Lipolytic activity assay

Abstract

Methods for rapidly measuring the level of active enzymes within an mAb sample, wherein the enzymes present in the sample cleave an ester group of the disclosed substrates and a fluorescence signal results upon cleavage.

Description

25737 LIPOLYTIC ACTIVITY ASSAY CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 519,626, filed August 15, 2023, the entire contents of which are incorporated by reference herein.  FIELD

[0002] This disclosure relates generally to the field of phospholipase substrates and enzymes for testing lipolytic activity within biotherapeutic protein downstream samples. BACKGROUND OF THE INVENTION

[0003] Non-ionic surfactant polysorbate is an excipient in formulation solutions used as a shear protectant to stabilize biotherapeutics, prevent agitation-induced aggregation, and minimize surface adsorption.1-3Polysorbate-80 (PS-80) and polysorbate-20 (PS-20) are the most widely used polysorbates in the biopharma industry.2-3The degradation of polysorbate in a formulation could result in turbidity and potential formation of sub-visible particles, which may have a direct impact on product quality.1-2Hence, polysorbate degradation can bring critical quality attribute (CQA) out of specification in several different ways. Polysorbate degradation is an industry-wide challenge for process and formulation development in biotherapeutics.1-10The challenge is increasing with higher titer fermentation batches and higher drug concentration in formulation development.10-11There are multiple mechanisms of polysorbate degradation, which can be grouped into two categories: oxidation and hydrolysis.1,3The enzymatic hydrolysis pathway is primarily mediated by cleavage of an ester bond through lipases, esterases or other host cell proteins (HCPs). Those enzymes can co-purify with the therapeutic protein via specific or non- specific interactions.12Several lipases or esterases, such as LPL and LPLA2, have been reported to degrade PS-80 or PS-20 in biotherapeutics.4-7,10

[0004] Multiple analytical tools have been developed as part of an overall HCP control strategy.12Total HCP testing via traditional ELISA is currently considered the “gold standard”. An ELISA assay usually provides one summed value for total HCP content. Specific antibodies have been produced against and purified for known HCPs present in a sample.13-14LC-MS based proteomics approach is evolving rapidly as an orthogonal assay for HCP characterization.15-19Proteomics can identify and quantify practically any individual HCP, present at high enough levels, in the entire secretome to guide process development. Absolute quantification ofindividual HCPs can be achieved by multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM) targeted proteomics approaches with suitable internal standards.6,13However, the existing proteomics approaches have two main analytical challenges for root cause investigation of PS-80 degradation.

[0005] First, the detection limit of current proteomics strategies depends on HCP abundances. Most reported proteomics-based approaches have detection limits at the sub-ppm level (HCP compared to drug substance, i.e., 6 orders of magnitude difference), which is limited by the dynamic range of the mass spectrometer. Some lipases, such as LPLA2, can significantly degrade polysorbate even below sub-ppm level,6which is around or beyond the threshold of most proteomics platforms. Thus research efforts have been focused on reducing the dynamic range of samples to improve assay sensitivity, such as limited native digestion17-18, molecular weight cut- off20or 2D-LC17,19.

[0006] Second, enzyme abundance cannot tell the whole story of polysorbate degradation. Different lipases / esterases may have different enzyme activity and impact on rate of polysorbate degradation. Determining the enzyme activity is key in understanding the root-cause of the degradation. There is huge analytical gap between enzyme abundance and the correlated activity for polysorbate degradation in biotherapeutics process and formulation development.

[0007] Serine hydrolases are one of the largest known enzyme classes, comprising approximately 200 enzymes or 1% of the genes in the human proteome.21-22Included in this enzyme superfamily are multiple lipases, esterases, thioesterase, amidases and peptidases,22which are all potentially harmful for biotherapeutic drug substances and formulation excipients.The defining characteristic of serine hydrolase superfamily members is the presence of a nucleophilic serine in the active site. This site can be covalently labelled with chemical probes for enrichment and subsequently identified by mass spectrometry.21-24This approach has been used to identify active serine hydrolases and screen inhibitors for serine hydrolases.25-28There is a need to explore and improve the application of this activity-based chemical proteomics approach for active enzyme profiling to investigate polysorbate degradation in biotherapeutic formulations.

[0008] Commercially available kits, like EnzChekTM, have also been reported to be highly sensitive against LPL and can be useful tool for monitoring lipase activity. EnzChekTM provides for measuring phospholipase A1(PLA1) and phospholipase A2(PLA2).29However, such commercially available kits have multiple steps that are laborious. Therefore, improved protocol methods for the rapid high-throughput detection of these enzymes are needed to investigate polysorbate degradation in biotherapeutic formulations.SUMMARY OF THE INVENTION

[0009] Disclosed are methods for rapidly measuring the level of active enzymes within a biotherapeutic protein sample. The enzymes present in the sample cleave an ester group of phospholipase A1 (PLA1) and phospholipase A2 (PLA2) substrates of the enzyme and a fluorescence signal results upon cleavage.

