyzer.

[0027] In some formats, mass spectrometry data is generated using electrospray ionization, atmospheric pressure ionization, atmospheric pressure chemical ionization, atmospheric pressure matrix-assisted laser desorption / ionization, and the viscosity can be determined simultaneously with the mass spectrometry data.

[0028] In some formats, in an analytical device that performs an analysis of a sample, optionally, one or more first acoustic signals transport the sample from a first and / or second location to the analytical device.

[0029] In some formats, the analytical device comprises a mass spectrometer, a liquid chromatography device, a spectrophotometric device, a glycoanalyzer, an infrared detector, a fluorescence plate reader, or a combination thereof.

[0030] In some formats, the acoustic liquid processor is configured to determine whether a sample meets or fails specifications based on the measured viscosity of the sample. Specifications refer to one or more specified parameters that indicate the acceptability of a sample if the sample characteristics (e.g., viscosity) fall within the specified parameters. If the sample characteristics do not fall within the specified parameters, the sample may be deemed to fail.

[0031] In some formats, the method and / or acoustic liquid processor can be performed on at least 100 additional samples within two hours.

[0032] The drawings described below illustrate various aspects of the systems and methods disclosed herein. It is understood that each drawing illustrates one embodiment of a particular aspect of the disclosed systems and methods, and that each drawing is intended to correspond to a possible embodiment. Furthermore, wherever possible, the following description will refer to elements with reference numbers included in the following drawings, and features shown in multiple drawings will be designated with consistent reference numbers. [Brief explanation of the drawings]

[0033] [Figure 1] 1 illustrates an example of an acoustic liquid processor configured to determine the viscosity of a liquid sample constructed in accordance with the principles of the present disclosure. [Figure 2A] 2 is a cross-sectional view of a portion of the acoustic liquid processor of FIG. 1 showing a first location containing a liquid sample and an acoustic signal emitter ejecting a droplet at a second location. [Figure 2B]2B is similar to FIG. 2A, but shows the liquid sample once fully ejected from the first location to the second location via the acoustic signal generator. [Figure 3A] 1 shows a graph depicting the correlation between the cone and plate method and the method of the present disclosure when used to determine the viscosity of liquid samples having concentrations between 100 and 165 mg / mL using an acoustic signal with a first output setting and a stepwise increase in acoustic signal frequency between replicates. [Figure 3B] 10 shows a graph depicting the correlation between the cone and plate method and the method of the present disclosure when used to determine the viscosity of liquid samples having concentrations between 100 and 165 mg / mL using an acoustic signal with a second output setting and a stepwise increase in acoustic signal frequency between replicates. [Figure 3C] 1 shows a graph depicting the correlation between the cone and plate method and the method of the present disclosure when used to determine the viscosity of liquid samples having concentrations between 100 and 165 mg / mL using an acoustic signal with a first output setting and a stepwise increase in frequency between repetitions reduced to 0.1 Hz. [Figure 3D] 10 shows a graph illustrating the correlation between the cone and plate method and the method of the present disclosure when used to determine the viscosity of liquid samples having concentrations between 100 and 165 mg / mL using an acoustic signal with a second output setting and a stepwise increase in frequency between repeats reduced to 0.1 Hz. [Figure 3E] 1 shows a graph depicting the correlation between the cone and plate method and the method of the present disclosure when used to determine the viscosity of liquid samples having concentrations between 100 and 165 mg / mL using an acoustic signal with a first output setting and a stepwise increase in frequency between repetitions reduced to 0.05 Hz. [Figure 3F] 10 shows a graph depicting the correlation between the cone and plate method and the method of the present disclosure when used to determine the viscosity of liquid samples having concentrations between 100 and 165 mg / mL using an acoustic signal with a second output setting and a stepwise increase in frequency between repetitions reduced to 0.05 Hz. [Figure 3G] 1 shows a graph depicting the correlation between the cone and plate method and the method of the present disclosure when used to determine the viscosity of liquid samples having concentrations between 135 and 165 mg / mL using an acoustic signal with a first output setting and a stepwise increase in frequency between repeats reduced to 0.1 Hz. [Figure 3H] 10 shows a graph illustrating the correlation between the cone and plate method and the method of the present disclosure when used to determine the viscosity of liquid samples having concentrations between 135 and 165 mg / mL using an acoustic signal with a second output setting and a stepwise increase in frequency between repeats reduced to 0.1 Hz. [Figure 3I] 1 shows a graph illustrating the correlation between the cone and plate method and the method of the present disclosure when used to determine the viscosity of liquid samples having concentrations between 135 and 165 mg / mL using an acoustic signal with a first output setting and a stepwise increase in frequency between repeats reduced to 0.05 Hz. [Figure 3J] 10 shows a graph illustrating the correlation between the cone and plate method and the method of the present disclosure when used to determine the viscosity of liquid samples having concentrations between 135 and 165 mg / mL using an acoustic signal with a second output setting and a stepwise increase in frequency between repeats reduced to 0.05 Hz. [Figure 3K] 10 shows a graph illustrating the correlation between the cone and plate method and the method of the present disclosure when used to determine the viscosity of liquid samples having concentrations between 135 and 165 mg / mL using an acoustic signal with a second output setting and a stepwise increase in frequency between repeats in CP mode reduced to 0.05 Hz. [Figure 3L] 10 shows a graph illustrating the correlation between the cone and plate method and the method of the present disclosure when used to determine the viscosity of liquid samples having concentrations between 135 and 165 mg / mL using an acoustic signal with a second output setting and a stepwise increase in frequency between repeats in CP mode reduced to 0.05 Hz. [Figure 3M] 10 shows a graph illustrating the correlation between the cone and plate method and the method of the present disclosure when used to determine the viscosity of liquid samples having concentrations between 135 and 165 mg / mL using an acoustic signal with a second output setting and a stepwise increase in frequency between repeats in CP mode reduced to 0.05 Hz. [Figure 3N] 10 shows a graph illustrating the correlation between the cone and plate method and the method of the present disclosure when used to determine the viscosity of liquid samples having concentrations between 135 and 165 mg / mL using an acoustic signal with a second output setting and a stepwise increase in frequency between repeats in CP mode reduced to 0.05 Hz. [Figure 3O]10 shows a graph illustrating the correlation between the cone and plate method and the method of the present disclosure when used to determine the viscosity of liquid samples having concentrations between 135 and 165 mg / mL using an acoustic signal with a second output setting and a stepwise increase in frequency between repeats in CP mode reduced to 0.05 Hz. [Figure 3P] 10 shows a graph illustrating the correlation between the cone and plate method and the method of the present disclosure when used to determine the viscosity of liquid samples having concentrations between 135 and 165 mg / mL using an acoustic signal with a second output setting and a stepwise increase in frequency between repeats in CP mode reduced to 0.05 Hz. [Figure 4] FIG. 1 is a block diagram of an example of a method for determining the viscosity of a liquid sample constructed in accordance with the principles of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0034] As mentioned above, there are cases where it is necessary to measure the viscosity of liquid samples containing proteins. Because very small (e.g., volume) liquid samples (in some cases, on the order of 10 μL) are often of interest in research, there are cases where it is necessary to measure the viscosity of these very small protein liquid samples. However, conventional viscosity measurement techniques have disadvantages, for example, in terms of throughput, speed, and sample consumption.

