Method and system for determining protein aggregation

Abstract

The present invention relates to a method and system (140) for determining the presence and / or quantity of protein aggregates in a liquid sample comprising a protein. The method comprises: a) measuring ultraviolet-visible spectroscopy (UV- Vis) absorbance of the sample to obtain a UV-Vis absorbance measurement; b) measuring index of refraction (IoR) of the sample to obtain an IoR measurement; c) determining a value for the UV-Vis absorbance measurement; d) determining a value for the IoR measurement; and e) determining the presence and / or quantity of protein aggregates in the sample based on a ratio between the value for the UV-Vis absorbance measurement and the value for the IoR measurement.

Description

[0001] METHOD AND SYSTEM FOR DETERMINING PROTEIN AGGREGATION

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to a methods and systems for determining the presence and / or quantity of protein aggregates in a liquid sample comprising a protein. The method and systems may for examples be implemented in a bioprocess system.

[0004] BACKGROUND

[0005] Biopharmaceutical manufacturers are under increasing pressure from regulators to ensure the safety and quality of their products. One of the key challenges in the development of biologic formulations is protein aggregation, which can significantly impact product quality in terms of efficacy and immunogenicity.

[0006] Proteins are inherently prone to aggregation due to the dynamic nature of their structures, which are stabilized by a combination of Van der Waals forces, hydrogen bonds, disulfide linkages, and hydrophobic interactions. Disruption of this delicate balance can expose internal hydrophobic regions of the polypeptide chain, leading to interactions with other proteins and the formation of larger complexes of misfolded proteins. Aggregation can be either “native”, where the protein structure is maintained and the aggregation is largely reversible, or “non-native”, where denaturation and structural changes result in largely irreversible aggregation. Aggregates can grow to a wide range of sizes, from soluble oligomers to visible precipitates.

[0007] Protein aggregation is a common consequence of various sample treatments and poses a significant problem for the biopharmaceutical industry. Such treatments include the addition of chemicals, improper reconstitution of lyophilized materials, mechanical stresses during manufacturing, freeze / thaw cycles, and prolonged storage. The presence of contaminating particles also promotes aggregation, with materials such as silicone oil (used as a lubricant in pre-filled syringes), silicone rubber from container-closure systems, glass particles from vials, and oxidized metal particles from filling lines acting as nucleation sites. Aggregation can occur at nearly every stage of a biopharmaceutical process, from development and formulation to manufacturing, storage, and point of use, leading to various deleterious consequences.

[0008] Understanding the pathway of aggregate formation at an early stage is crucial to developing processes that minimize aggregation. The aggregation behavior of two proteins in similar processes can differ significantly, underscoring the need for tailored mitigation strategies.

[0009] Among the various forms of protein degradation, the presence of aggregates in biopharmaceutical formulations remains a primary quality and safety concern. While not all aggregates compromise the protein's functionality, continued aggregation can diminish or completely lose the protein's activity, reducing therapeutic efficacy. More critically, numerous studies have shown that protein aggregates in therapeutic formulations destined for parenteral administration can trigger unwanted and potentially dangerous immune responses in recipients.

[0010] Efficacy can be affected in different ways, from no impact to rendering a drug completely ineffective. For instance, large aggregates formed during formulation may be filtered out later in production, reducing the concentration of the active molecule in the final product. The most serious safety issue is the potential for protein aggregates to trigger a life-threatening immune response, such as anaphylactic shock. Less severe immune responses can also have significant consequences, such as reduced drug efficacy due to the immune system's elimination of the therapeutic protein, leaving the patient without effective treatment options. Administration of proteins designed to supplement endogenous levels can trigger immune responses that destroy both the therapeutic and intrinsic proteins, leading to additional clinical complications, as seen in the case of Eprex-associated Pure Red Cell Aplasia (PRCA).

[0011] The FDA's "Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products," issued in August 2014, underscores the importance of minimizing protein aggregation and developing minimization strategies early in product development. The guidance recommends employing methods that enhance the detection of protein aggregates to characterize distinct species of aggregates in a product.

[0012] Current regulations require the quantification of subvisible particles >10 pm and >25 pm using light obscuration and flow imaging techniques. Smaller aggregates, typically caused by oligomerization, are characterized using size exclusion chromatography (SEC). However, characterizing smaller aggregates and soluble oligomers remains challenging, necessitating the use of multiple orthogonal analytical approaches to cover the full measurement range required. Regulatory authorities strongly encourage the use of orthogonal approaches to obtain a more detailed and comprehensive view of the product and avoid reliance on single technologies. Combining multiple techniques enhances understanding of the complex nature of protein therapeutics and ensures more robust product characterization.

[0013] In conclusion, protein aggregation is a consequence of protein degradation and poses significant challenges in biopharmaceutical development. A thorough understanding of aggregation pathways and risk factors is essential for devising effective mitigation strategies, aligning manufacturing processes with Quality by Design (QbD) principles, and ensuring the safety and efficacy of biopharmaceutical products. As analytical technologies advance, their implementation in GMP- compliant environments and QC activities will be crucial for better particle quantification and characterization, ultimately enhancing product quality and patient safety.

[0014] SUMMARY OF THE INVENTION

[0015] An object of embodiments of the present invention is to provide a solution which mitigates or solves drawbacks and problems with existing methods which allow for determination of the presence and / or quantity of protein aggregates in liquid samples. A further object of embodiments of the present invention is to provide a method and a system which allow for non-destructive determination of the presence and / or quantity of protein aggregates in a liquid sample comprising a protein.

[0016] A further object of embodiments of the present invention is to provide a method and a system which allow for detection and quantification of smaller protein aggregates, typically caused by oligomerization.