[0010] In a first aspect, the disclosure provides a method for rapidly measuring the level of active phospholipase enzyme within a biotherapeutic protein sample comprising a) forming a solution comprising: dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidglycerol (DOPG), and a substrate for phospholipase having a cleavable fluorescent marker linked via an ester bond; b) vortexing the solution; and c) adding the vortexed solution to a biotherapeutic protein sample in a vessel suitable for measurement of fluorescence and measuring the fluorescence.

[0011] In an embodiment of the first aspect, the fluorescence is measured using a microplate reader. In one class of this aspect, the microplate reader has excitation 482 nm, emission 515 nm and cutoff 495 nm.

[0012] In another embodiment of the first aspect, the phospholipase enzyme is selected from LPL enzyme and PLA2G7 enzyme.

[0013] In another embodiment of the first aspect, the substrate is selected from (A) and (B).the substrate is vortexed for 10-30 seconds.

[0015] In another embodiment of the first aspect, the biotherapeutic protein samples are stored frozen at -80 ^C.

[0016] In another embodiment of the first aspect, the biotherapeutic protein samples are thawed and stored for up to 28 days before measuring the level of lipolytic activity within the sample.

[0017] In another embodiment of the first aspect, the biotherapeutic protein samples are thawed and stored at 4 ^C before measuring the lipolytic activity within the sample.

[0018] The summary of the technology described above is non-limiting and other features and advantages of the technology will be apparent from the following detailed description, and from the claims.BRIEF DESCRIPTION OF THE FIGURES

[0019] Figure 1 of the example shows the PLA1and PLA2enzyme activity standard curves across 4 separate runs.

[0020] Figure 2 of the example shows activity measurement using the PLA1 kit across two batches of mAb 10 UFP (no PS-80) and BRFL (PS-80 present).

[0021] Figure 3 of the example shows the measured slope across the PLA1 and PLA2 kits using various buffers as an assay diluent. Across the typical downstream buffers provided a wide range of impact is seen.

[0022] Figure 4 of the example provides an example sample set for how the PLA1and PLA2kit generated data was evaluated to determine if both kits are required for routine testing.

[0023] Figure 5 of the example shows the measured activity across mAb6 samples over 28 days storage at 4°C. mAb1 PAP is included as a control and was freshly thawed on each day.

[0024] Figure 6 of the example shows the measured activity for mAb6 samples across multiple freeze / thaw cycles. DETAILED DESCRIPTION OF THE PRESENT LIPOLYTIC ASSAY

[0025] To improve existing lipolytic activity assays, the inventors adjusted the steps of existing lipolytic activity assay standards. The inventors designed the lipolytic activity assay to rapidly determine lipolytic activity of mAb samples by combining protocol steps. The resultant assay was then validated by calculating S / N and % recovery of each standard. Definitions

[0026] Listed below are definitions of various terms used herein. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

[0028] As used herein, the articles “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0029] As used herein, the term “comprising” may include the embodiments “consisting of” and “consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “may,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions or processes as “consisting of” and “consisting essentially of” the enumerated components, which allows the presence of only the named components or compounds, along with any acceptable carriers or fluids, and excludes other components or compounds.

[0030] Reference throughout this specification to “embodiment” or "an embodiment," means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Abbreviations 4PL 4-parameter logistics BRFL bioburden reduction filtration load CV coefficient of variance DMSO dimethyl sulfoxide DOPC dioleoylphosphatidylcholine DOPG dioleoylphosphatidglycerol F / T freeze / thaw FNVIP filtered neutralized viral-inactivation pool FRET forster resonance energy transfer HCCF host-cell culture fluid LAA lipolytic activity assay LPL lipoprotein lipase mAb monoclonal antibody NaCl sodium chloride NVIP neutralized viral-inactivation pool PAP protein A poolPBS phosphate buffered saline PLA2G7 phospholipase A2 group VII PS-80 polysorbate-80 S / N signal-to-noise ratio SST system suitability test Tris tris(hydroxymethyl)aminomethane UFP ultrafiltration pool REFERENCE EXAMPLE 1

[0031] Commercially available EnzChekTM PLA1Assay Kit is useful for monitoring PLA1activity. PLA1 represents a family of enzymes that hydrolyze the sn-1 ester linkage of phospholipids and fatty acids. The Phospholipase A1substrate PED-A1 (A): , provides sensitiveactivities.

[0032] The EnzChekTM Phospholipase A1substrate is specific for PLA1with fluorescence dye-labeled acyl chain at the sn-1 position and dinitrophenyl quencher-modified head group. Quenching efficiency is decreased by cleavage of the acyl chain at the sn-1 position. The result is a PLA1-dependent increase in fluorescence emission detected at approximately 515 nm. Specificity is imparted by the placement of the acyl chain in the sn-1 position and by incorporation of an acyl group with an enzymatic resistant (non-cleavable) ether linkage in the sn-2 position. Preparing solutions