[0035] For example, the conventional cone and plate viscosity measurement method described above typically requires 80 μL of liquid sample to perform an accurate viscosity measurement. Therefore, if less than 80 μL of liquid sample is available, the cone and plate method may be inoperable or inaccurate for measuring the viscosity of a liquid sample. The cone and plate method is also a destructive measurement method in that it is typically difficult or impossible to retrieve the liquid sample after the viscosity measurement is complete. This is because the cone and plate measurement process involves spinning the liquid sample on a rotating cone while tracking the rotational shear stress and shear rate to determine the viscosity. As the cone rotates, the liquid sample rotates and forms a pool on the plate with a high surface area-to-volume ratio. Portions of the liquid sample may detach from the plate due to the rotation and infiltrate other areas or components of the cone and plate tester. Furthermore, even if the liquid sample can be retrieved, the fidelity of the liquid sample may be affected (e.g., due to sample contamination and / or damage), and the retrieved liquid sample may be unsuitable for use in subsequent studies. Therefore, even if a liquid sample greater than 80 μL is available, the destructive nature of the cone and plate method means that all or a large portion of the liquid sample may be destroyed during the measurement process. This destruction consumes limited resources and hinders the repeatability of the study. Another problem is that the traditional cone and plate method is manual. Therefore, while the cone and plate method is capable of high-precision measurements, this only occurs under ideal operation. If the operator does not use proper technique when performing the cone and plate method, the viscosity of the measured liquid sample may be distorted, compromising the comparison between assays.

[0036] Other methods for measuring the viscosity of small liquid samples also have problems. For example, the Rheosense Initium method does not demonstrate high precision and accuracy for highly viscous liquid samples, requires that the general range of viscosity be known prior to measurement to function as intended, and does not account for shear rate. As another example, the Malvern Viscosizer method also uses glycerol standards and also does not account for shear rate. Furthermore, the Rheosense Initium and Malvern Viscosizer instruments are each serial instruments (processing one sample at a time), which are susceptible to capillary blockage and can require extensive cleaning and drying procedures (it is known that residual liquid in the capillaries of these instruments can dilute the sample and distort viscosity readings). Extensive cleaning and drying procedures can result in run times of more than one hour per sample.

[0037] The present disclosure aims to alleviate the above-mentioned problems by providing a highly accurate and precise method and system for determining the viscosity of a liquid protein sample using an acoustic liquid processor that moves the liquid sample from a first location to a second location. The present method and system nondestructively measure the viscosity of a liquid sample smaller than that measurable using conventional methods, such as the cone-and-plate method. In fact, the disclosed method and system can accurately determine the viscosity of a liquid sample as small as 10 μL. Furthermore, because the disclosed method and system are nondestructive, the liquid sample can be retrieved from the second location without affecting the fidelity of the liquid sample. Therefore, the liquid sample can be reused, maintaining the repeatability of the study and conserving resources.For example, after viscosity determination, additional analyses may be performed, such as, but not limited to, high-throughput dynamic light scattering viscosity (see He, F.; Becker, GW; Litowski, JR; Narhi, LO; Brems, DN; Razinkov, VI, High-throughput dynamic light scattering method for measuring viscosity of concentrated protein solutions. Anal Biochem 2010, 399(1), 141-3), colloidal stability measurements (see He, F.; Woods, CE; Becker, GW; Narhi, LO; Razinkov, VI, High-throughput assessment of thermal and colloidal stability parameters for monoclonal antibody formulations. J Pharm Sci 2011, 100(12), 5126-41), and size-exclusion chromatography for the analysis of large molecular weight biotherapeutics (see Hong, P.; Koza, S.; Bouvier, ES, Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and their Aggregates. J Liq Chromatogr Relat Technol 2012,35(20),2923-2950) and high-throughput mass spectrometry (Campuzano, ID; San Miguel, T.; Rowe, T.; Onea, D.; Cee, VJ; Arvedson, T.; McCarter, JD, High-Throughput Mass Spectrometric Analysis of Covalent Protein-Inhibitor Adduct for the Discovery of Irreversible Inhibitor: A Complete Workflow. J Biomol Screen 2016,21(2),136-44).The disclosed methods and systems are also substantially, if not fully, automated so that performance is not dependent on operator skill. The automated methods and systems are also significantly faster than the cone and plate method, providing results on a timescale of seconds per liquid sample rather than minutes per liquid sample (as required by the cone and plate method).

[0038] FIG. 1 illustrates an example of a system 100 for determining the viscosity of a liquid protein sample constructed in accordance with the teachings of the present disclosure. As shown in FIG. 1, system 100 includes an acoustic liquid processor 101 having a first position 105 adapted to receive the liquid sample and a second position 109 adapted to receive the liquid sample from first position 105. As discussed in more detail below, acoustic liquid processor 101 is configured to apply one or more first acoustic signals to the liquid sample at first position 105 until a specified amount of sample is transferred from first position 105 to second position 109. System 100 is then configured to determine the viscosity of the sample based on the number of one or more first acoustic signals required to transfer the specified amount of sample from first position 105 to second position 109.