[0017] A further object of embodiments of the present invention is to provide a method and a system which allow for determination of the presence and / or quantity of protein aggregates in a liquid sample comprising a protein in-line or on-line in a bioprocess system, preferably in a bioprocess purification system.

[0018] A further object of embodiments of the present invention is to provide a method and a system which allow determination of the presence and / or quantity of protein aggregates in a sample instantaneously, and using the information obtained to control a bioprocess purification system in real time.

[0019] In view of the above, the present disclosure provides an improved or alternative technology having the features set out in the independent claims. Further embodiments of the disclosure are set out in the independent claims.

[0020] According to a first aspect, the above mentioned and other objects are achieved by a method for determining the presence and / or quantity of protein aggregates in a liquid sample comprising a protein, the method comprising: a) measuring ultraviolet-visible spectroscopy (UV-Vis) absorbance of the sample to obtain a UV-Vis absorbance measurement; b) measuring index of refraction (loR) of the sample to obtain an loR measurement; c) determining a value for the UV-Vis absorbance measurement; d) determining a value for the loR measurement; and e) determining the presence and / or quantity of protein aggregates in the sample based on a ratio between the value for the UV-Vis absorbance measurement and the value for the loR measurement.

[0021] According to a second aspect, the above mentioned and other objects are achieved by a system for determining the presence and / or quantity of protein aggregates in a liquid sample comprising a protein. The system comprises: an ultraviolet-visible spectroscopy (UV-Vis) absorbance detector configured to obtain a UV-Vis absorbance measurement of the sample; an index of refraction (loR) detector configured to obtain an loR measurement of the sample; and a data processing unit comprising one or more processors and one or more non- transitory computer-readable media storing instructions executable by the one or more processors, wherein the instructions, when executed, cause the system to perform operations comprising: determining a value for the UV-Vis absorbance measurement; determining a value for the loR measurement; and determining the presence and / or quantity of protein aggregates in the sample based on a ratio between the value for the UV-Vis absorbance measurement and the value for the loR measurement.

[0022] The protein in the present disclosure may be any protein prone to formation of protein aggregates. In some embodiments, the protein is a monoclonal antibody (mAb) or a protein associated with a neurodegenerative disease. The method and system of the present disclosure may be especially useful in the manufacture of monoclonal antibodies. The method and system of the present disclosure may be particularly useful for the detection and quantification of smaller protein aggregates, typically caused by oligomerization. Thus, in some embodiments, the protein is a protein prone to formation of protein oligomers, such as dimers, trimers, or other multimers.

[0023] Protein aggregates are clusters of misfolded or partially folded proteins that have come together to form complexes. These aggregates can be considered impurities, particularly in the context of pharmaceutical manufacturing and biotechnology, where the presence of unwanted protein aggregates can affect the quality, efficacy, and safety of therapeutic protein products. The method and system of the present disclosure may be particularly useful for the detection and quantification of smaller protein aggregates, typically caused by oligomerization. Thus, in some embodiments, the protein aggregates are protein oligomers, such as dimers, trimers, or other multimers.

[0024] The present disclosure is based on the inventive realization that a predictable relationship between measured UV-Vis absorbance and loR values can be used for determining the presence and / or quantity of protein aggregates in a liquid sample comprising a protein.

[0025] An advantage of embodiments according to the aspects is that it allows for nondestructive determination of the presence and / or quantity of protein aggregates in a liquid sample comprising a protein.

[0026] A further advantage is that it allows for determination of the presence and / or quantity of protein aggregates in a liquid sample comprising a protein in-line or on-line in a bioprocess system, preferably in a bioprocess purification system.

[0027] The method and system of the present disclosure can be used for monitoring, and optionally also controlling, the progress and efficiency of various separation and purification steps of a bioprocess purification system. The presence and / or quantity of protein aggregates in a sample can be determined instantaneously, and the information obtained can be used to control the bioprocess purification system in real time. The real-time data can be fed into a control system that adjusts process parameters dynamically, such as by extending or shortening purification steps, switching to different buffers, adjusting column conditions, or controlling elution pool collection, to maintain optimal purification conditions. By continuously optimizing the process, real-time methods can reduce the use of reagents, minimize product loss, and decrease the overall cost of purification. Incorporating real-time analytical methods into bioprocess purification improves efficiency, product quality, and regulatory compliance while reducing costs and development time. These methods transform purification from a static, batch-based approach to a dynamic, data-driven process, aligning with modern bioprocessing paradigms like continuous manufacturing and Quality by Design (QbD).

[0028] Further applications and advantages of embodiments of the invention will be apparent from the following detailed description. The second aspect may generally have the same features and advantages as the first aspect. It is further noted that the disclosure relates to all possible combinations of features, unless explicitly stated otherwise.

[0029] BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Fig. 1 shows a bioprocess system comprising a UV-Vis absorbance detector and an loR detector in series.

[0031] Figs. 2a-2c are diagrams showing the relationship between the UV / loR signal ratio and mAb aggregate concentration.

[0032] It should be appreciated that like reference numerals are used to identify like elements illustrated in one or more of the figures.

[0033] DETAILED DESCRIPTION

[0034] The present disclosure provides a method and a system for determining the presence and / or quantity of protein aggregates in a liquid sample comprising a protein. The method and system of the present disclosure may be implemented in any application where determining the presence and / or quantity of protein aggregates in a liquid sample comprising a protein is required or desired. The method and system of the present disclosure may be especially useful in applications where detection and quantification of smaller protein aggregates, typically caused by oligomerization is required or desired. One application where the method and system of the present disclosure is in a bioprocess system, and preferably in a bioprocess purification system. The method and system of the present disclosure will be described in more detail herein in the context of a bioprocess system, but it is understood that the method and system of the present disclosure may be equally useful in many other applications.

