METHOD FOR THE DETERMINATION OF HYDROLYTIC ACTIVITY

Abstract

Herein is reported a method for determining the presence of hydrolase activity in a sample by incubating the sample in a buffered solution, which comprises an artificial hydrolase substrate that comprises an ester bond covalently linking an alcohol residue, which is conjugated to a label for removal, and a carboxylic acid residue, which is conjugated to a capture and detection label, as well as bovine serum albumin, and thereafter determining or quantifying, respectively, the released carboxylic acid in the free alcohol depleted incubation mixture.

Claims

1. A method for determining hydrolase activity in a sample, comprising the following steps: a) incubating the sample or an aliquot thereof and a buffered solution to form an incubated sample, wherein the buffered solution comprises a buffer substance, an artificial hydrolase substrate, a salt and bovine serum albumin, wherein the sample comprises a therapeutic polypeptide, wherein the artificial hydrolase substrate comprises an ester bond covalently linking a carboxylic acid residue, which is conjugated to a first label, and an alcohol residue, which is conjugated to a nucleic acid tag and a second label, which is different from the first label, wherein the ester bond in the artificial hydrolase substrate is cleaved into a free alcohol residue and a free carboxylic acid residue if hydrolase activity and/or a hydrolase is present in the sample, b) removing the free carboxylic acid residue and artificial hydrolase substrate from the incubated sample to obtain a depleted sample, wherein the removing is by contacting the incubated sample with a first solid phase, which specifically binds to the first label, and separating the free carboxylic acid- and artificial hydrolase substrate-depleted incubation mixture from the first solid phase thereafter, c) determining hydrolase activity and/or the presence of a hydrolase in the sample by determining the second label in the depleted sample, wherein the determining is by capturing the free alcohol residue on a second solid phase via the nucleic acid tag followed by detecting the solid-phase captured free alcohol residue with an antibody specifically binding to the second label, which antibody is conjugated to one or more detectable labels.

2. The method according to claim 1, wherein the hydrolase is an esterase.

3. The method according to claim 1, wherein the detection is with an electrochemiluminescense immunoassay and the second label is a ruthenium label.

4. The method according to claim 1, wherein the detection is with an antibody specifically binding to the second label, wherein the antibody is conjugated to one or more ruthenium labels.

5. The method according to claim 3 or claim 4, wherein the ruthenium label is a detectable ruthenium label.

6. The method according to claim 1, wherein the first and the second label are selected independently of each other from the group of specific binding pairs.

7. The method according to claim 1, wherein the nucleic acid tag is a locked nucleic acid.

8. The method according to claim 1, wherein the nucleic acid tag has the sequence in 3′- to 5′-direction TGGTTG.

9. The method according to claim 1, wherein the alcohol of the artificial hydrolase substrate and the carboxylic acid of the artificial hydrolase substrate each comprises independently of each other one or more ethylene oxide (CH.sub.2—CH.sub.2—O) units.

10. The method according to claim 1, wherein the buffered solution has a pH value of about 7.

11. The method according to claim 1, wherein the incubating is at a temperature of about 37° C.

12. The method according to claim 1, wherein the incubating is for up to 22 hours.

13. The method according to claim 1, wherein incubation mixture comprises a final concentration of 3 μM to 10 μM bovine serum albumin.

14. The method according to claim 1, wherein the incubation mixture comprises a final concentration of about 150 mM MOPS, about 5 mM MgCl.sub.2, about 25 mM NaCl and about 6 μM Cys34 oxidized bovine serum albumin.

15. The method according to claim 1, wherein the first label is biotin or a variant thereof and the second label is digoxygenin or a variant thereof.

16. The method according to claim 1, wherein the incubation mixture further comprises a final concentration of about 0.05% to 0.15% (w/v) of a detergent.

17. The method according to claim 16, wherein the detergent is Octoxinol-9.

Description

DESCRIPTION OF THE FIGURES

[0436] FIG. 1 Idealized polysorbate structure with non-polar fatty acids at one end and polar sorbitol and its anhydrides at the other end. Here, w+x+y+z indicates the total number of moles of ethylene oxide per mole of sorbitol and ethoxylated sorbitol anhydride, and the sum must not exceed 20.