[0039] The acoustic liquid processor 101 shown in FIG. 1 is a standalone scientific instrument, such as the Labcyte Echo550 acoustic liquid processor or the EDC Biosystems ATS-100 instrument, although in other examples, the acoustic liquid processor 101 may be incorporated into a broader range of scientific instruments. The acoustic liquid processor 101 generally enables non-contact and highly accurate transfer of a small volume of liquid sample from a first location 105 to a second location 109 using an ADE process in which ultrasonic pulses are applied to eject droplets of the liquid sample. The acoustic liquid processor 101 may be used to transfer small volumes (e.g., small volumes) of liquid sample, such as milliliters, microliters, nanoliters, picoliters, or other small volumes or small amounts of liquid sample. While the acoustic liquid processor 101 is used in this example with a protein sample, such as a therapeutic protein, the acoustic liquid processor 101 may alternatively be used to determine the sample viscosity of nucleic acids (e.g., DNA and / or RNA), surfactants, serum, cultured cells, or other liquid samples of interest. Examples of therapeutic proteins include antibodies (such as monoclonal antibodies), antigen-binding antibody fragments, antibody protein products, hormones, growth factors, cytokines, cell surface receptors or their ligands, fusion proteins, chimeric proteins, PEGylated proteins, peptides, protein fragments, or protein-containing conjugates (e.g., antibody-drug conjugates or antibody-nucleic acid conjugates). Antibody protein products also include those based on the complete antibody structure and antibody fragments that retain complete antigen-binding function, such as scFvs, Fabs, and VHH / VH mimics. Non-limiting examples of antibody protein products include single-chain antibodies (SCAs), nanobodies, bispecific T-cell engager molecules, diabodies, triabodies, tetrabodies, and multispecific antibodies (such as bispecific or triabodies). In some embodiments, the antibody protein product comprises or consists of a bispecific T-cell engager (BiTE®) molecule. BiTE® molecules refer to engineered bispecific antibody protein product formats, see Huehls et al., Immunol Cell Biol 93(3):290-296 (2015).These contain two single-chain variable fragments (scFvs) of different antibodies, or the fusion of amino acid sequences from four different genes, into a single peptide chain, typically about 55 kilodaltons in size. One of the scFvs binds to T cells via the CD3 receptor, while the other binds to tumor cells via a tumor-specific molecule.

[0040] In this example, first location 105 is a plate or tray with multiple wells that facilitate high-throughput liquid transfer of samples. More specifically, first location 105 is a plate with 384 wells, each configured to receive a portion of a sample. Alternatively, the plate may have 96 wells, 1536 wells, 6144 wells, or any other number of wells that facilitate high-throughput liquid transfer. In other examples, first location 105 may be a single well. In still other examples, the plate at first location 105 may be replaced with one or more jars, beakers, troughs, pens, flasks, test tubes, cylinders, burets, microfluidic or nanofluidic arrays, or any other receptacle suitable for holding a liquid sample. In this example, the plate defining first location 105 is removable from acoustic liquid processor 101 to facilitate dispensing a liquid sample into the well or wells at first location 105. The plate can then be mounted to the acoustic liquid processor 101 in preparation for ADE operation of the acoustic liquid processor 101. However, in other examples, the plate (or other element defining the first position 105) can be an integral part of the acoustic liquid processor 101.

[0041] 1, the second position 109 is inverted relative to the first position 105 and positioned above the first position 105. The liquid sample transferred from the first position 105 to the second position can be retained at the second position by any other suitable method of retaining the liquid sample at the second position, such as by surface tension of the liquid sample or by use of an electric field. However, in other examples, the second position 109 can be positioned at a different location relative to the first position 105.

[0042] 2A and 2B, there is shown the interior of the acoustic liquid processor 101 and acoustic signal generator 201, and controller 202 for determining the viscosity of a protein liquid sample 213 at a first location 105. As noted above, the amount of liquid sample 213 required to determine viscosity using system 100 is small. For example, the volume of liquid sample 213 may be, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μL or less of sample, or any other suitable volume or amount of liquid sample 213.

[0043] 2A and 2B, there is shown the interior of the acoustic liquid processor 101 and acoustic signal generator 201, and controller 202 for determining the viscosity of a protein-containing liquid sample 213 at a first location 105. Note that the illustrated liquid sample 213 may include one or more samples, each of which may have the same or a different viscosity than another sample. As discussed above, the amount of liquid sample 213 required to determine viscosity using system 100 is small. For example, the volume of liquid sample 213 may be, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μL or less of sample, or any other suitable volume or amount of liquid sample 213.

[0044] The acoustic liquid processor 101 is configured to apply one or more first acoustic signals to the liquid sample at the first location 105 via an acoustic signal generator 201. The acoustic signal generator 201 focuses each of the first acoustic signals on the surface of the liquid sample in the well of the plate at the first location 105, causing a mound (sometimes called a "displacement") to form on the surface of the liquid sample and ejecting a droplet from the liquid sample at the first location 105 into the second location 109. The volume of the ejected droplet can be determined based on a set of parameters of the first acoustic signal. These parameters can include one or more of frequency, power, amplitude, wavelength, bandwidth, period, or other parameters.

[0045] 2A, acoustic signal generator 201 applies one or more acoustic signals 217 to liquid sample 213 contained in the left-most well of the group of wells of the plate defining first location 105, thereby generating mound 225 and ejecting droplet 221 upward into the left-most well of the group of wells of the plate defining second location 109. It will be appreciated that other groups of wells in first location 105 may contain additional samples from liquid sample 213, each of which may contain more of the same sample (e.g., for running samples in duplicate, triplicate, etc.) and / or different samples (e.g., containing different proteins, concentrations, and / or formulation compositions). The acoustic signal generator 201 is shown with a right-pointing arrow to indicate that after a specified volume of liquid sample 213 is ejected from the left-most well of the group of wells at the first position 105 into the left-most well of the group of wells at the second position 109, the acoustic signal generator 201 moves rightward to similarly apply one or more acoustic signals 217 to each liquid sample contained in each well of the other group of wells at the first position 105. It should be noted that while Figure 2A shows only a single row of wells for each of the first position 105 and the second position 109, multiple rows laid out on a grid, such as the first position 105 and the second position 109 shown in Figure 1, or some other arrangement is also possible.