[0033] 2 mM EnzChekTM PLA1 Substrate was prepared by allowing one vial of PLA1 substrate and DMSO to warm to room temperature. The contents of one vial of PLA1substrate were dissolved in 40 μL DMSO.1X EnzChekTM PLA1 reaction buffer was prepared by adding 4 mL of 5X PLA1reaction buffer to 16 mL deionized water.10 mM DOPC (Dioleoylphosphatidylcholine) was prepared by dissolving the contents of the DOPC vial in 100 μL ethanol.10 mM DOPG (Dioleoylphosphatidylglycerol) was prepared by dissolving thecontents of the DOPG vial in 100 μL ethanol.500 Units / mL PLA1stock solution was prepared by dissolving the contents of the PLA1 vial in 100 μL of 1X PLA1 reaction buffer. Assay protocol The assay was performed comprising the steps of:

[0034] 1) Preparing a PLA1standard curve by diluting the appropriate amount of 500 Units / mL PLA1 stock solution to 10 Units / mL in 1X PLA1 reaction buffer to produce PLA1 concentrations of 0–10 Units / mL, each in a volume of 50 μL.

[0035] 2) the PLA1-containing samples were then diluted in PLA1 reaction buffer.50 μL sample was required for each reaction.

[0036] 3) 50 μL of the controls and experimental samples were pipetted into individual wells of a microplate.

[0037] 4) The Lipid Mix was prepared by mixing together 30 μL 10 mM DOPC, 30 μL 10 mM DOPG, and 30 μL 2 mM PLA1substrate.

[0038] 5) 5 mL 1X PLA1 reaction buffer was added to a 20 mL beaker containing a small magnetic stir bar and the beaker was placed on a magnetic stirrer to form a vortex. To prepare 5 mL substrate-liposome for 100 assays, slowly and steadily (over about 1 minute) 50 μL of Lipid Mix was injected into the side of the vortex using a pipettor fitted with a narrow orifice gel- loading tip.

[0039] 6) 50 μL of the substrate-liposome mix was added to each microplate well containing standards, controls, and samples to start the reaction.

[0040] 7) The microplate was incubated at room temperature for 30 minutes, protected from light. Fluorescence was measured at multiple time points to follow the kinetics of the reactions.

[0041] 8) The fluorescence was measured using a microplate reader equipped for excitation in the range of 450–490 nm and fluorescence emission at ~515 nm.

[0042] 9) For each point, the value derived from the no-PLA1 control was subtracted to correct for background fluorescence. REFERENCE EXAMPLE 2

[0043] The EnzChekTM Phospholipase A2 Assay Kit provides a fluorometric method designed for continuous monitoring of phospholipase A2(PLA2). PLA2represents a family of enzymes that hydrolyze the sn-2 ester linkage of phospholipids. The enzyme assay can be used in an intensity or ratiometric based detection mode providing a simple method with low background, high sensitivity, and high specificity for PLA2. The Phospholipase A2 substrate (B):, provides sensitive and enzyme activities.Preparing Solutions

[0044] 1 mM EnzChekTM PLA2 Substrate was prepared by allowing one vial of PLA2 substrate and DMSO to warm to room temperature. The contents of one vial of PLA2substrate were dissolved in 40 µL DMSO. The stock solution was stored and frozen at ≤–20°C, protected from light.1X EnzChekTM PLA2reaction buffer was prepared by adding 4 mL of 5X PLA2reaction buffer to 16 mL deionized water.10 mM DOPC was prepared by dissolving the contents of the DOPC vial in 100 μL ethanol. The solution was then stored at ≤–20°C.10 mM DOPG was prepared by dissolving the contents of the DOPG vial in 100 μL ethanol. The solution was then stored at ≤–20°C.500 Units / mL PLA2 stock solution was prepared by dissolving the contents of the PLA2 vial in in 80 μL of 1X PLA2 reaction buffer prepared above. The stock solution was then stored at 4°C. Assay Protocol The assay was performed comprising the steps of:

[0045] 1) Preparing a PLA2 standard curve by diluting the appropriate amount of 500 Units / mL PLA2stock solution to 10 Units / mL in 1X PLA2reaction buffer to produce PLA2concentrations of 0–10 Units / mL, each in a volume of 50 μL.

[0046] 2) The PLA2-containing samples were then diluted in PLA2reaction buffer.50 μL sample was required for each reaction.

[0047] 3) 50 μL of the controls and experimental samples were pipetted into individual wells of a microplate.

[0048] 4) The Lipid Mix was prepared by mixing together 30 μL 10 mM DOPC, 30 μL 10 mM DOPG, and 30 μL 1 mM PLA2 substrate.

[0049] 5) 5 mL 1X PLA2reaction buffer was added to a 20 mL beaker containing a small magnetic stir bar and place the beaker on a magnetic stirrer to form a vortex. To prepare 5 mL substrate-liposome for 100 assays, slowly and steadily (over about 1 minute) 50 μL of Lipid Mix25737 was injected into the side of the vortex using a pipettor fitted with a narrow orifice gel-loading tip.

[0050] 6) 50 μL of the substrate-liposome mix was added to each microplate well containing standards, controls, and samples to start the reaction.

[0051] 7) The microplate was incubated at room temperature for 10 minutes, protected from light.