[0035] The term “bioprocess” refers to a series of techniques and methods used to produce biological products, such as drugs, enzymes, proteins, nucleotides, oligonucleotides, VP (viral particles), AAV’s (adeno associated virus) or nanoparticles, using living organisms or their components. Bioprocesses involve the use of microorganisms, animal or plant cells, or other biological systems to produce and modify products, often through genetic engineering, fermentation, or other techniques. Bioprocesses range from small scale processes, e.g. performed for experimental or bioanalytical purposes in a laboratory, to large scale industrial manufacturing. Bioprocesses are used in a wide range of industries, including pharmaceuticals, biotechnology, food production, and agriculture.

[0036] The initial stages of the bioprocess, also referred to as the upstream phase, are typically carried out in a “bioreactor” and involve culturing or growing the organism or cells that produce the desired product. The upstream phase may involve selecting and optimizing the growth conditions, such as temperature, pH, and nutrients, to ensure maximum productivity and yield. The upstream phase may also include genetic engineering, which can be used to modify and improve the organism's ability to produce the desired product.

[0037] The upstream phase is typically followed by purification and processing of the product produced during the upstream phase. This purification and processing stage, also referred to as the downstream phase, is carried out in a bioprocess purification system. The goal of the downstream phase is to isolate and purify the desired product from the complex mixture of other components produced in the upstream phase. The bioprocess purification system often involves a series of separation and purification steps, such as filtration and chromatography, to obtain a highly pure and concentrated product.

[0038] The opportunity to remove unwanted protein aggregates is typically given during downstream phase, however the lack of in-line protein aggregate sensors in the prior art has resulted in conservative UV-based pooling strategies that lead to lower process yield.

[0039] The method of the present disclosure comprises: a) measuring ultraviolet-visible spectroscopy (UV-Vis) absorbance of the sample to obtain a UV-Vis absorbance measurement; b) measuring index of refraction (loR) of the sample to obtain an loR measurement; c) determining a value for the UV-Vis absorbance measurement; d) determining a value for the loR measurement; and e) determining the presence and / or quantity of protein aggregates in the sample based on a ratio between the value for the UV-Vis absorbance measurement and the value for the loR measurement.

[0040] Step a) comprises measuring ultraviolet-visible spectroscopy (UV-Vis) absorbance of the sample to obtain a UV-Vis absorbance measurement.

[0041] The UV-Vis absorbance can be measured using an ultraviolet-visible spectroscopy (UV-Vis) absorbance detector. An ultraviolet-visible spectroscopy (UV-Vis) absorbance detector as used in the present disclosure is an analytical instrument designed for the measurement of the absorbance or transmittance of light of a liquid sample within the ultraviolet and visible regions of the electromagnetic spectrum, typically from 200 nm to 800 nm. UV-Vis absorbance detectors and their implementation in bioprocess systems is well known in the art. Any suitable UV-Vis absorbance detector known in the art may be used. A UV-Vis absorbance detector typically comprises several key components: a light source, a wavelength selection system, a sample compartment, a photodetector array, and a data processing unit. The light source typically includes two types of lamps to cover the full UV-Vis range, for example a deuterium lamp providing UV light from 200 nm to about 400 nm and a tungsten-halogen lamp providing light in the visible spectrum from about 350 nm to 800 nm. The wavelength selection system typically comprises either a monochromator or a diffraction grating. The sample compartment typically comprises a flow cell with a well-defined optical path length. The flow cell is typically made of transparent material such as quartz or glass to minimize interference with the light path. The photodetector array converts the transmitted or absorbed light into an electrical signal, e.g. using photodiodes or CCDs. The data processing unit includes hardware and software components designed for signal processing and analysis.

[0042] The UV-Vis absorbance of the sample can be measured instantaneously or over a period of time.

[0043] In some embodiments, the UV-Vis absorbance of the sample is measured instantaneously to obtain an instantaneous UV-Vis absorbance measurement. An instantaneous measurement refers to the real-time, immediate acquisition of data, without any significant delay, providing near-real-time insights into the conditions of the bioprocess. A series of instantaneous measurements can be performed in order to follow a process over a period of time. This allows for rapid detection of any changes or deviations in the process, enabling prompt adjustments to maintain optimal conditions and ensure consistent product quality.

[0044] In some embodiments, the UV-Vis absorbance of the sample is measured over a period of time to obtain a UV-Vis absorbance vs time measurement. A UV-Vis absorbance vs time measurement can be obtained by directing the liquid sample through a flow cell of the UV-Vis absorbance detector. The flow cell is designed to allow the liquid sample to pass through continuously while the measurement is being taken. The UV-Vis absorbance detector continuously monitors the sample as it flows through the cell, taking measurements at regular intervals. This creates a real-time data stream of absorbance values. The measurement is performed over a period of time, resulting in a UV-Vis absorbance vs time measurement.

[0045] Step b) comprises measuring index of refraction (loR) of the sample to obtain an loR measurement.

[0046] The loR can be measured using an index of refraction (loR) detector.

[0047] An index of refraction (loR) detector as used in the present disclosure is an analytical instrument designed to measure the refractive index of a liquid sample. Any suitable loR detector known in the art may be used. An loR detector typically comprises several key components: a light source, a sample compartment, an optical system for light refraction measurement, a photodetector, and a data processing unit. The light source in the loR detector is designed to emit a stable and monochromatic beam of light, for example using an LED or laser diode. Commonly used wavelengths are 589 nm (sodium D-line) or 632.8 nm (He-Ne laser). The sample compartment typically comprises a flow cell, for example a refractometer cell, with a well-defined optical path length. The flow cell is typically made of transparent material such as quartz or glass to minimize interference with the light path. The optical system is arranged to accurately measure the angle of refraction of the light passing through the liquid sample. It may for example include a collimating lens and a refractometer cell. The photodetector converts the refracted light into an electrical signal, e.g. using photodiodes or CCDs, and the signal is then processed to determine the refractive index.