[0046] In this example, the controller 202 is located remotely from the acoustic liquid processor 101 but is communicatively connected to the acoustic liquid processor 101. However, in other examples, the controller 202 may be part of the acoustic liquid processor 101 or may be located proximate to the acoustic liquid processor 101. As shown in FIG. 2A , the controller 202 generally includes a processor 206, a memory 210, a communication interface 214, and computational logic 218. Those skilled in the art will appreciate that the controller 202 may also include additional elements, such as analog-to-digital converters, digital-to-analog converters, amplifiers, sensors, and gauges not explicitly shown herein. Optionally, the controller 202 is calibrated based on a predetermined relationship between the viscosity of a standard substance, such as a known substance (e.g., a standard cone-and-plate derived cP viscosity), and the number of first acoustic signals (e.g., iterations) required to transfer a specified amount of the standard substance. The R in FIGS. 3H-3J 2A linear fit between 0.89 and 0.90 suggests that the controller 202 can be reliably calibrated based on the relationship between viscosity and the number of first acoustic signals, including for determining the viscosity of a sample of unknown viscosity and / or composition. The calibrated controller 202 can be used to determine the viscosity of one or more samples, including samples of unknown composition and / or viscosity. The calibrated controller 202 can determine the viscosity of one or more samples without further calibration. In some examples, the calibration is based on a linear relationship between the viscosity of a standard or known substance and the number of first acoustic signals required to transfer a specified amount of the standard or known substance. In some examples, the calibration is based on a predetermined mathematical relationship between the viscosity of a standard or known substance and the number of first acoustic signals required to transfer a specified amount of the standard or known substance, which mathematical relationship can be linear or nonlinear. In some examples, calibration may be performed based on a linear relationship between the viscosity of the standard or known substance and any of: (i) the number of first acoustic signals required to transfer a specified amount of the standard or known substance, (ii) the SubEject output (dB) required to transfer a specified amount of the standard or known substance, (iii) the SubEject amplitude (volts) required to transfer a specified amount of the standard or known substance, (iv) the NewEject amplitude (volts) required to transfer a specified amount of the standard or known substance, (v) the NewEject amplitude + ThreshdB (volts) required to transfer a specified amount of the standard or known substance, or (vi) the output difference (volts) required to transfer a specified amount of the standard or known substance.

[0047] Processor 206 may be a general-purpose processor, a digital signal processor, an ASIC, a field-programmable gate array, a graphics processing unit, an analog circuit, a digital circuit, or any other known or later-developed processor. Processor 206 operates according to instructions in memory 210. Memory 210 may be volatile or non-volatile memory. Memory 210 may include one or more of read-only memory (ROM), random-access memory (RAM), flash memory, electronically erasable programmable read-only memory (EEPROM), or other types of memory. Memory 210 may include optical, magnetic (hard drive), or any other form of data storage.

[0048] Communication interface 214 may be, for example, a HART® interface, a FOUNDATION™ Fieldbus interface, a PROFIBUS® interface, or any other port or interface, provided to enable or facilitate electronic communication between acoustic liquid treatment device 101 (e.g., acoustic signal generator 201) and controller 202, as well as any other elements of system 100. Such electronic communication may occur via any known communication protocol, such as, for example, a HART® communication protocol, a FOUNDATION™ Fieldbus communication protocol, a PROFIBUS® communication protocol, or any other suitable communication protocol.

[0049] Logic 218 includes one or more routines and / or one or more subroutines implemented as computer-readable instructions stored in memory 210. Controller 202, and in particular its processor 206, executes logic 218 to cause processor 206 to perform operations related to the operation (e.g., control, regulation), maintenance, diagnosis, and / or troubleshooting of acoustic liquid treater 101 and any elements within acoustic liquid treater 101 (e.g., acoustic signal emitter 201), as described in more detail below.

[0050] More specifically, the controller 202 is generally configured to control the operation of the acoustic signal generator 201. In particular, the controller 202 is configured to instruct the acoustic signal generator 201 to (i) emit one or more acoustic signals 217 of a set of parameters toward a first well in the first position 105 until a specified amount of liquid sample 213 in the first well has been transferred to the second position 109, and (ii) move the acoustic signal generator 201 to be positioned beneath a second well in the first position 105. The controller 202 is configured to repeat these two steps until a specified set of conditions is met. These conditions may be that a specified number of wells in the first position 105 have transferred a specified amount of liquid sample to corresponding wells in the second position 109. The controller 202 may be configured to instruct the acoustic signal generator 201 to repeatedly emit one or more acoustic signals 217 while varying a specified set of parameters until the specified amount of liquid sample 213 has been transferred. These parameters may include one or more of frequency, power, amplitude, wavelength, bandwidth, period, or other parameters.

[0051] In some examples, the controller 202 may also be configured to determine when a specified amount of the liquid sample 213 has been transferred from the first position 105 to the second position 109. In one example, the controller 202 may be configured to directly or indirectly measure the amount of the liquid sample 213 transferred from the first position 105 to the second position 109 using a fluid measurement technique. For example, the volume loss at the first position 105 (such as the volume loss in a well) may be measured to calculate the amount of the liquid sample 213 transferred. For example, the fluid displacement per ping may be measured and multiplied by the number of pings. For example, the volume of the droplets may be calculated based on one or more parameters described herein. The amount of fluid transferred may be calculated from the number of droplets transferred. As an example, the diameter of the droplets may be determined from the diameter of the beam of the acoustic signal 217. As an example, the diameter and / or volume of the droplets may be determined optically. The controller may further determine whether the amount of the liquid sample 213 transferred is the specified amount of the liquid sample 213 to be transferred. The controller may determine whether a specified amount of liquid sample 213 has been transferred by (i) instructing the acoustic signal generator 201 to apply one or more second acoustic signals from the first location to a first portion of the liquid sample 213 that was not transferred, (ii) determining the amount of the first portion of the liquid sample 213 that was not transferred based on the application of the second acoustic signals, and (iii) determining the amount of the second portion of the liquid sample 213 that was transferred based on the determination of the amount of the first portion of the liquid sample 213 that was not transferred. After determining the amount of liquid sample 213 that has been transferred, the controller 202 may compare the amount of the second and / or first portion of the sample to a specified amount of the liquid sample 213 that is to be transferred. If the specified amount of liquid sample 213 has been transferred, the controller may instruct the acoustic signal generator 201 to stop applying the acoustic signal 217 to the current well and move to a different well.