[0052] 8) Fluorescence was measured using a microplate reader equipped for excitation in the range of 450–490 nm and fluorescence emission at ~515 nm and ~575 nm. For ratiometric (FRET) detection, the ratio of the emission 515 nm / 575 nm was used. For single wavelength intensity detection, the emission at ~515 nm was used.

[0053] 9) For each point, the value derived from the no-PLA2 control was subtracted to correct for background fluorescence. EXAMPLE

[0054] The following example is meant to be illustrative and should not be construed as further limiting. The contents of the figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference. Improved protocol methods for routine testing of lipolytic activity within mAb downstream samples.

[0055] To rapidly measure the level of active enzymes within an mAb sample, the steps of the commercial EnzChekTM protocols were adjusted so that the substrate / DOPC / DOPG was added all at once, followed by vortexing for 10-30 seconds at high speeds, resulting in improved protocol methods. Table 2 provides a comparison of the kit protocol and Internal protocol for combining substrate / DOPC / DOPG with 1X reaction buffer. To evaluate, the S / N and % recovery of each standard was calculated. For mAb1 PAP, the measured activity was calculated using the kit and the improved protocol methods. Both approaches gave consistent performance across both standards with respect to S / N. The same result is seen for % recovery for the standards, with the improved protocol methods showing better % Recovery at the lower end of the PLA2 standard curve. The measured PLA1 activity for mAb1 PAP was not impacted by the approach taken for mixing the substrate / DOPC / DOPG solution with 1X reaction buffer. Based on this set of data, the improved protocol methods were implemented by mixing and vortexing to combine substrate / DOPC / DOPG with 1X substrate buffer.25737

[0056] Performance of the different microplate reader wavelength settings are summarized in Table 3. S / N, % Recovery, and measured activity are evaluated across the standard curve along with using mAb1 PAP as the test sample. For S / N, the improved protocol methods gave noticeable improvement for PLA1 with minimal improvement for PLA2. The % recovery for both PLA1 and PLA2 showed comparable performance between the kit and improved protocol methods. Combined with no significant difference in measured activity for the mAb1 PAP sample, the improved protocol methods were implements for all future routine testing. Materials and methods

[0057] Lab reagents included ethanol (Sigma-Aldrich), 1x PBS pH 7.4 (Gibco), and Milli-Q grade water (Millipore Sigma). The EnzChek PLA1 and PLA2 Assay kits were purchased from Invitrogen, components listed in Table 1. The EnzChekTM Lipase Substrate, an alternative substrate to PLA1 and PLA2substrates, was also purchased from Invitrogen. In addition to the kit- included enzymes, purified enzymes lipoprotein lipase (LPL, R&D Systems) and phospholipase A2 group VII (PLA2G7, Abcam) were purchased. Other common lab supplies include black 384 well, clear bottom plates (Thermo Scientific) and MicroAmp Optical Adhesive Film (Applied Biosystems). A SpectraMax M5e (Molecular Devices) along with SoftMax Pro (v6, Molecular Devices) were used to collect and evaluate data. Table 1: The components for the EnzChekTM PLA1and PLA2kits. Component A1Kit A2Kit A Ph h li A Ph h li AMeasuring Lipolytic Activity

[0058] The lipolytic activity assay quantified the level of active enzymes within a sample that can cleave the ester group of the substrate within the PLA1 kit and PLA2 kit. Upon cleavage of the ester group, the fluorescence dyed moiety and quencher / FRET moiety separate, leading to fluorescence signal. The PLA1 substrate has an ester bond in the sn-1 position, making it more specific toward PLA1class enzymes. Similarly, the PLA2substrate has an ester bond in the sn-2 position, making it more specific toward PLA2 class enzymes30.25737

[0059] Reagents found in the EnzChekTM PLA1and PLA2kits were used for the improved protocol methods. Milli-Q grade water was used to dilute the 5X reaction buffer. Ethanol was used to reconstitute DOPC and DOPG.1x PBS pH 7.4 was used to pre-dilute samples having activity above the assay range to ensure measured activity is within assay range. Quantitation was performed by generating a standard curve using serial dilutions of a provided enzyme with a known activity.

[0060] Samples were either plated neat or diluted along with the serially-diluted standard on a preparation plate. The 1X reaction buffer was mixed 1:1 with samples and standards in the plate. A mixture of reconstituted DOPC, DOPG, substrate, and 1X reaction buffer is then added 1:1 with samples and standards in the preparation plate. Sample and standard solutions are transferred from the preparation plate (single well) to a 384-well plate (duplicate wells) for data acquisition. Datum from each well was collected using bottom-reading fluorescence with the following parameters: Excitation 482 nm, Emission 515 nm, Cutoff 495 nm. The microplates are read every 10 minutes over a 15 hour period for a total of 91 data points. The microplate reader is kept at room temperature during this 15 hour period. The slope of each standard is measured across these 91 data points and is used to generate a standard curve, linking slope to activity. Based off the slope across the 91 points for each sample, an activity value is back-calculated using the standard curve.

[0061] The structure of the (A) PLA1 substrate and (B) PLA2 substrate are illustrated below. Location of the ester bond is switched between sn-1 and sn-2 to alter specificity of the two substrates.

