Process for Purifying C1-INH

Abstract

The present invention relates to a process for purifying C1-esterase inhibitor (C1-Inh), and more in particular a Cl-Inh concentrate.

Claims

1. Process for the depletion of 1-antichymotrypsin (ACT) from a C1-INH preparation obtained from blood plasma by means of a preceding process involving several steps including, but not limited to hydrophobic interaction chromatography, wherein the depletion of ACT from the C1-INH preparation is carried out by anionic exchange chromatography and comprising the following steps: (i) loading an anionic exchange chromatography column comprising a stationary phase with the C1-INH preparation under first conditions under which C1-INH and ACT bind to the stationary phase; (ii) an optional step of washing the charged column; (iii) application of second conditions so as to elute ACT by means of a mobile phase; (iv) application of third conditions so as to elute C1-INH by means of a mobile phase, characterized in that the second condition consists in the use of an elution buffer of an ionic strength A and the third condition consists in the use of an elution buffer of an ionic strength B, wherein ionic strengths A and B are different, and wherein transition from ionic strength A to ionic strength B is achieved by means of a salt concentration gradient, or by means of a step elution using elution buffers EB.sub.A and EB.sub.B with different salt concentrations c.sub.A and c.sub.B and wherein (i) elution buffer EB.sub.A has a conductivity of 18.7 to 20.2 mS/cm at 25° C., preferably of 18.9 to 19.8 mS/cm at 25° C., most preferably of 19.2 mS/cm at 25° C. (ii) elution buffer EB.sub.B has a conductivity being higher than 21.6 mS/cm at 25° C. wherein elution buffer EB.sub.B is eluting C1-INH still bound to the anionic exchange chromatography column after elution step (i).

2. Process according to one or more of the preceding claims, wherein (i) elution buffer EB.sub.A consists of 10 mM Tris, 175-190 mM NaCl, preferably 175-185 mM NaCl, most preferably 180 mM NaCl, pH 7.2, and (ii) elution buffer EB.sub.B has a conductivity being higher than 25.3 mS/cm at 25° C. wherein elution buffer EB.sub.B is eluting C1-INH still bound to the anionic exchange chromatography column after elution step (i).

3. Process according to one or more of the preceding claims, wherein (i) elution buffer EB.sub.A consists of 10 mM Tris, 175-190 mM NaCl, preferably 175-185 mM NaCl, most preferably 180 mM NaCl, pH 7.2, and (ii) elution buffer EB.sub.B consists of 10 mM Tris, 1M NaCl, pH 7.2

4. Process according to one or more of the preceding claims, wherein the stationary phase material belongs to the type of weak anion exchangers, such as Capto® DEAE (sold by GE, using diaminoethyl as a functional group) or preferably of strong anion exchangers, such as Q HP resin, Capto® Q Impres resin, Capto® Q resin (all sold by GE, all with quaternary ammonium as a functional group) or Fractogel® TMAE, Eshmuno® H (sold by Merck, with trimethylamonethyl as a functional group).

5. Process according to any one of the preceding claims, wherein the C1-INH preparation is derived from human blood plasma.

6. Process according to any one of the preceding claims, wherein the C1-INH preparation consists essentially of C1-INH and ACT dissolved in a medium.

7. C1-INH preparation that can be obtained by using a process according to any one of the preceding claims.

Description

[0056] In the following, the present invention will be described in more details by means of figures and examples, wherein the figures depict the following:

[0057] FIG. 1: electrophoresis gels showing the presence of ACT in commercially available C1-INH preparations according to the prior art;

[0058] FIG. 2: a chromatogram of an AEX carried out in a bind/elute mode on a C1-INH preparation obtained by an established industrial process and SDS-page gel of eluate samples obtained in the same experiment;

[0059] FIG. 3: an SDS-page gel of eluate samples obtained using AEX carried out in a bind/elute mode on a C1-INH preparation obtained by an established industrial process;

[0060] FIG. 4: SDS-page gels of eluate samples obtained from AEX carried out in a bind/elute mode on a C1-INH preparation obtained by an established industrial process comparing various elution buffers;

[0061] FIG. 5: a diagram summarising purity and extent of C1-INH recovery in a second AEX chromatography eluate using a buffer of high ionic strength C1-INH depending on the salt content of eluent buffer used in a a first elution from the same AEX matrix using a low ionic strength buffer at NaCl concentrations from 170 to 195 mM, corresponding to buffers having a conductivity from 18.7 to 20.7 mS/cm at 25° C.

[0062] FIG. 6: a diagram depicting the amount of C1-INH and ACT in a second AEX chromatography eluate using a buffer of high ionic strength depending on the salt content of eluent buffer used in a a first elution from the same AEX-matrix using a low ionic strength buffer at NaCl concentrations from 170 to 195 mM, corresponding to buffers having a conductivity from 18.68 to 20.7 mS/cm at 25° C.