[0052] In one example, the designated amount of liquid sample 213 to be transferred can be all of liquid sample 213. In another example, the designated amount of liquid sample 213 to be transferred can be a majority, a substantial portion (but not all), or some other portion of liquid sample 213. As used herein, a "substantial portion" of a liquid sample refers to a volume for which the number of acoustic signal repetitions required to move that volume is reproducible. When attention is given to additional numerical details, a substantial portion may refer to at least 20%, 30%, 40%, or 50% of the sample. As used herein, "substantially all" refers to a portion of the liquid sample that is insufficient in volume to move the remainder of the liquid sample with a reproducible number of acoustic signal repetitions. When attention is given to additional numerical details, substantially all of the liquid sample may refer to at least 80%, 90%, 95%, 97%, or 99% of the sample. In another example, the specified amount of sample to be transferred is sufficient to cause a displacement along one or more axes on the meniscus of a portion of the liquid sample 213 remaining at the first location 105. The displacement may be referred to as a mound, such as mound 225 shown in FIG. 2A, which is formed when acoustic energy focused on the liquid sample 213 in the first location 105 exceeds an ejection threshold determined by the surface tension of the liquid sample 213, causing the ejection of a droplet, such as droplet 221, due to Rayleigh-Taylor instability, as described in the Background section above.

[0053] The controller 202 may iteratively vary parameters of the acoustic signal 217 until a specified amount of liquid sample 213 is transferred. The controller 202 may, for example, adjust the frequency of the acoustic signal 217, iteratively increasing the frequency by 0.1 Hz or less, or by 0.05 Hz or less, with each iteration. In some examples, the applied acoustic signal 217 may have a frequency in the range of 1 mHz to 5 mHz, e.g., 1 to 4 mHz or 1.5 to 3 mHz. In other examples, the frequency range may be significantly wider than the 1 mHz to 5 mHz range, depending on the equipment used and the intended application. The controller 202 may alternatively or additionally adjust other acoustic signal parameters, including power, amplitude, wavelength, bandwidth, period, or combinations thereof. Power may be measured directly in decibels of the output acoustic signal 217, or indirectly, for example, by measuring the voltage applied to a transducer of the acoustic signal generator 201. In some examples, the power of the acoustic signal 217 may be in the range of 0.5 dB to 2.5 dB, such as 1 to 2 dB or 1 to 1.5 dB. In other examples, the power range may be significantly wider than the 0.5 dB to 2.5 dB range depending on the equipment used and the intended application.

[0054] 2B , which illustrates the entire liquid sample 213 being transferred from the first position 105 to the second position 109, the controller 202 is further configured to determine the viscosity of the liquid sample 213 based on the number (or amount) of acoustic signals 217 applied by the acoustic signal generator 201 required to transfer the specified amount of liquid sample 213 from the first position 105 to the second position 109. For example, the controller 202 may determine that a first portion of the liquid sample 213 has a first viscosity if 50 acoustic signals 217 are required to transfer the specified amount of liquid sample 213 from the first position 105 to the second position 109, but that a second portion of the liquid sample 213 has a second viscosity that is higher than the first viscosity if 100 acoustic signals 217 are required to transfer the specified amount of liquid sample 213 from the first position 105 to the second position 109. Applicant has discovered that there is a correlation between the viscosity of the liquid sample 213 and the number of acoustic signals 217 required to transfer a specified amount of the liquid sample 213 from the first location 105 to the second location 109. This correlation may be a 1:1 correlation, a 2:1 correlation, or some other correlation. In any case, the controller 202 executes logic 218 to cause the processor 206 to determine the viscosity of the sample 213 by comparing the number of acoustic signals 217 applied to a predetermined relationship between the number of acoustic signals applied and viscosity stored in memory 210. The stored predetermined relationship may be for one or more of the parameters described herein.

[0055] In some examples, the controller 202 may determine the viscosity of the liquid sample 213 based on the number of acoustic signals 217 required to transfer a specified amount and other factors or data. As an example, the viscosity determination may be further based on one or more parameters of the acoustic signals 217, such as the frequency (or frequencies) of the applied acoustic signals 217. Furthermore, in some examples, as the controller 202 makes the viscosity determination, the controller 202 may update the predetermined relationship stored in the memory 210 to include the viscosity determination (e.g., by updating the predetermined relationship based on the number of acoustic signals 217 required to transfer a specified amount and other known factors or data). That is, the predetermined relationship may be further refined based on additional empirical data.

[0056] In some examples, once the viscosity of a portion of the liquid sample 213 or all of the liquid sample 213 has been determined, the controller 202 may make a determination as to whether the measured viscosity is acceptable (e.g., to enable analysis of the liquid sample 213). The measured viscosity may be determined to be acceptable if it is within a predetermined range of values, equal to a predetermined value within a predetermined tolerance range, less than a predetermined threshold, greater than a predetermined threshold, or meets some other suitable criteria. For example, the controller 202 may be calibrated based on a determined relationship between the viscosity of a standard or known substance (e.g., cP from a cone and plate) and the number of first acoustic signals required to transfer a specified amount of the standard or known substance. For example, the controller 202 can be calibrated based on a determined relationship between the viscosity of a standard or known substance and any of the following: (i) the number of first acoustic signals required to transfer a specified amount of the standard or known substance; (ii) the SubEject output (dB) required to transfer a specified amount of the standard or known substance; (iii) the SubEject amplitude (volts) required to transfer a specified amount of the standard or known substance; (iv) the NewEject amplitude (volts) required to transfer a specified amount of the standard or known substance; (v) the NewEject amplitude + ThreshdB (volts) required to transfer a specified amount of the standard or known substance; or (vi) the output difference (volts) required to transfer a specified amount of the standard or known substance. For example, the determined relationship can be a linear relationship, although some nonlinear mathematical relationships are also suitable. The calibrated controller 202 can determine the viscosity of one or more samples of unknown composition and / or viscosity. The calibrated controller can determine the viscosity of one or more samples without further calibration. The measured viscosity may also be determined to be acceptable if it is determined to be a viscosity that does not inhibit or interfere with the normal operation of a high performance liquid chromatography (HPLC) or ultra performance liquid chromatography (UPLC) applied to the portion of the liquid sample. If a portion of the liquid sample 213 or all of the liquid sample 213 is determined to be unacceptable, the portion of the liquid sample 213 or all of the liquid sample 213 may be discarded, or the sample 213 may be diluted to bring the viscosity to a level acceptable for the HPLC or UPLC.The maximum viscosity tolerated by instruments such as HPLC and UPLC instruments can depend on several factors, including the degree to which the sample is diluted during and after injection, the injection needle and tubing diameter, and the autosampler and system temperature. Accordingly, a predetermined threshold can be set for the maximum viscosity tolerated by a particular instrument, such as an HPLC or UPLC instrument. If a system or method described herein determines that the viscosity of the sample 213 exceeds the predetermined threshold (i.e., if the viscosity of the sample 213 is determined to be too high), the sample 213 may not be injected into the instrument, or the sample 213 may be diluted to bring the viscosity to a level acceptable for the instrument. For example, the predetermined threshold can be set such that a viscosity of the liquid sample 213 determined to be less than 500 cP has an acceptable viscosity for use in an HPLC or UPLC. In such an example, if the controller 202 determines that the viscosity of the liquid sample 213 is 600 cP, the liquid sample may be discarded or diluted to an acceptable viscosity.