[0048] The index of refraction (loR) of the sample can be measured instantaneously or over a period of time. In some embodiments, the loR of the sample is measured instantaneously to obtain an instantaneous loR measurement. An instantaneous measurement refers to the real-time, immediate acquisition of data, without any significant delay, providing near-real-time insights into the conditions of the bioprocess. A series of instantaneous measurements can be performed in order to follow a process over a period of time. This allows for rapid detection of any changes or deviations in the process, enabling prompt adjustments to maintain optimal conditions and ensure consistent product quality.

[0049] In some embodiments, the loR of the sample is measured over a period of time to obtain an loR vs time measurement. An loR vs time measurement can be obtained by directing the liquid sample through a flow cell of the loR detector. The flow cell is designed to allow the liquid sample to pass through continuously while the measurement is being taken. The loR detector continuously monitors the sample as it flows through the cell, taking measurements at regular intervals. This creates a real-time data stream of loR values. The measurement is performed over a period of time, resulting in an loR vs time measurement.

[0050] Step c) comprises determining a value for the UV-Vis absorbance measurement. The value may for example be the value as obtained from the measurement, or as subjected to conversion or correction.

[0051] In some embodiments, wherein the UV-Vis absorbance is measured instantaneously, step c) comprises determining a value for the instantaneous UV- Vis absorbance measurement.

[0052] In some embodiments, wherein the UV-Vis absorbance is measured over time, step c) comprises determining a peak value or an area under the curve (AUC) value for the UV-Vis absorbance vs time measurement.

[0053] Step d) comprises determining a value for the loR measurement. The value may for example be the value as obtained from the measurement, or as subjected to conversion or correction. In some embodiments, wherein the loR is measured instantaneously, step d) comprises determining a value for the instantaneous loR measurement.

[0054] In some embodiments, wherein the loR is measured over time, step d) comprises determining a peak value or an area under the curve (AUC) value for the loR vs time measurement.

[0055] The peak value is the maximum absorbance recorded during the measurement period. This can be determined manually from a plot or using software algorithms that search for the maximum value in the dataset.

[0056] The area under the curve (AUC) value can be determined by: i) If necessary, performing baseline correction to account for any drift or background absorbance or loR. This can for example be done by subtracting a baseline absorbance or loR measured before the sample introduction from the absorbance or loR data, or by other suitable baseline subtraction methods known to the skilled person and often implemented in chromatography system software; and ii) Numerically integrating the absorbance or loR versus time data to calculate the AUC. This can be done using various numerical integration methods, such as the trapezoidal rule or Simpson's rule.

[0057] Step e) comprises determining the presence and / or quantity of protein aggregates in the sample based on a ratio between the value for the UV-Vis absorbance measurement and the value for the loR measurement.

[0058] In some embodiments, wherein the UV-Vis absorbance and loR are measured instantaneously, step e) comprises determining the presence and / or quantity of protein aggregates in the sample based on a ratio between the value for the instantaneous UV-Vis absorbance measurement and the value for the instantaneous loR measurement.

[0059] In some embodiments, wherein the UV-Vis absorbance and loR are measured over time, step e) comprises determining the presence and / or quantity of protein aggregates in the sample based on a ratio between the peak value for the UV-Vis absorbance vs time measurement and the peak value for the loR vs time measurement or a ratio between the AUC value for the UV-Vis absorbance vs time measurement and the AUC value for the loR vs time measurement.

[0060] The ratio between the value for the UV-Vis absorbance measurement and the value for the loR measurement is calculated. This ratio correlates with the quantity of protein aggregates in the sample, and can be used to determine the presence or quantity of protein aggregates in the sample. The presence of protein aggregates in the sample can be assessed against a standard curve.

[0061] In some embodiments, step e) comprises determining the presence and / or quantity of protein aggregates in the sample based on a ratio between the peak value for the UV-Vis absorbance vs time measurement and the peak value for the loR vs time measurement.

[0062] In some embodiments, step e) comprises determining the presence and / or quantity of protein aggregates in the sample based on a ratio between the AUC value for the UV-Vis absorbance vs time measurement and the AUC value for the loR vs time measurement.

[0063] In some embodiments, step e) comprises determining the quantity of protein aggregates in the sample.

[0064] The quantity of protein aggregates in the sample may preferably be determined as High Molecular Weight Percentage (HMW%). HMW% is defined as the proportion of protein molecules or aggregates in a sample that have a molecular weight higher than the expected or monomeric form of the protein. This percentage is calculated by comparing the quantity, for example in terms of mass, of these higher molecular weight species to the total protein content in the sample. The ratio between the value for the UV-Vis absorbance measurement and the value for the loR measurement depends on the fraction of aggregates while both UV and loR are affected by the total protein concentration. Thus, in some embodiments, step e) comprises determining the HMW% in the sample. One or more of steps c) to e) may in principle be performed manually by a user, however in preferred embodiments the steps one or more of c) to e) are performed by a data processing unit comprising one or more processors and one or more non- transitory computer-readable media storing instructions executable by the one or more processors. The data processing unit receives the data from the UV-Vis absorbance measurement and the data from the loR measurement and perform the necessary operations to determine the presence and / or quantity of protein aggregates in the sample based on a ratio between the value for the UV-Vis absorbance measurement and the value for the loR measurement.