[0062] It was determined that after extended use of both the PLA1and PLA2kits, two steps could be adjusted to improve in-lab execution and assay performance. The step of the protocol that added the most effort and time during in-lab execution, was the addition of the substrate / DOPC / DOPG solution into the 1X reaction buffer. To streamline this step, an experiment was performed to evaluate the impact of adding the substrate / DOPC / DOPG all at once, followed by vortexing at high speeds for 10-30 seconds had on assay performance. In addition, the excitation wavelength used to collect data was investigated. The kit suggested25737 parameters were Excitation 460 nm / Emission 515 nm while the newly proposed parameters are (Excitation 482 nm / Emission 515 nm). For both microplate reader settings, a cutoff of 495 nm was used.

[0063] The comparison of the kit protocol and improved protocol for combining substrate / DOPC / DOPG with 1X reaction buffer are provided in Table 2. To evaluate if any difference between approaches exists, the S / N and % recovery of each standard was calculated. For mAb1 PAP the measured activity was calculated using the kit and improved protocol methods (vortexing). With respect to S / N, both approaches give consistent performance across both standards. The same result was seen for % recovery for the standards, with the improved protocol methods showing slightly better % Recovery at the lower end of the PLA2standard curve. The measured PLA1 activity for mAb1 PAP was not impacted by the approach taken for mixing the substrate / DOPC / DOPG solution with 1X reaction buffer. Based on this set of data it was proposed that future testing would use the improved protocol methods by simply mixing and vortexing to combine substrate / DOPC / DOPG with 1X substrate buffer.

[0064] Performance of the different microplate reader wavelength settings are summarized in Table 3. S / N, % Recovery, and measured activity are evaluated across the standard curve along with using mAb1 PAP as the test sample. For S / N, the improved protocol method parameters give noticeable improvement for PLA1with minimal improvement for PLA2. Looking at % recovery for both PLA1 and PLA2 there was comparable performance between the kit and improved protocol method parameters. Combined with no significant difference in measured activity for the mAb1 PAP sample, this led to selection of the newly proposed parameters for routine testing. Table 2A-2B. Summary of data collected comparing the kit protocol method for mixing substrate / DOPC / DOPG and 1X reaction buffer with using the Internal protocol. Table 2A Standards Signal to Noise % Recovery d25737 0.103 1 1.1 1.5 1.2 N / A N / A N / A N / A Table 2B Standards Measured Activity & Samplesata collected comparing the kit protocol wavelength parameters with Internal wavelength parameters. Internal parameter provides improved S / N for both PLA1 and PLA2. Table 3A Standards Signal to Noise % Recovery & dTable 3B Standards Measured Activity & dSubstrate Specificity

[0065] Confirmation of the specificity of the substrate was performed using commercially available LPL and PLA2G7. The kit substrate is designed to be preferential toward either the PLA1 or PLA2 cleavage mechanism30. The two commercial enzymes are preferential toward25737 either the PLA1(LPL) or PLA2(PLA2G7) cleavage31,32. Each enzyme was diluted from 10-fold to 106-fold dilution using a 10-fold serial dilution. Each dilution of enzyme was tested against both A1and A2substrate and measured activity was calculated as previously discussed.

[0066] Results are outlined in Table 4, showing the dilutions that gave signal across either substrate. Dilutions with signal outside both PLA1 and PLA2 standard curves were not included. The LPL enzyme produces signal with the PLA1substrate but no signal with the PLA2substrate. The PLA2G7 enzyme produces signal with both PLA1 and PLA2 substrates but with higher measured activity for the PLA2substrate at 1,000-fold dilution. The lack of signal for LPL with PLA2 substrate demonstrates the specificity between both substrates with alignment that LPL should cleave PLA1substrate preferentially. For PLA2G7, higher activity is measured at 1,000- fold dilution for the PLA2 substrate, highlighting the higher specificity compared to the PLA1 substrate.

[0067] However, when observing typical assay performance, the PLA1 standard curve provided higher signal and thus higher sensitivity than the PLA2standard curve (Figure 1). Due to this higher sensitivity for the PLA1 kit, measurable signal was seen for PLA2G7 with the PLA1 substrate at 10,000-fold dilution while the PLA2substrate does not produce a measurable signal. Based on this result, the PLA1 kit performed better than the PLA2 kit. Table 4: Shows the measured activity for LPL and PLA2G7 enzymes at various dilutions for both PLA1and PLA2substrates. LPL digests PLA1substrate at a significantly higher rate than PLA2. PLA2G7 digests PLA2 substrate at a significantly higher rate than PLA1. Enzyme Fold nA1 substrate A2 substrate Dilutiong / mL(mU / mL) (mU / mL)Excipient and Matrix Interference

[0068] Interference of the excipient polysorbate-80 (PS-80), along with impact of various matrices was evaluated for both PLA1and PLA2kits. To determine if PS-80 impacted the assay, two mAb10 samples were tested: one without PS-80 (UFP) and one with PS-80 (BRFL). While there are other minor differences between UFP and BRFL, these are not considered critical for the assay. To gauge the impact of different matrices, 10 commonly used downstream purification matrices were obtained and run as the assay diluent for both PLA1and PLA2kits. The different25737 matrices were evaluated based on the intensity of slope across recorded data points with higher slope being indicative of higher activity.