[0057] Additionally, applicants have determined that the methods and systems described herein effectively determine the viscosity of a liquid sample while overcoming problems associated with the cone and plate method and other prior art techniques. Figures 3A-3J show the correlation between the viscosity of a liquid sample 213 measured by the cone and plate method and the viscosity determined by the methods and systems described herein as a function of the total number of iterations. The correlation coefficient (R 2) shows a linear regression calculated from the BP2 signal. Figure 3A shows the correlation when the liquid sample 213 was tested under conditions where the concentration was 100-165 mg / mL and the acoustic signal 217 had a first power setting and the frequency increased stepwise with each iteration according to the instrument's default BP2 setting. Note that the default BP2 setting was configured with a larger step size per iteration than the adjusted settings in Figures 3C and 3G (0.1 Hz) and Figures 3E and 3I (0.05 Hz). It is further contemplated that a constant frequency or a linear increase in frequency with each iteration may be appropriate in some instances. Note also that different instruments may have different power settings. For example, the software for LabCyte's Echo550 automated liquid processing machine has low, medium, and high power settings. Figure 3B shows the correlation between the liquid sample 213 and the acoustic signal 217 when the liquid sample 213 had a concentration of 100-165 mg / mL and the acoustic signal 217 had a second power setting different from the first power setting, with the frequency stepwise increase between repeats according to the instrument's default CP setting (the CP setting utilizes dynamic fluid analysis). Note that the default CP setting was configured with a larger step size per repeat than the adjusted settings shown in Figures 3D and 3H (0.1 Hz) and Figures 3F and 3J (0.05 Hz). Figure 3C shows the correlation between the liquid sample 213 and the acoustic signal 217 when the liquid sample 213 had a concentration of 100-165 mg / mL and the acoustic signal 217 had a first power setting with a frequency stepwise increase of only 0.1 Hz between repeats. Figure 3D shows the correlation between the liquid sample 213 and the acoustic signal 217 when the liquid sample 213 had a concentration of 100-165 mg / mL and the acoustic signal 217 had a second power setting with a frequency stepwise increase of only 0.1 Hz between repeats. 3E shows the correlation when the liquid sample 213 is tested at a concentration of 100-165 mg / mL and the acoustic signal 217 has a first power setting with only a 0.05 Hz frequency step increase between repeats. As described herein, it has been observed that the relatively small magnitude of the frequency increase between repeats results in a superior (stronger) correlation coefficient compared to larger frequency step increases. Note the relatively small step increases, within the 0.05-0.1 Hz range.Figure 3F shows the correlation when both liquid sample 213 and acoustic signal 217 are tested under conditions where the concentration of the liquid sample 213 is 100-165 mg / mL, acoustic signal 217 has a second output setting, and the frequency is increased in smaller increments of 0.05 Hz between repeats. Figure 3G shows the correlation when both liquid sample 213 and acoustic signal 217 are tested under conditions where the concentration of the liquid sample 213 is 135-165 mg / mL, acoustic signal 217 has a first output setting, and the frequency is increased in stepwise increments of 0.1 Hz between repeats. Figure 3H shows the correlation when both liquid sample 213 and acoustic signal 217 are tested under conditions where the concentration of the liquid sample 213 is 135-165 mg / mL, acoustic signal 217 has a first output setting, and the frequency is increased in stepwise increments of 0.1 Hz between repeats. Figure 3I shows the correlation when liquid sample 213 is tested under conditions where the concentration is 135-165 mg / mL and acoustic signal 217 has a first output setting and the frequency is increased stepwise by a relatively small increment of 0.05 Hz between repeats. Figure 3J shows the correlation when liquid sample 213 is tested under conditions where the concentration is 135-165 mg / mL and acoustic signal 217 has a second output setting and the frequency is increased stepwise by a relatively small increment of 0.05 Hz between repeats.

[0058] Further analysis showed that the additional power required to transfer the liquid sample correlated with viscosity and the total number of iterations outlined above ( Figure 3K, R 2 =0.896908), the specified amount (Fig. 3L, R 2 = 0.84986), (iii) the SubEject power (dB) required to transfer the sample by the specified amount (Figure 3M, R 2 = 0.851302), (iv) the SubEject amplitude (volts) required to transfer the sample by the specified amount (Figure 3N, R 2 = 0.873549), (v) the NewEject amplitude (volts) required to transfer the sample by the specified amount (Figure 3O, R 2 = 0.853561) required to transfer the sample by the specified amount (Figure 3P, R 2=0.859205) of sample. Thus, in the methods and systems described herein, in addition to the total number of iterations required to transfer a specified amount of sample from a first location to a second location, it is contemplated that viscosity may be determined based on any of the following: the SubEject power (dB) required to transfer a specified amount of sample (e.g., to the second location), the SubEject amplitude (Volts) required to transfer a specified amount of sample (e.g., to the second location), the NewEject amplitude (Volts) required to transfer a specified amount of sample (e.g., to the second location), the NewEject amplitude + ThreshdB (Volts) required to transfer a specified amount of sample (e.g., to the second location), or the power difference (Volts) required to transfer a specified amount of sample (e.g., to the second location). Thus, when viscosity determination is based on the total number of iterations required to transfer a specified amount of sample, it is contemplated that viscosity may also be determined based on any of the following: The SubEject power (dB) required to transfer a specified amount of sample, the SubEject amplitude (volts) required to transfer a specified amount of sample, the NewEject amplitude (volts) required to transfer a specified amount of sample, the NewEject amplitude + ThreshdB (volts) required to transfer a specified amount of sample to a second location, and / or the power difference (volts) required to transfer a specified amount of sample.