[0437] FIG. 2 Oxidative and hydrolytic degradation pathways of polysorbate 20 [44]. Long-chain fatty acids are released through hydrolytic cleavage of the fatty acid ester bond (red bolt icon). Oxidative cleavage (blue bolt icons) leads to a mixture of degradation products.

[0438] FIG. 3 Ester cleavage of the 4-methylumbelliferyl fatty acid ester into the fluorophoric head group 4-methylumbelliferone and a fatty acid.

[0439] FIG. 4 Schematic representation of the chemical structure of the artificial substrate according to the invention with three different residues. R3 is an antigen of a specific cardenolide, e.g. digoxygenin. R2 is a class of high-affinity RNA analogues and R1 is part of a non-covalent, biological binding pair, e.g. biotin (biotin/avidin pair)). The thunder-like arrow shows an ester bond. Hydrolytically active HCPs cleave this ester bond, as result of which an alcohol and an acid are released from the substrate.

[0440] FIG. 5 Graphical representation of the factors tested for influence on the enzyme activity. (A) Half-normal probability plot. (B) Pareto plot.

[0441] FIG. 6 Graphical representation of the factors tested for influence on the enzyme activity in the pH range between pH 6-7. (A) Half-normal probability plot. (B) Pareto plot.

[0442] FIG. 7 Graphic representation of the detergent effect.

[0443] FIG. 8 Effect of temperature on the method according to the invention.

[0444] FIG. 9 Scheme of an exemplary assay according to the invention.

[0445] FIG. 10 Bar graph of percent error for FAMS assay for in process samples of exemplary therapeutic polypeptide 3.

[0446] FIG. 11 Bar graph of percent error for the method according to the invention for in process samples of exemplary therapeutic polypeptide 3.

[0447] FIG. 12 Bar graph showing the amount of generated hydrolysate in a method according to the current invention depending on the employed buffer: TRIS-based buffer on the left, MOPS-based buffer on the right.

[0448] FIG. 13 Effect of BSA and various grades of BSA on the method according to the current invention.

DESCRIPTION OF THE EXAMPLES

Example 1

[0449] Design of Experiments Using Plackett-Burman Design

[0450] In the buffer screening process factors that have an impact on the enzyme activity have been identified.

[0451] For this experiment, a host cell culture fluid (HCCF) was used.

[0452] Two categorical factors, i.e. the buffer salts and the type of bivalent cation, as well as seven continuous factors—pH, temperature, concentrations of buffer, NaCl, methionine, BSA and the concentration of the bivalent salts were used. The conditions are summarized in Table 8 below.

TABLE-US-00010 TABLE 8 Conditions of the DoE; E = acid conditions; F = basic conditions. Codierte Level Faktor Symbol − + Temperatur (° C.) A 25 37 Konzentration Puffer (mM) B 20 50 Puffer.sup.+ C Citrat His-Acetat Puffer.sup.− D Tris-HCl HEPES pH-Wert.sup.+ E 5 6 pH-Wert.sup.− F 7 8 NaCl (mM) G 25 1000 Methionin (mM) H 0 7 BSA (mM) I 0 0.1 Bivalente Salze J CaCl.sub.2 MgCl.sub.2 Konz. bivalente Salze (mM) K 5 10

[0453] For the selection of the experimental conditions, a Plackett-Burman Design was used. This design is a two-level partial factorial design in which all factors have only two values. The low (−) and high (+) values of these factors to be tested are listed in Table 8 above.

[0454] Based on these factors, two plans for the Plackett-Burman design were created (see Table 9 below). One plan served as an acidic buffer template and the other as an alkaline buffer template.

[0455] To be able to exclude chemical cleavage of the substrate by the buffer compositions, a sample in the absence of hydrolytic enzyme was also measured, a so-called buffer blank. This value was then subtracted from the respective sample value in order to give only the value of the enzyme activity

TABLE-US-00011 TABLE 9 Plans for the Plackett-Burman design. Run A E/F B C/D G H I J K 1 + − + − + − − − − 2 + − − − − + + − − 3 − + − + − − − − + 4 − + − − − − + + − 5 − − + + − − + − − 6 − − − + + − − + − 7 − − − − + + − − + 8 + + − + − + − − − 9 + − + − − − − + + 10 − + + − − + − − + 11 + + + − + − + − − 12 + + − − + − − + + 13 + − − + − − + + + 14 − + − − + + + + − 15 − − + + + + − + − 16 − − + − − + + + + 17 + + + + − + − + − 18 + − − + + + + − + 19 − + + + + − + − + 20 + + + + + + + + +

[0456] Each pattern was created as a duplicate on a 96-well plate. The Experiment was repeated twice under the same conditions.