[0069] In Figure 2, two different batches of mAb10 UFP were tested and showed measurable activity for PLA1. However, when these two samples are formulated with PS-80 (BRFL) and tested there was no longer any measurable activity. This confirms that the presence of PS-80 competitively inhibits the assay. This is expected based on the proposed mechanism of the assay and helps to inform on what type of samples can be tested. While the PLA2 kit was also used during testing, no sample gave any measurable activity. The results from the evaluation of various matrices are presented in Figure 3. Across the provided downstream purification buffers, the highest activity for both A1and A2is with 25 mM tris pH 7.5. However, the best result for both A1 and A2 is the kit provided buffers. Across the downstream purification matrices there is a dramatic and varying impact on assay performance (example: 20 mM sodium acetate pH 4.6 vs 25 mM tris pH 7.5). To reduce the potential impact during routine testing, all samples will be diluted at least 4-fold prior to collecting data using the kit provided reaction buffer. Substrate Selection

[0070] To streamline routine testing using this assay, evaluation of the PLA1and PLA2kits was performed to determine if only a single substrate could be used without loss of information. Measured data (slope across assay runtime) using both kits was generated for 9 different mAbs (mAbs1-9) across 3 different cell lines. The measured slope values generated for a sample set using the PLA1 kit were plotted against the values generated using the PLA2 kit, as demonstrated in Figure 4. A linear regression was performed to evaluate the overall trend agreement between the two sets of results. From this graph an R2value of 0.7-1.0 was deemed as not gaining additional information from running both kits. In this situation the data-driven decisions are the same regardless of which kit is used to generate data. An R2value of 0.0-0.6 was seen as gaining additional information from running both kits. In this situation the data-driven decisions could vary depending on what kit was used to generate data. Through this we evaluated if both kits were needed to support routine testing.

[0071] Table 6 shows PLA1 and PLA2 trending across up to 24 data sets. Considering all the data sets, there was a portion (38%) where results from PLA1kit and PLA2kit did not provide a comparable trend (R2of 0.0-0.6), meaning there would still be value in generating data using both PLA1and PLA2kits. However, most of these instances are due to HCCF-related samples. If HCCF-related data sets are removed, we see only 1 (6%) data set that gives R2below 0.7.25737 Looking specifically at this data set, the low R2is due to a single sample, which when removed results in R2= 0.99. Overall, this gives high confidence that only one kit needs to be used for routine testing, which would not regularly run HCCF-related samples.

[0072] The PLA1 substrate provided higher sensitivity associated with both PLA1-specific and PLA2-specific enzymes than the PLA2 substrate. The improved robustness and sensitivity of the PLA1kit is why it was chosen for routine testing. Table 6. Shows the number of data sets fitting between R2= 0.7-1.0 and 0.0-0.6 when plotting PLA1 slope against PLA2 slope. This is further broken down depending on if HCCF samples are included or excluded. Results R2= 0.7-1.0 R2= 0.0-0.6 HCCF Included (n=24) 15 9o age e a ee e aw a y

[0073] To evaluate both storage stability and freeze / thaw (F / T) cycle impact on measurements, intermediates across the downstream process for mAb6 were tested. mAb1 PAP was tested fresh as a control across each day of analysis for storage stability. For storage stability, samples were stored at 4 ^C with timepoints taken at t= 0, 14, and 28 days. The day 0 measurement is from newly generated material which had not been previously stored. To test F / T cycle, the day 0 material was aliquoted and serial thawed at room temperature / re-frozen at -80 ^C up to n=4 times.

[0074] Generally, one month of storage stability at 4 ^C is preferred for method ATP to ensure flexibility for routine testing. The sample storage stability study for the improved protocol methods was designed to support this requirement. Figure 5 provides the measured activity for each intermediate stored at 4 ^C, up to 28 days. % change on day 14 and day 28 compared to day 0 are summarized in Table 7. While there is variability across the trends (up to ±50% change), there does not seem to be any consistent change across the storage time. The data demonstrates that storage at 4 ^C for up to 28 days does not have a significant impact on measured activity. Based on this result, it is proposed that samples to be tested for LAA can be stored at 4 ^C up to 4 weeks.