[0065] The inventive method may advantageously be used for monitoring, and optionally controlling a bioprocess. In such cases the method for determining the presence and / or quantity of protein aggregates may preferably be repeated continuously over a period of time, such that a series of measurements is obtained. The series of measurements may preferably be a series of instantaneous measurements, but may also be a series of measurements over time to obtain a series of peak values or AUC values. Thus, in some embodiments, the method for determining the presence and / or quantity of protein aggregates in a liquid sample comprising a protein further comprises the step: f) repeating steps a)-e) to monitor the presence and / or quantity of protein aggregates in the liquid sample over a period of time.

[0066] The nature of the UV-Vis absorbance and loR detection allows for the method to be performed on-line or in-line in a bioprocess system.

[0067] On-line measurement refers to a setup where the measurement is taken from the process stream, but not directly within the process stream. Instead, a sample is diverted from the main process stream to an external analytical instrument and then returned to the process or discarded. This allows for continuous or near-continuous monitoring of the process without direct interference with the main process stream.

[0068] In-line measurement refers to the integration of the measurement device directly into the process stream, without diverting the flow. The instrument is typically installed in such a way that the process fluid flows directly through the measurement device, allowing for real-time monitoring.

[0069] In some embodiments, the measurement is performed in-line in a bioprocess system, preferably in a bioprocess purification system.

[0070] In some embodiments, the measurement is performed on-line in a bioprocess system, preferably in a bioprocess purification system.

[0071] The UV-Vis absorbance measurement and the loR measurement may preferably be performed in the downstream phase of a bioprocess to allow measurement on a fully purified or partially purified sample, or more preferably after the downstream phase of a bioprocess to allow measurement on a fully purified sample. The bioprocess system may also be provided with two or more sets of UV-Vis absorbance detectors and loR detectors to allow a change in the presence and / or quantity of protein aggregates in a sample between two different stages of the bioprocess to be measured.

[0072] The UV-Vis absorbance measurement and the loR measurement are preferably performed on the same sample volume. This prevents changes in the liquid sample from affecting or biassing the measurement results. In practice, this means that the UV-Vis absorbance detector and the loR detector are preferably arranged such that the UV-Vis absorbance and loR measurement can be performed on the same sample volume.

[0073] More preferably, the UV-Vis absorbance measurement and the loR measurement are performed in such a manner that the composition of the liquid sample is unchanged between the UV-Vis absorbance measurement and the loR measurement. In practice, this means that the UV-Vis absorbance detector and the loR detector are preferably arranged in such a manner that the composition of the liquid sample is unchanged between the UV-Vis absorbance measurement and the loR measurement. In other words, the UV-Vis absorbance detector and the loR detector are preferably not separated by purification or other treatment steps performed on the liquid sample that would change the composition of the liquid sample.

[0074] In some embodiments, the UV-Vis absorbance measurement and the loR measurement are obtained in series.

[0075] In some embodiments, the UV-Vis absorbance measurement and the loR measurement are obtained simultaneously.

[0076] In some embodiments, the UV-Vis absorbance vs time measurement and the loR vs time measurement are obtained in the same flow cell.

[0077] The inventive method and system allow for the presence and / or quantity of protein aggregates in the sample to be continuously monitored in real time. In addition to monitoring a process, the obtained data can also be used to control the process in real time.

[0078] Fig. 1 shows a bioprocess system 100 comprising a bioreactor 110 and a bioprocess purification system 120. In some embodiments, the bioprocess purification system 120 is a chromatography system or a filtration system. A process stream flows from the bioreactor 110 through the bioprocess purification system 120 and the product 130 is collected.

[0079] The bioprocess system further comprises a protein aggregate detection system 140 for determining the presence and / or quantity of protein aggregates in a liquid sample comprising a protein. In various embodiments, the protein aggregate detection system 140 can be provided as an integrated single use (SU) component. Such a SU component may be formed using medical grade materials that are readily sterilisable (e.g. using gamma radiation).

[0080] The system for determining the presence and / or quantity of protein aggregates in a liquid sample comprising a protein comprises: an ultraviolet-visible spectroscopy (UV-Vis) absorbance detector 150 configured to obtain a UV-Vis absorbance vs time measurement of the sample; an index of refraction (loR) detector 160 configured to obtain an loR vs time measurement of the sample; and a data processing unit (DPU) 170 comprising one or more processors and one or more non-transitory computer-readable media storing instructions executable by the one or more processors.

[0081] The nature of the UV-Vis absorbance and loR detection allows for the UV-Vis absorbance detector and the loR detector to be arranged in-line or on-line in a bioprocess system, preferably in the bioprocess purification system.

[0082] In the embodiment of Fig. 1 , the UV-Vis absorbance detector 150 and the loR detector 160 are arranged on-line in the bioprocess purification system 110 of the bioprocess system 100. This means that the liquid sample is taken from the process stream, but not directly within the process stream. Instead, the liquid sample is diverted from the main process stream to the protein aggregate detection system 140 and then returned to the bioprocess purification system 110. This allows for continuous or near-continuous monitoring of the process without direct interference with the main process stream.

[0083] In alternative embodiments, the UV-Vis absorbance detector and the loR detector can be arranged in-line in a bioprocess system.

[0084] In the embodiment of Fig. 1 , the UV-Vis absorbance detector 150 and the loR detector 160 are arranged in the bioprocess purification system 120 to allow measurement on a fully purified or partially purified sample.

[0085] In an alternative embodiment the UV-Vis absorbance detector and the loR detector may be arranged after the bioprocess purification system to allow measurement on a fully purified sample. The bioprocess system may also be provided with two or more sets of UV-Vis absorbance detectors and loR detectors to allow a change in the presence and / or quantity of protein aggregates in a sample between two different stages of the bioprocess to be measured.