[0457] The results for the alkaline conditions are presented in Table 10 and that for the acidic conditions in Table 11.

TABLE-US-00012 TABLE 10 Alkaline DoE results. Relative #1 #2 Mittelwert CV Enzymaktivität Run Pattern (pg/mL) (pg/mL) (pg/mL) (%) (%) Kontrolle 593 593 593 0 100 (25° C.) Kontrolle 619 499 559 0.15 100 (37° C.) 1 + − + − + − − − − 261 23 142 1.19 25 2 + − − − − + + − − 5339 8529 6934 0.33 1240 3 − + − + − − − − + 803 118 461 1.05 78 4 − + − − − − + + − 9562 7799 8681 0.14 1464 5 − − + + − − + − − 7851 6163 7007 0.17 1182 6 − − − + + − − + − 116 47 82 0.60 14 7 − − − − + + − − + 41 37 39 0.07 7 8 + + − + − + − − − 2225 1790 2008 0.15 359 9 + − + − − − − + + 175 −172 −174 −0.01 −31 10 − + + − − + − − + 80 122 101 0.29 17 11 + + + − + − + − − 1319 4179 2749 0.74 492 12 + + − − + − − + + −369 −59 −214 −1.02 −38 13 + − − + − − + + + 5086 8708 6897 0.37 1234 14 − + − − + + + + − 3745 2978 3362 0.16 567 15 − − + + + + − + − 135 23 79 1.00 13 16 − − + − − + + + + 7361 6155 6758 0.13 1140 17 + + + + − + − + − −19 −122 −71 −1.03 −13 18 + − − + + + + − + 1654 3807 2731 0.56 488 19 − + + + + − + − + 4257 2724 3491 0.31 589 20 + + + + + + + + + 1798 4923 3361 0.66 601

TABLE-US-00013 TABLE 11 Acidic DoE results. Relative #1 #2 Mittelwert CV Enzymaktivität Run Pattern (pg/mL) (pg/mL) (pg/mL) (%) (%) Kontrolle 593 593 593 0 100 (25° C.) Kontrolle 619 499 559 0.15 100 (37° C.) 1 + − + − + − − − − −31 −233 −132 −1.08 −24 2 + − − − − + + − − 466 1553 1010 0.76 181 3 − + − + − − − − + −2 0 −1 −1.41 0 4 − + − − − − + + − 7051 4306 5679 0.34 958 5 − − + + − − + − − 123 111 117 0.07 20 6 − − − + + − − + − 20 28 24 0.24 4 7 − − − − + + − − + 91 27 59 0.77 10 8 + + − + − + − − − 332 362 347 0.06 62 9 + − + − − − − + + −95 132 19 8.68 3 10 − + + − − + − − + 847 111 479 1.09 81 11 + + + − + − + − − 612 1029 821 0.36 147 12 + + − − + − − + + 0 344 172 1.41 31 13 + − − + − − + + + −757 2393 818 2.72 146 14 − + − − + + + + − 1228 122 675 1.16 114 15 − − + + + + − + − 13 26 20 0.47 3 16 − − + − − + + + + 1170 674 922 0.38 155 17 + + + + − + − + − −12 −11 −12 −0.06 −2 18 + − − + + + + − + −149 −176 −163 −0.12 −29 19 − + + + + − + − + 1728 2037 1883 0.12 317 20 + + + + + + + + + 256 691 474 0.65 85

Example 2—Comparative Example

[0458] LEAP Assay

[0459] A non-limiting example for measuring, determining or quantifying hydrolase/hydrolase activity (hydrolytic activity) is an assay to detect lipolytic activity or, in other words, a lipase activity assay (e.g. as described by Jahn et al.), herein also termed LEAP (lipase enzymatic assay for polysorbates) assay (Jahn (2020) Pharm. Res. 37: 118).

[0460] This assay allow measuring, determining or quantifying hydrolase/hydrolase activity (hydrolytic activity) is a lipase activity assay (or in other words an assay to detect lipolytic activity) measuring the conversion of 4-methylumbelliferone caprylate (4-MUCA) to 4-methylumbelliferone (4-MU). An assay using 4-MUCA as a substrate may be performed as described in the following.