[0075] Figure 6 provides measured activity for each intermediate at F / T cycles n=0 (never frozen), 2, 3, and 4 and % change from F / T 0 to F / T 4 are also summarized in Table 8. There is inherent variability with the assay, however there was no significant decrease in measured25737 activity across F / T cycles. For the HCCF sample the % change from F / T 0 ^2 was more significant. Based on this, there was a potential true change in activity with increased F / T cycles for the HCCF sample. Based on this and other results, routine testing was suggested to not include HCCF. Information gathered across other intermediates provided the necessary data needed to properly inform downstream process understanding and development. Table 7. Shows the % change of measured activity from day 0 for both days 14 and 28. Intermediate% Change% Change Day 14Day 28. g ivity from freeze / thaw cycle 0 to 4. Most changes in measured activity falls within expected assay variation without a consistent trend. However, the HCCF change from F / T 0 ^ 2 may be due to other variables. % Change Intermediate F / T 0 ^ F / T 2 ^ F / T 3 ^25737 References 1. Dwivedi, M.; Blech, M.; Presser, I.; Garidel, P., Polysorbate degradation in biotherapeutic formulations: Identification and discussion of current root causes. Int J Pharm 2018, 552 (1-2), 422-436. 2. Kerwin, B. A., Polysorbates 20 and 80 used in the formulation of protein biotherapeutics: structure and degradation pathways. J Pharm Sci 2008, 97 (8), 2924-35. 3. Kishore, R. S.; Kiese, S.; Fischer, S.; Pappenberger, A.; Grauschopf, U.; Mahler, H. C., The degradation of polysorbates 20 and 80 and its potential impact on the stability of biotherapeutics. Pharm Res 2011, 28 (5), 1194-210. 4. Dixit, N.; Salamat-Miller, N.; Salinas, P. A.; Taylor, K. D.; Basu, S. K., Residual Host Cell Protein Promotes Polysorbate 20 Degradation in a Sulfatase Drug Product Leading to Free Fatty Acid Particles. J Pharm Sci 2016, 105 (5), 1657-1666. 5. Chiu, J.; Valente, K. N.; Levy, N. E.; Min, L.; Lenhoff, A. M.; Lee, K. H., Knockout of a difficult-to-remove CHO host cell protein, lipoprotein lipase, for improved polysorbate stability in monoclonal antibody formulations. Biotechnol Bioeng 2017, 114 (5), 1006-1015. 6. Hall, T.; Sandefur, S. L.; Frye, C. C.; Tuley, T. L.; Huang, L., Polysorbates 20 and 80 Degradation by Group XV Lysosomal Phospholipase A2 Isomer X1 in Monoclonal Antibody Formulations. J Pharm Sci 2016, 105 (5), 1633-1642. 7. McShan, A. C.; Kei, P.; Ji, J. A.; Kim, D. C.; Wang, Y. J., Hydrolysis of Polysorbate 20 and 80 by a Range of Carboxylester Hydrolases. PDA J Pharm Sci Technol 2016, 70 (4), 332-45. 8. Tomlinson, A.; Demeule, B.; Lin, B.; Yadav, S., Polysorbate 20 Degradation in Biopharmaceutical Formulations: Quantification of Free Fatty Acids, Characterization of Particulates, and Insights into the Degradation Mechanism. Mol Pharm 2015, 12 (11), 3805-15. 9. Cao, X.; Fesinmeyer, R. M.; Pierini, C. J.; Siska, C. C.; Litowski, J. R.; Brych, S.; Wen, Z. Q.; Kleemann, G. R., Free fatty acid particles in protein formulations, part 1: microspectroscopic identification. J Pharm Sci 2015, 104 (2), 433-46. 10. Labrenz, S. R., Ester hydrolysis of polysorbate 80 in mAb drug product: evidence in support of the hypothesized risk after the observation of visible particulate in mAb formulations. J Pharm Sci 2014, 103 (8), 2268-77. 11. Tran, B.; Grosskopf, V.; Wang, X.; Yang, J.; Walker, D., Jr.; Yu, C.; McDonald, P., Investigating interactions between phospholipase B-Like 2 and antibodies during Protein A chromatography. J Chromatogr A 2016, 1438, 31-8.25737 12. Bracewell, D. G.; Francis, R.; Smales, C. M., The future of host cell protein (HCP) identification during process development and manufacturing linked to a risk-based management for their control. Biotechnol Bioeng 2015, 112 (9), 1727-37. 13. Gao, X.; Rawal, B.; Wang, Y.; Li, X.; Wylie, D.; Liu, Y. H.; Breunig, L.; Driscoll, D.; Wang, F.; Richardson, D. D., Targeted Host Cell Protein Quantification by LC-MRM Enables Biologics Processing and Product Characterization. Anal Chem 2019. 14. Fischer, S. K.; Cheu, M.; Peng, K.; Lowe, J.; Araujo, J.; Murray, E.; McClintock, D.; Matthews, J.; Siguenza, P.; Song, A., Specific Immune Response to Phospholipase B-Like 2 Protein, a Host Cell Impurity in Lebrikizumab Clinical Material. AAPS J 2017, 19 (1), 254-263. 15. Vanderlaan, M.; Zhu-Shimoni, J.; Lin, S.; Gunawan, F.; Waerner, T.; Van Cott, K. E., Experience with host cell protein impurities in biopharmaceuticals. Biotechnol Prog 2018, 34 (4), 828-837. 16. Walker, D. E.; Yang, F.; Carver, J.; Joe, K.; Michels, D. A.; Yu, X. C., A modular and adaptive mass spectrometry-based platform for support of bioprocess development toward optimal host cell protein clearance. MAbs 2017, 9 (4), 654-663. 17. Kufer, R.; Haindl, M.; Wegele, H.; Wohlrab, S., Evaluation of Peptide Fractionation and Native Digestion as Two Novel Sample Preparation Workflows to Improve HCP Characterization by LC-MS / MS. Anal Chem 2019, 91 (15), 9716-9723. 18. Huang, L.; Wang, N.; Mitchell, C. E.; Brownlee, T.; Maple, S. R.; De Felippis, M. R., A Novel Sample Preparation for Shotgun Proteomics Characterization of HCPs in Antibodies. Anal Chem 2017, 89 (10), 5436-5444. 19. Yang, F.; Walker, D. E.; Schoenfelder, J.; Carver, J.; Zhang, A.; Li, D.; Harris, R.; Stults, J. T.; Yu, X. C.; Michels, D. A., A 2D LC-MS / MS Strategy for Reliable Detection of 10- ppm Level Residual Host Cell Proteins in Therapeutic Antibodies. Anal Chem 2018, 90 (22), 13365-13372. 20. Chen, I. H.; Xiao, H.; Daly, T.; Li, N., Improved Host Cell Protein Analysis in Monoclonal Antibody Products through Molecular Weight Cutoff Enrichment. Anal Chem 2020, 92 (5), 3751-3757. 21. Simon, G. M.; Cravatt, B. F., Activity-based proteomics of enzyme superfamilies: serine hydrolases as a case study. J Biol Chem 2010, 285 (15), 11051-5. 22. Long, J. Z.; Cravatt, B. F., The metabolic serine hydrolases and their functions in mammalian physiology and disease. Chem Rev 2011, 111 (10), 6022-63. 23. Adam, G. C.; Sorensen, E. J.; Cravatt, B. F., Chemical strategies for functional proteomics. Mol Cell Proteomics 2002, 1 (10), 781-90.25737 24. Sanman, L. E.; Bogyo, M., Activity-based profiling of proteases. Annu Rev Biochem 2014, 83, 249-73. 25. Zweerink, S.; Kallnik, V.; Ninck, S.; Nickel, S.; Verheyen, J.; Blum, M.; Wagner, A.; Feldmann, I.; Sickmann, A.; Albers, S. V.; Brasen, C.; Kaschani, F.; Siebers, B.; Kaiser, M., Activity-based protein profiling as a robust method for enzyme identification and screening in extremophilic Archaea. Nat Commun 2017, 8, 15352. 26. Lentz, C. S.; Sheldon, J. R.; Crawford, L. A.; Cooper, R.; Garland, M.; Amieva, M. R.; Weerapana, E.; Skaar, E. P.; Bogyo, M., Identification of a S. aureus virulence factor by activity-based protein profiling (ABPP). Nat Chem Biol 2018, 14 (6), 609-617. 27. Niphakis, M. J.; Cravatt, B. F., Enzyme inhibitor discovery by activity-based protein profiling. Annu Rev Biochem 2014, 83, 341-77. 28. Liu, Y.; Patricelli, M. P.; Cravatt, B. F., Activity-based protein profiling: the serine hydrolases. Proc Natl Acad Sci U S A 1999, 96 (26), 14694-9. 29. Basu, D.; Manjur, J.; Jin, W. Determination of lipoprotein lipase activity using a novel fluorescent lipase assay. J. Lipid Res.2011, 52, 826-832. 30. Richmond, G.S.; Smith T.K. Phospholipase A1. Int. J. Mol. Sci.2011, 12, 588- 612. 31. Morley, N.; Kuksis, A. Positional specificity of lipoprotein lipase. J. Biol. Chem. 1972, 247 (20), 6389-6393. 32. Six, D.A.; Dennis, E.A. The expanding superfamily of phospholipase A2enzymes: classification and characterization. Biochim. Biophys. Acta 2000, 1488, 1-19.