[0086] In the embodiment of Fig. 1 , the UV-Vis absorbance detector and the loR detector are arranged such that the UV-Vis absorbance and loR measurement can be performed on the same sample volume. The UV-Vis absorbance detector and the loR detector are also arranged in such a manner that the composition of the liquid sample is unchanged between the UV-Vis absorbance measurement and the loR measurement. In other words, the UV-Vis absorbance detector and the loR detector are not separated by purification or other treatment steps performed on the liquid sample that would change the composition of the liquid sample.

[0087] In the embodiment of Fig. 1 , the UV-Vis absorbance detector and the loR detector are arranged in series. In alternative embodiments, the UV-Vis absorbance detector and the loR detector can be arranged in parallel.

[0088] An advantage of the system of the present disclosure is that both of the measurements techniques relied upon are optical methods performed in a flow cell. In some embodiments, the UV-Vis absorbance detector and the loR detector are arranged to use the same flow cell.

[0089] The UV-Vis absorbance detector is configured to obtain a UV-Vis absorbance vs time measurement of the sample. A UV-Vis absorbance vs time measurement can be obtained by directing the liquid sample through a flow cell of the UV-Vis absorbance detector. The flow cell is designed to allow the liquid sample to pass through continuously while the measurement is being taken. The UV-Vis absorbance detector continuously monitors the sample as it flows through the cell, taking measurements at regular intervals. This creates a real-time data stream of absorbance values. The measurement is performed over a period of time, resulting in a UV-Vis absorbance vs time measurement.

[0090] The index of refraction (loR) detector is configured to obtain an loR vs time measurement of the sample. An loR vs time measurement can be obtained by directing the liquid sample through a flow cell of the loR detector. The flow cell is designed to allow the liquid sample to pass through continuously while the measurement is being taken. The loR detector continuously monitors the sample as it flows through the cell, taking measurements at regular intervals. This creates a real-time data stream of loR values. The measurement is performed over a period of time, resulting in an loR vs time measurement.

[0091] The protein aggregate detection system 140 further comprises a data processing unit 170 comprising one or more processors and one or more non-transitory computer-readable media storing instructions executable by the one or more processors.

[0092] Generally, the data processing unit 140 may comprise circuitry which is configured to implement (using one or more non-transitory computer-readable media) the functionality described herein. Suitable processors for the execution of a program of instructions include, by way of example, both general and special purpose microprocessors, and the sole processor or one of multiple processors or cores, of any kind of computer. The processors can be supplemented by, or incorporated in, ASICs (application-specific integrated circuits). Those skilled in the art will understand that the above-described exemplary embodiments may be implemented in any suitable software, hardware, or firmware configuration or combination thereof. An exemplary hardware platform for implementing the exemplary embodiments may include, for example, an Intel x86 based platform with compatible operating system, a Windows OS, a Mac platform and MAC OS, a mobile device having an operating system such as iOS, Android, etc. In a further example, the exemplary embodiments of the above-described method may be embodied as a program containing lines of code stored on a non-transitory computer readable storage medium that, when compiled, may be executed on a processor or microprocessor.

[0093] The instructions, when executed, cause the system to perform operations comprising: determining a peak value or an area under the curve (AUC) value for the UV-Vis absorbance vs time measurement; determining a peak value or an area under the curve (AUC) value for the loR vs time measurement; and determining the presence and / or quantity of protein aggregates in the sample based on a ratio between the peak value for the UV-Vis absorbance vs time measurement and the peak value for the loR vs time measurement or a ratio between the AUC value for the UV-Vis absorbance vs time measurement and the AUC value for the loR vs time measurement.

[0094] The peak value is the maximum absorbance recorded during the measurement period. This can be determined manually from a plot or using software algorithms that search for the maximum value in the dataset.

[0095] The area under the curve (AUC) value can be determined by: i) If necessary, performing baseline correction to account for any drift or background absorbance or loR. This can for example be done by subtracting a baseline absorbance or loR measured before the sample introduction from the absorbance or loR data, or by other suitable baseline subtraction methods known to the skilled person and often implemented in chromatography system software; and ii) Numerically integrating the absorbance or loR versus time data to calculate the AUC. This can be done using various numerical integration methods, such as the trapezoidal rule or Simpson's rule.

[0096] A ratio between the peak value for the UV-Vis absorbance vs time measurement and the peak value for the loR vs time measurement or a ratio between the AUC value for the UV-Vis absorbance vs time measurement and the AUC value for the loR vs time measurement is then determined. This ratio correlates with the quantity of protein aggregates in the sample and can be used to determine the presence or quantity of protein aggregates in the sample. The presence of protein aggregates in the sample can be assessed against a known ratio for an aggregate free sample. The quantity of protein aggregates in the sample can be assessed against a standard curve. The quantity of protein aggregates in the sample may preferably be determined as High Molecular Weight Percentage (HMW%). HMW% is defined as the proportion of protein molecules or aggregates in a sample that have a molecular weight higher than the expected or monomeric form of the protein. This percentage is calculated by comparing the quantity, for example in terms of mass, of these higher molecular weight species to the total protein content in the sample. The ratio between the value for the UV-Vis absorbance measurement and the value for the loR measurement depends on the fraction of aggregates while both UV and loR are affected by the total protein concentration. Thus, in some embodiments, the system determines the HMW% in the sample.

[0097] In some embodiments, the instructions, when executed, cause the system to perform operations comprising: determining the presence and / or quantity of protein aggregates in the sample based on a ratio between the peak value for the UV-Vis absorbance vs time measurement and the peak value for the loR vs time measurement.