[0059] From Figures 3A-3P, the strongest correlation (R 2 It can be seen that the R values ​​(shown in red) of about 0.9 or more for the liquid sample 213 were observed in the stepwise increase in frequency shown in FIGS. 3H and 3J. 2 The majority of the liquid samples 213 at the first power setting and the second power setting, including the 2 It will be appreciated that a correlation (R ) between the cone and plate method and the method of the present disclosure was observed. 2The difference in the frequency (shown in values) is generally stronger when higher concentrations of protein are used (compare 135-165 mg / mL in Figures 3G-3J with 100-165 mg / mL in Figures 3C-3F). It is further contemplated that adjusting the step size to vary the frequency between repeats can affect the acoustic measurement of viscosity (as shown by the correlation with cone and plate viscosity). In some embodiments, the step size for varying the frequency between repeats is reduced from a default or baseline value (i.e., the step size is made smaller). However, a trade-off may be made in that a smaller step size requires more acoustic signals 217 to be tested, thereby increasing the time required to transfer a specified volume of liquid sample 213 from first position 105 to second position 109, thus potentially reducing the throughput of the disclosed methods. However, it should be noted that even with a relatively lower throughput for smaller step sizes (e.g., about 0.05 Hz per step), the methods and systems described herein are still significantly faster than traditional cone and plate assays. In any event, the correlation data obtained, especially for smaller step sizes and highly concentrated liquid samples 213, demonstrates that the accuracy of the methods and systems described herein can compete with conventional cone and plate methods.

[0060] For the data shown in Figures 3A-3J, the 81 BTI mAbs were initially received in A52SuT at approximately 10 mg / mL and concentrated to a target of 150 mg / mL ±10% using a 30 kDa MW cutoff filter. Concentration was measured using SoloVPE and the respective extinction coefficients. Viscosity measurements were performed with an Anton Paar MCR Rheometer set to the following geometry: 20 mm, 1.988° cone-plate, Peltier steel plate -990918. Viscosity was measured using a flow sweep setup from shear rates 10 to 1000 s. Viscosity values ​​in this study are based on 1000 s -1It has been reported that 80 μL was loaded onto a steel plate for each measurement. For Echo viscosity measurements, 30 μL of each sample was loaded into a 384-well plate compatible with the Echo 550. The plate was loaded into the "source plate" position of the Echo 550. The Echo 550 was set up to transfer 50 nL of material from the source plate to the destination plate (optionally, the source plate can be sealed and the contents transferred to the sealed section, eliminating the need to transfer the sample to a new plate). The number of iterations or "pings" required to form a "mound image print" (MIP) on the surface of the sample was recorded. The Echo 550 settings conform to the following fluid class nomenclature: B - buffer only; BP - buffer and protein (a detergent-free, medium-viscosity liquid) | minimum well fluid volume 15 μL | maximum 65 μL; GP - glycerol and protein (a detergent-free, high-viscosity liquid) | minimum well fluid volume 15 μL | maximum 65 μL; CP - protein crystallization reagent (a high-viscosity fluid with low levels of detergent) | minimum well fluid volume 25 μL | maximum 50 μL. Two protocols, BP and CP, were evaluated for material transfer, both of which incrementally increase the material transfer frequency. Note that the CP mode dynamically adjusts the power output based on measurements of the viscosity and surface tension of the fluid in the well using dynamic fluid analysis and is considered applicable to the transfer of protein crystallization reagents and other aqueous liquids that cannot be transferred using other techniques.

[0061] 4 is a block diagram of an example iterative method 400 for determining the viscosity of a liquid sample (e.g., liquid sample 213) using an acoustic liquid processor (e.g., acoustic liquid processor 101) having an ADE. In the illustrated method 400, the liquid sample is placed at a first position of the acoustic liquid processor (block 401). A parameter set of acoustic signals is initialized (block 402), and a designated number of acoustic signals of the parameter set are applied to a portion of the liquid sample in a well at the first position (block 403). Next, the method 400 includes determining (block 404) the amount of the portion of the liquid sample that has been transferred from the well at the first position to the well at the second position. The amount is compared to a designated amount of the portion of the liquid sample to be transferred (block 405). If an insufficient portion of the liquid sample has been transferred, the set of acoustic signal parameters may or may not be changed (blocks 406, 407), but the designated number of acoustic signals are again applied (block 403), and the transferred portion of the liquid sample is again determined (block 404). If the designated portion of the liquid sample has been transferred (block 405), the method 400 includes determining whether the designated number of wells at the first location have transferred the designated portion of the liquid sample (block 408). If the designated number of wells at the first location have not transferred the designated portion of the liquid sample, the acoustic liquid processor is adjusted to be positioned to apply acoustic signals to different wells at the first location (block 409), the designated number of acoustic signals are again applied (block 403), and the transferred portion of the liquid sample is again determined (the transferred portion of the liquid sample may be a sample having the same or a different composition and / or viscosity as the previously transferred portion) (block 404). If the specified number of wells at the first location have transferred a specified amount of the liquid sample, the method 400 includes determining the viscosity of the liquid sample based on the set of parameters and the number of acoustic signals required to transfer the specified amount of the liquid sample (block 410).