[0461] 10 μL of the composition comprising the protein in which hydrolase/hydrolase activity (hydrolytic activity) is to be determined may be mixed with 80 μL of reaction buffer (150 mM Tris-HCl pH 8.0, 0.25% (w/v) Triton X-100 and 0.125% (w/v) Gum Arabic) and 10 μL 4-MUCA substrate (1 mM in DMSO). The reactions may be set up in 96-well half-area polystyrene plates (black with lid and clear flat bottom, Corning Incorporated) and the increase of fluorescent signal (excitation at 355 nm, emission at 460 nm) may be monitored every 10 min by incubating the reaction plate for two hours at 37° C. for example in an Infinite 200Pro plate reader (Tecan Life Sciences) to derive the 4-MU production rate. The 4-MU production rate of the composition comprising the protein may be compared with the starting composition comprising a protein.

Example 3—Comparative Example

[0462] FAMS Assay

[0463] The following paragraph describes an exemplary sample preparation and analytical procedure for performing a FAMS assay.

[0464] The FAMS assay measures the lipase activity by quantifying the accumulation of free fatty acids through the hydrolysis of Polysorbate 20.

[0465] All samples (antibody solutions and buffer controls) may be supplemented with a stock solution of 1% (w/v) super-refined Polysorbate 20 (Croda Health Care) and 0.25 ML-Methionine (Sigma Aldrich, Art. No. M5308) to obtain a final concentration per sample of 0.04% (w/v) super-refined Polysorbate 20 and 0.01 M L-Methionine. For each spiked sample, aliquots of 190 μL may be transferred in five capped glass vials (termed t0, t1, t2, t3, t4), which may be used for sample incubation. The glass vials t0 may be frozen immediately at −70° C. after their preparation until analysis. The glass vials t1, t2, t3 and t4 may be incubated at 25° C. upright and protected from light in an incubator. Incubation of the glass vials may be stopped one at a time over a time period between the next five day and 14 days the glass vials may be subsequently frozen at −70° C. until analysis.

[0466] For analysis, frozen samples may be brought to ambient temperature for 1 hour. Stock solutions of stable isotope labelled fatty acids may be prepared by dissolving 50 mg of the respective fatty acid, lauric acid .sup.2d.sub.23 (Sigma Aldrich, Cat. No. 451401) and myristic acid .sup.13C.sub.14 (Sigma Aldrich, Cat. No. 605689) in 50 mL 80% acetone/20% methanol to obtain the precipitation reagent with a final concentration of 1 μg/mL for each investigated fatty acid. 50 μL of the sample solution may be added to 200 μL of the precipitation reagent in a reaction tube, mixed by vortexing and kept by room temperature for 1 h to allow the protein to precipitate. The precipitate may be spun down by centrifugation at 20° C. with 15,000×g for 15 min. 100 μL of the supernatant may be transferred in a fresh reaction tube and mixed with 100 μL of Mobile phase A (20 mM ammonium acetate). After spinning down at 20° C. with 15,000×g for 15 min, 50-100 μL of the solution may be transferred into LC-MS vials (300 μL Fixed Insert Vial (Clear, Screw Top), Thermo Scientific, Cat. No. 03-FISV).

[0467] Separation of fee fatty acids (FFAs) may be achieved on an ACQUITY UPLCH-Class System (Waters Corp. Milford, MA, US) equipped with a temperature-controllable auto sampler and column compartment by using a Jupiter® C4 RP column (300 Angstroms, 2×50 mm, 5 μm) (Phenomenex, Cat. No. 00B-4167-BO). Mobile Phase A may be 20 mM ammonium acetate (Sigma Aldrich, Cat. No. 73594) and Mobile Phase B may be 100% methanol (Merck, Cat. No. 1.06007.2500), which may be rum for 4 min. at a flow rate of 0.4 mL/min. by applying the following gradient: initial conditions: 70% Mobile Phase B, 0.5 min-3.4 min: gradient may be changed linearly from 70% to 85% Mobile Phase B; 3.5-4.0 min: 70% Mobile Phase B. The auto sampler may be maintained at 20° C. and the column compartment at 60° C. The injection volume may be set to 8 μL. Detection may be performed on a connected QDa Performance mass spectrometer (Waters) equipped with an external backing pump in negative ion mode. The MS settings may be as follows, cone voltage 15 V, source temperature 120° C., capillary voltage 800 V, probe temperature 600° C., mass range 50-1000 m/z and sampling frequency 2 Hz. All samples may be analyzed in triplicates.