[0063] In the context of the present invention, the following definitions apply:

[0064] In the claims and in the description of the invention “C1-INH”, C1-INH″, “C1-INH preparation” and “C1-INH preparation” are concurrently used to designate concentrates containing the protein C1-esterase inhibitor and in particular liquid concentrates containing the protein C1-esterase inhibitor. When referring to the technical background and/or prior art, “C1-INH” may also mean the protein as such, e.g. in the context of discussing C1-INH deficiency.

[0065] Throughout the present application/patent [0066] “HIC” means hydrophobic interaction chromatography; [0067] “AEX” means anion exchange chromatography; [0068] “AEX resin” means a resin used as stationary phase in AEX; [0069] “strong AEX resin” means a highly ionized AEX resin that can be used over a broad pH range; [0070] “weak AEX resin” means a resin of which the degree of ionization strongly depends on pH; [0071] “BC” means binding capacity of a chromatography column; [0072] “negative mode” or “flow through mode”, or “flow through” designates a way of carrying out a chromatography under conditions under which a target compound (e.g. C1-INH) does not bind to the stationary phase of a chromatography column; [0073] “binding mode”, “binding and elution” or “positive mode” stands for a chromatography first carried out under conditions under which a target compound (e.g. C1-INH) binds to the stationary phase of a chromatography column and then under conditions under which the same compound is eluted from the chromatography column; [0074] when a “compound binds to the stationary phase”, this is intended to mean is adsorbed by or retained on the stationary phase without the structural integrity of the compound being affected, preferably not by covalent bonds or chemisorption, but rather by physisorption; [0075] “WFI” means “water for injection”; [0076] “concentration gradient” designates the gradual variation of the concentration of a dissolved substance in a solution from a higher concentration to a lower concentration, [0077] “step elution” means a sudden transition from the first to the second concentration instead of a continuous transition as in a concentration gradient, wherein the concentration is gradually lowered; [0078] “%” means “% by weight” unless otherwise stated; [0079] “precipitant” is an agent triggering precipitation of proteins; [0080] “eluate fraction” designates a fraction of the mobile phase stream emerging from a chromatographic column irrespective of whether specific analytes comprised therein were previously bound to or retained by the stationary phase (as in a positive mode as mentioned herein) or not (as in a negative mode as mentioned herein).

[0081] In the following, the present invention will be explained in more detail by making reference to the figures.

[0082] FIG. 1 shows an electrophoresis gel of commercially available C1-INH preparations known from prior art. The dotted line indicates the molecular weight of C1-INH (105 kD). The differences between commercially available C1-INH preparations derived from blood plasma known under the tradenames Berinert®, Cetor® and Cinryze® can clearly be distinguished. Lanes 1 to 5 in this gel show that Berinert®, Cetor® and Cinryze® comprise traces of ACT, wherein Berinert® contains the smallest amount thereof (FIG. 1, 1), as discussed in more detail by Feussner et al. Berinert®, Cetor® and Cinryze®, but also Haegarda® are, respectively, C1-INH preparations obtained from blood plasma by means of a process involving several steps. The steps involved in the manufacture of Berinert®, Cetor® and Cinryze® have been described previously (Feussner et al., Transfusion 2014 October;54(10):2566-73, doi: 10.1111/trf.12678).

[0083] FIG. 2 represents a chromatogram of an AEX chromatography experiment according to the invention and an SDS-page gel analysing elute samples from that experiment; column load was a C1-INH concentrate taken from the production of C1-INH preparation Berinert®, i.e. the eluate of the last hydrophobic interaction chromatography step in the preparation of Berinert® (diluted 1:25); binding capacity BC was 15 mg protein/mL resin; separation was carried out by means of a salt gradient from 30 mM to 1000 mM NaCl. The pre-peak eluate sample clearly comprises ACT as can be seen on the SDS-page gel in FIG. 2 (cf. lane 6). Thus, the chromatogram and SDS-page gel of FIG. 2 demonstrate the depletion of ACT from a C1-INH preparation obtained by a process involving multiple steps and comprising already a very low concentration of ACT by using AEX chromatography.

[0084] FIG. 3 represents an SDS-page gel of eluate samples obtained from an AEX chromatography experiment with the same column load and binding capacity as in the experiment represented in FIG. 2, but using a less steep salt gradient, i.e. 30 mM to 515 mM NaCl. The SDS-page gel of FIG. 3 demonstrates the depletion of ACT from a C1-INH preparation by using AEX chromatography.