[0062] While the systems and methods described herein are used in the context of ADE, it will be understood that the systems and methods may also be used in conjunction with analytical devices performing downstream analyses (e.g., mass spectrometry, high-throughput dynamic light scattering viscosity, colloid stability measurements, and / or biotherapeutic high molecular weight analysis by size-exclusion chromatography). In a first example, a liquid sample is introduced into a mass spectrometer (e.g., a high-resolution accurate-mass (HRAM) mass spectrometer) using acoustic waves, applying principles similar to Rayleigh-Taylor instability to electrospray ionization. The disclosed systems and methods can then measure viscosity and generate mass spectrometry data. Thus, the systems can simultaneously generate viscosity data and mass spectrometry data. However, the systems and methods do not necessarily employ strict electrospray operation. In such an example, the systems and methods can generate electrospray ionization through atmospheric pressure ionization by passing an electric field through droplets, rather than applying an electric field to a liquid sample confined in a narrow capillary. In another example, atmospheric pressure chemical ionization can be used. In yet another example, atmospheric pressure matrix-assisted laser desorption / ionization (MALDI) ionization can be used. The system and method can deposit a droplet onto a MALDI target plate for subsequent atmospheric pressure MALDI mass analysis. In this manner, the system or method can determine the viscosity of the sample and then transfer the sample, or a portion thereof, to a mass spectrometer for further mass analysis.

[0063] In a second example, the liquid sample can be introduced into an analytical device different from the acoustic liquid processor, such as a liquid chromatography device (e.g., an ion exchange chromatography column, a cation exchange chromatography column, an anion exchange chromatography column), a high performance liquid chromatography device, an ultra-high performance liquid chromatography device, a spectrophotometric device (e.g., a UV detector), a glycoanalysis device, an infrared detector, a fluorescence plate reader, another type of analytical device, or a combination thereof. The liquid sample can be introduced into the different analytical device via acoustic movement of fluid by the acoustic liquid processor. The different analytical device can be in fluid communication with the acoustic liquid processor. The disclosed systems and methods can then measure viscosity to generate downstream analytical data.

Claims

1. 1. A method for measuring the viscosity of a liquid sample of protein, comprising: placing the liquid sample of the protein at a first location in an acoustic liquid processor; applying one or more first acoustic signals with the acoustic liquid processor until a designated volume of the liquid sample is transferred from a first position to a second position of the acoustic liquid processor; determining a viscosity of the liquid sample based on i) a number of the one or more first acoustic signals required to transfer the specified volume of the liquid sample from the first location to the second location, and (ii) one or more of a set of parameters of the first acoustic signals; A method comprising:

2. 2. The method of claim 1, wherein applying the one or more first acoustic signals comprises applying the one or more first acoustic signals until all or substantially all of the liquid sample is transferred from the first location to the second location.

3. 3. The method of claim 1 or 2, wherein the specified amount of the liquid sample to be transferred is sufficient to cause a displacement along one or more axes on the meniscus of the portion of the liquid sample remaining at the first position.

4. 4. The method of claim 1, further comprising measuring the amount of the liquid sample transferred between each of the one or more first acoustic signals using a fluid transfer measurement technique.

5. The method of any one of claims 1 to 4, wherein the set of parameters includes at least two of the following: frequency, power, amplitude, wavelength, bandwidth, and period.

6. 6. The method of any one of claims 1 to 5, wherein the one or more first acoustic signals each have a frequency in the range of 1 mHz to 5 mHz, such as 1 to 4 mHz or 1.5 to 3 mHz, and the one or more first acoustic signals each have a power in the range of 0.5 dB to 2.5 dB, such as 1 to 2 dB or 1 to 1.5 dB.

7. 7. The method of claim 1, wherein applying the one or more first acoustic signals comprises varying the set of parameters until the specified volume of the liquid sample is transferred.

8. The method of any one of claims 1 to 7, further comprising determining whether the specified volume of the liquid sample has been transferred.

9. determining whether the specified volume of the liquid sample has been transferred; applying one or more second acoustic signals to a first portion of the liquid sample that has not been transferred from the first location; determining an amount of the untransferred first portion of the liquid sample based on the application of the one or more second acoustic signals; determining an amount of the transferred second portion of the liquid sample based on the determination of the amount of the untransferred first portion of the liquid sample; comparing the amount of the first portion and / or the second portion of the liquid sample to the designated amount of the liquid sample to be transferred; The method of claim 8, comprising:

10. The method of any one of claims 1 to 9, wherein the liquid sample has a volume of at least 12 μL and no more than 80 μL, preferably about 30 μL, more preferably 20 μL.

11. 11. The method of any one of claims 1 to 10, further comprising generating mass spectrometry data relating to the liquid sample, optionally wherein the one or more first acoustic signals transport the liquid sample or a portion of the liquid sample to a mass spectrometer.

12. 12. The method of any one of claims 1 to 11, further comprising carrying out the method on at least 100 additional liquid samples within 2 hours.

13. A method according to any one of claims 1 to 12, wherein the applying step includes repeatedly applying the one or more first acoustic signals to the liquid sample at the first position using the acoustic liquid processing device until it is determined that the specified amount of the liquid sample has been transferred from the first position to the second position.

14. 1. A system for determining the viscosity of a liquid sample of protein, comprising: an acoustic liquid processor having a first location adapted to receive the liquid sample of protein, the acoustic liquid processor configured to apply one or more first acoustic signals to the liquid sample of protein at the first location until a designated amount of the liquid sample is transferred from the first location to a second location; a controller configured to determine a viscosity of the liquid sample based on (i) the number of first acoustic signals required to transfer the specified volume of the liquid sample from the first location to the second location, and (ii) one or more of a set of parameters of the first acoustic signals; A system including:

15. 15. The system of claim 14, wherein the controller is configured to vary the set of parameters of the acoustic liquid processor until the specified volume of the liquid sample is transferred.

16. The controller determines whether the specified amount of the liquid sample has been transferred by: applying one or more second acoustic signals to a first portion of the liquid sample that has not been transferred from the first location; determining an amount of the untransferred first portion of the liquid sample based on the application of the one or more second acoustic signals; determining an amount of the transferred second portion of the liquid sample based on the determination of the amount of the untransferred first portion of the liquid sample; comparing the amount of the second portion and / or the first portion of the liquid sample to the designated amount of the liquid sample to be transferred; 16. The system of claim 14 or 15, comprising:

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