[0098] In some embodiments, the instructions, when executed, cause the system to perform operations comprising: determining the presence and / or quantity of protein aggregates in the sample based on a ratio between the AUC value for the UV-Vis absorbance vs time measurement and the AUC value for the loR vs time measurement.

[0099] In some embodiments, the instructions, when executed, cause the system to perform operations comprising: determining the quantity of protein aggregates in the sample based on a ratio between the peak value for the UV-Vis absorbance vs time measurement and the peak value for the loR vs time measurement or a ratio between the AUC value for the UV-Vis absorbance vs time measurement and the AUC value for the loR vs time measurement.

[0100] Although the embodiment in Fig. 1 is described with reference to measuring UV-Vis absorbance and loR over time, and determining a peak value or an AUC value for the measurement, it is understood that the UV-Vis absorbance and loR may also be measured instantaneously and the ratio between the values for the instantaneous UV-Vis absorbance and loR measurements be used for determining the quantity of protein aggregates in the sample.

[0101] The system of the present disclosure may advantageously be used for monitoring, and optionally controlling a bioprocess. In such cases the steps for determining the presence and / or quantity of protein aggregates may preferably be repeated continuously over a period of time, such that a series of measurements is obtained. The series of measurements may preferably be a series of instantaneous measurements, but may also be a series of measurements over time to obtain a series of peak values or AUC values.

[0102] It is understood that all the embodiments described with reference to various embodiments of the present disclosure may be combined in any combination without departing from the present disclosure.

[0103] Additionally, variations to the disclosed embodiments can be understood and effected by the skilled person in practising the claimed invention, from a study of the drawing, the disclosure, and the appended claims. Moreover, in the drawings and specification, there have been disclosed preferred examples of the invention and, although specific terms are employed, they are used in a generic and descriptive sense only and not for the purpose of limitation. The scope of the invention is set forth in the following claims, in which the word ‘comprising’ does not exclude other elements or steps, and the indefinite article ‘a’ or ‘an’ does not exclude a plurality.

[0104] Example - Correlation between UV / loR signal ratio and mAb aggregate concentration

[0105] It was hypothesized that the ratio of UV absorption to the index of refraction (loR) of a monoclonal antibody (mAb) sample will differ depending on the concentration of mAb aggregates (HMW%). This experiment aimed to test this hypothesis.

[0106] Four samples of mAb10 in PBS buffer (HyClone, Cytiva) with differing aggregate concentrations were prepared. The aggregate concentrations (HMW%) were 0%, 4%, 10%, and 25%, respectively. In-line measurements of UV absorption and loR were conducted to assess the correlation between UV / loR signal ratio and mAb aggregate concentration.

[0107] An autosampler (Alias autoinjector) was used to inject 25 pl of each sample. The following instruments were used for the measurements:

[0108] • Akta pure 25 (Cytiva): for measuring UV absorbance at 280 nm.

[0109] • VE 3580 Refractive Index (Rl) detector (Viscotek): for measuring loR.

[0110] The flow path for the experiment was configured as follows:

[0111] • Autosampler — Injection valve — UV detector — Conductivity meter — Rl detector.

[0112] Connections between these components were made using tubing with an inner diameter of 0.5 mm. The internal temperature of the Rl detector was maintained at 35°C, with signal smoothing turned OFF. For the UV detector, the signal averaging time was set to 2.5 seconds. The flow rate throughout the experiment was maintained at 0.5 ml / min, with PBS buffer (HyClone, Cytiva) as the running buffer.

[0113] To ensure the Rl detector reached a stable baseline, it was flushed with 100 ml of PBS buffer before the first injection. Each injection was eluted with 10 ml of the running buffer.

[0114] The ratios between UV absorption at 280 nm and loR signal for the mAb samples containing different aggregate concentrations were calculated. The results are presented in Figures 2a (peak values), 2b (AUC values), and 2c (AUC values, logarithmic scale). These figures clearly illustrate the correlation between UV / loR signal ratio and mAb aggregate concentration.

[0115] The experiment confirmed a clear correlation between the UV / loR signal ratio and the concentration of mAb aggregates (HMW%), supporting the initial hypothesis.

Claims

CLAIMS:1 . A method for determining the presence and / or quantity of protein aggregates in a liquid sample comprising a protein, the method comprising: a) measuring ultraviolet-visible spectroscopy (UV-Vis) absorbance of the sample to obtain a UV-Vis absorbance measurement; b) measuring index of refraction (loR) of the sample to obtain an loR measurement; c) determining a value for the UV-Vis absorbance measurement; d) determining a value for the loR measurement; and e) determining the presence and / or quantity of protein aggregates in the sample based on a ratio between the value for the UV-Vis absorbance measurement and the value for the loR measurement.

2. The method according to claim 1 , wherein the protein is a monoclonal antibody (mAb) or a protein associated with a neurodegenerative disease, preferably a mAb.

3. The method according to any one of the preceding claims, wherein the measurement is performed in-line in a bioprocess system, preferably in a bioprocess purification system.

4. The method according to any one of the preceding claims, wherein the UV-Vis absorbance measurement and the loR measurement are obtained in series.

5. The method according to any one of the claims 1 -3, wherein the UV-Vis absorbance measurement and the loR measurement are obtained simultaneously.

256. The method according to any one of the preceding claims, wherein the UV-Vis absorbance measurement and the loR measurement are obtained in the same flow cell.

7. The method according to any one of the preceding claims, the method comprising: a) measuring ultraviolet-visible spectroscopy (UV-Vis) absorbance of the sample instantaneously to obtain an instantaneous UV-Vis absorbance measurement; b) measuring index of refraction (loR) of the sample instantaneously to obtain an instantaneous loR measurement; c) determining a value for the instantaneous UV-Vis absorbance vs time measurement; d) determining a value for the instantaneous loR measurement; and e) determining the presence and / or quantity of protein aggregates in the sample based on a ratio between the value for the instantaneous UV-Vis absorbance measurement and the value for the instantaneous loR measurement.