[0468] Data evaluation may be performed with Target Lynx as part of the Mass Lynx Software Version 4.1 (SCN781) (Waters Corp. Milford, MA, US). The content of lauric and myristic acid may be determined by comparing the peak area of the fatty acid with the respective internal labelled standard by using the following formula (3):

[00003]$\begin{array}{cc}\mathrm{Conc}.\left(FFA\right)=\frac{.Math.\mathrm{Peak}\mathrm{areas}\mathrm{of}FFA}{.Math.\mathrm{Peak}\mathrm{areas}\mathrm{of}\mathrm{Internal}\mathrm{standard}}*4*1\mu g/\mathrm{mL}& \left(3\right)\end{array}$

[0469] where ΣPeak areas of FFA and ΣPeak areas of Internal standard refer to the sum of the monoisotopic peak and the isotopic peaks at +1/+2 or −1/−2, respectively.

[0470] For determining the fatty acid release rate, the free fatty acid concentration for each sample may be plotted against the incubation time and the degradation may be extracted from the slope of the linear regression

Example 4

[0471] Assay According to the Invention

[0472] An exemplary method including sample preparation and its measurement for performing an Elecsys based Polysorbase Activity (EPA) assay according to the current invention is presented in this Example.

[0473] EPA assay determines hydrolase/hydrolase activity (hydrolytic activity) in samples. The source or matrix of the sample does not influence the EPA. Thus, samples from various different stages of purification (from HCCF until UFDF pool) can be analyzed.

[0474] An artificial hydrolase substrate according to the invention (FIG. 4) is used in this Example comprising: [0475] 1) A biotin moiety as R1, coupled via an ester bond, which is susceptible to cleavage by hydrolases present in samples; [0476] 2) L-Locked Nucleic Acid (LNA) as R2, used for immobilization of the cleaved substrate onto a solid surface; [0477] 3) Digoxygenin as R3, used for detection of immobilized cleaved substrate using Ruthenium labeled anti-digoxygenin antibody.

[0478] Sample and analysis buffer are mixed to obtain a mixture comprising final concentrations of 150 mM MOPS, 5 mM MgCl.sub.2, 6 μM BSA, 25 mM NaCl, 0.1% (w/v) Triton X-100 and 0.1-250 mg/mL protein at a pH value of 7. This reaction mixture is incubated at 37° C. for the respective time, i.e. 1, 3, 5, 7, 22 or 48 hours.

[0479] A standard EPA reaction is performed in duplicates where the substrate is incubated with samples (antibody solutions and buffer controls) to be measured for various time points (up to 48 hours).

[0480] In next steps, the digested biotin moieties along with undigested substrate is removed from the reaction mix using magnetic Streptavidin beads (Cytiva, Art. No. 28985799) according to the manufacturer's instructions.

[0481] The corresponding duplicates are combined (pooled) and the content of released alcohol part of the artificial hydrolase substrate is determined using L-LNA capture and Digoxygenin determination (as a readout) on a Cobas e411 analyzer according to the manufacturer's instructions. The readout is the amount of hydrolysate (in pg) detected per ml sample.

[0482] The amount of hydrolysate detected (pg/ml) in the corresponding sample is corrected for the amount of hydrolysate detected (pg/ml) in a blank sample (using e.g. water instead of the protein sample) according to equation (3).

Blank corrected value=hydrolysate detected in sample (pg/ml)−hydrolysate detected in blank (pg/ml)  (3)

[0483] The blank corrected value for each sample is normalized to its protein concentration, giving a final readout in pg of hydrolysate detected per mg of protein according to equation (4):

Normalized hydrolytic activity (pg/mg)=Blank corrected value (pg/mL)*1000 mL/protein per well (μg)  (4)

[0484] To calculate the rate at which hydrolases present in a sample digest the artificial hydrolase substrate, rate of hydrolysis is determined and is represented in pg of hydrolysate detected per mg of protein per hour according to equation (5):

Rate of hydrolysis (pg/mg/h)=normalized activity (pg/mg)/incubation time (h)   (5)