Claims

25737 WHAT IS CLAIMED IS:

1. A method of measuring of lipolytic activity within biotherapeutic protein downstream samples, comprising the steps of: a) forming a solution comprising: dioleoylphosphatidylcholine, dioleoylphosphatidglycerol, and a substrate for phospholipase comprising a cleavable fluorescent marker linked via an ester bond; b) vortexing the solution; and c) adding the vortexed solution to a biotherapeutic protein sample in a vessel suitable for fluorescence measurement and measuring the fluorescence.

2. The method of claim 1, wherein the fluorescence is measured using a microplate reader having excitation 482 nm, emission 515 nm, and cutoff 495 nm.

3. The method of claim 2, wherein the fluorescence measurements are collected every 10 minutes for 15 hours.

4. The method of claim 2, wherein the microplate reader is kept at room temperature during the measurement reading.

5. The method of claim 1, wherein the PLA substrate is selected from (A) and (B) .dioleoylphosphatidglycerol, and substrate solution is vortexed for 10-30 seconds.

7. The method of Claim 1, wherein the biotherapeutic protein samples are stored frozen at -80oC.25737 8. The method of claim 7, wherein the frozen biotherapeutic protein sample is thawed and stored for up to 28 days at 4oC before measuring the lipolytic activity within the sample.

Images