8. The method according to any one of claims 1 -6, the method comprising: a) measuring ultraviolet-visible spectroscopy (UV-Vis) absorbance of the sample over a period of time to obtain a UV-Vis absorbance vs time measurement; b) measuring index of refraction (loR) of the sample over a period of time to obtain an loR vs time measurement; c) determining a peak value for the UV-Vis absorbance vs time measurement; d) determining a peak value for the loR vs time measurement; ande) determining the presence and / or quantity of protein aggregates in the sample based on a ratio between the peak value for the UV-Vis absorbance vs time measurement and the peak value for the loR vs time measurement.

9. The method according to any one of claims 1 -6, the method comprising: a) measuring ultraviolet-visible spectroscopy (UV-Vis) absorbance of the sample over a period of time to obtain a UV-Vis absorbance vs time measurement; b) measuring index of refraction (loR) of the sample over a period of time to obtain an loR vs time measurement; c) determining an area under the curve (AUC) value for the UV-Vis absorbance vs time measurement; d) determining an area under the curve (AUC) value for the loR vs time measurement; and e) determining the presence and / or quantity of protein aggregates in the sample based on a ratio between AUC value for the UV-Vis absorbance vs time measurement and the AUC value for the loR vs time measurement.

10. The method according to any one of the preceding claims, wherein e) comprises determining the quantity of protein aggregates in the sample.

11. A system (140) for determining the presence and / or quantity of protein aggregates in a liquid sample comprising a protein, comprising: an ultraviolet-visible spectroscopy (UV-Vis) absorbance detector (150) configured to obtain a UV-Vis absorbance measurement of the sample; an index of refraction (loR) detector (160) configured to obtain an loR measurement of the sample; anda data processing unit (170) comprising one or more processors and one or more non-transitory computer-readable media storing instructions executable by the one or more processors, wherein the instructions, when executed, cause the system to perform operations comprising: determining a value for the UV-Vis absorbance measurement; determining a value for the loR measurement; and determining the presence and / or quantity of protein aggregates in the sample based on a ratio between the value for the UV-Vis absorbance measurement and the value for the loR measurement.

12. The system (140) according to claim 11 , wherein the UV-Vis absorbance detector (150) and the loR detector (160) are arranged in-line in a bioprocess system (100), preferably in a bioprocess purification system.

13. The system (140) according to claim 11 , wherein the UV-Vis absorbance detector (150) and the loR detector (160) are arranged on-line in a bioprocess system (100), preferably in a bioprocess purification system.

14. The system (140) according to any one of claims 11 -13, wherein the UV-Vis absorbance detector (150) and the loR detector (160) are arranged in series.

15. The system (140) according to any one of claims 11 -13, wherein the UV-Vis absorbance detector (150) and the loR detector (160) are arranged in parallel.

16. The system (140) according to any one of claims 11 -15, wherein the UV-Vis absorbance detector (150) and the loR detector (160) are arranged to use the same flow cell, optionally wherein said UV-Vis absorbance detector (150) and the loR detector (160) are integrated together into a single use (SU) flow cell.

17. The system (140) according to any one of claims 11-16, comprising:28an ultraviolet-visible spectroscopy (UV-Vis) absorbance detector configured to obtain an instantaneous UV-Vis absorbance measurement of the sample; an index of refraction (loR) detector configured to obtain an instantaneous loR measurement of the sample; and a data processing unit (170) comprising one or more processors and one or more non-transitory computer-readable media storing instructions executable by the one or more processors, wherein the instructions, when executed, cause the system to perform operations comprising: determining a value for the instantaneous UV-Vis absorbance measurement; determining a value for the instantaneous loR measurement; and determining the presence and / or quantity of protein aggregates in the sample based on a ratio between the value for the instantaneous UV-Vis absorbance measurement and the value for the instantaneous loR measurement.

18. The system (140) according to any one of claims 11-16, comprising: an ultraviolet-visible spectroscopy (UV-Vis) absorbance detector configured to obtain an UV-Vis absorbance vs time measurement of the sample; an index of refraction (loR) detector configured to obtain an instantaneous loR vs time measurement of the sample; and a data processing unit comprising one or more processors and one or more non- transitory computer-readable media storing instructions executable by the one or more processors, wherein the instructions, when executed, cause the system to perform operations comprising: determining a peak value for the UV-Vis absorbance vs time measurement;29determining a peak value for the loR vs time measurement; and determining the presence and / or quantity of protein aggregates in the sample based on a ratio between the peak value for the UV-Vis absorbance vs time measurement and the peak value for the loR vs time measurement.

19. The system (140) according to any one of claims 11-16, comprising: an ultraviolet-visible spectroscopy (UV-Vis) absorbance detector configured to obtain an UV-Vis absorbance vs time measurement of the sample; an index of refraction (loR) detector configured to obtain an instantaneous loR vs time measurement of the sample; and a data processing unit comprising one or more processors and one or more non- transitory computer-readable media storing instructions executable by the one or more processors, wherein the instructions, when executed, cause the system to perform operations comprising: determining an area under the curve (AUC) value for the UV-Vis absorbance vs time measurement; determining an area under the curve (AUC) value for the loR vs time measurement; and determining the presence and / or quantity of protein aggregates in the sample based on a ratio between AUC value for the UV-Vis absorbance vs time measurement and the AUC value for the loR vs time measurement.

20. The system (140) according to any one of claims 11 -19, wherein the instructions, when executed, cause the system to perform operations comprising: determining the quantity of protein aggregates in the sample.30

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