[0085] FIG. 4 represents SDS-page gels of eluate samples obtained from AEX chromatography experiments using step elution with varying salt concentrations of the first elution buffer destined to elute ACT from the stationary phase. “E1” designates a lane corresponding to a sample of the respective eluate 1, “E2” designates a lane corresponding to a sample of the respective eluate 2. It can clearly be seen that the extent of depletion of ACT from C1-INH preparations increases with increasing salt concentration, whereas the extent of C1-INH recovery in eluate 2 decreases with increasing salt concentration.

[0086] FIG. 5 shows the purity and extent of C1-INH recovery in eluate 2 depending on the salt content of eluent buffer 1 for different eluate buffers 1 in comparison. As can be inferred from FIG. 5, eluent buffer 1 with NaCl concentration in the range of 175-190, preferably 175-185, most preferable 180 mM NaCl is best suited for maximum depletion of ACT without essential loss of C1-INH from C1-INH preparations.

[0087] FIG. 6 shows the amount of C1-INH and ACT in in eluate 2 depending on the salt content of eluent buffer 1 for different eluate buffers 1 in comparison. FIG. 6 shows that at 175 mM NaCl or above in the buffer used for the first elution a much better depletion of ACT is achieved in the final product compared to a lower ionic strength in eluent buffer 1 and that at an ionic strength of above 190 mM of eluent buffer 1 the yield of C1-INH becomes too low to be commercially viable.

[0088] C1-INH preparations derived from animal blood, and in particular human blood, are nowadays obtained by various multi-step processes. The established processes departing from human blood plasma include steps of cryoprecipitation, ion-exchange chromatography, quaternary aminoethyl adsorption, ammonium sulphate precipitations, pasteurization and hydrophobic interaction chromatography (process steps included in the manufacture of Berinert®, see Feussner et al., or EP 0 101 935) or cryoprecipitation, various steps of ion-exchange chromatography, precipitation with PEG (in particular PEG-4000) and pasteurization (process steps included in the manufacture of Cinryze®/Cetor®). These processes have in common that they comprise precipitation steps. Additional last steps are filtration and lyophilisation. The present invention proposes to add the additional step of anion exchange chromatography to further purify C1-INH preparations obtained by multi-step processes like the aforementioned ones, thus providing a “polishing” step to enhance the safety of existing products even further. That polishing step may take place before filtration and lyophilisation, but otherwise it is the last step following a sequence of other steps yielding a C1-INH concentrate or C1-INH preparation which nearly corresponds to the final product.

[0089] Surprisingly, the use of AEX chromatography in an additional polishing step enables still further depletion of ACT from C1-INH preparations without essential loss of C1-INH and without unnecessary dilution. Although AEX chromatography has been known for a long time, and even though it's use in the preparation of C1-INH concentrates has occasionally been mentioned, also in the context of separating C1-INH from accompanying proteins, it has so far not been used to the specific aim of depleting ACT from C1-INH preparations obtained by multiple step processes, wherein ACT still subsists as an impurity despite considerable efforts having been made in the past to obtain essentially pure C1-INH preparations.

[0090] That this is possible at all could not have been expected in view of this background. Quite surprisingly, otherwise well-established processes enabling production of C1-INH preparations on an industrial scale may still be improved via the inclusion of a corresponding polishing step. i.e. at a comparably late stage of the respective process.

[0091] In the following, the invention will be described in more detail by referring examples.

Examples

Example 1

[0092] A C1-INH concentrate taken from the production of C1-INH preparation Berinert®, i.e. the eluate of the last hydrophobic interaction chromatography (HIC) step in the preparation of Berinert® was diluted 1:25 to decrease the concentration of the chaotropic agent ammonium sulphate (AS) employed in the preceding HIC so as to enable protein binding. An AEX chromatography was then carried out in bind/elute mode, i.e. the starting material was loaded onto a column using a binding buffer, subsequently washed with a wash buffer, and lastly eluted by applying a salt gradient. Composition of buffers and gradient and further details are disclosed in Table a-1, the corresponding chromatogram and SDS page gel are shown in FIG. 2.

TABLE-US-00002 TABLE a-1 Binding buffer 10 mM Tris, 32 mM AS, pH 7.2 BC 15 mg protein/mL resin Wash buffer 10 mM Tris, 30 mM NaCl, pH 7.2 Elution gradient from 10 mM Tris, 30 mM NaCl, pH 7.2, to 10 mM Tris, 1M NaCl, pH 7.2

[0093] Example 1 thus demonstrates the depletion of ACT from a C1-INH preparation obtained by a process involving multiple steps and comprising already a very low concentration of ACT by using AEX chromatography.

Example 2

[0094] An AEX chromatography was carried out as described in example 1, yet with an elution gradient from 10 mM NaCl to 515 mM NaCl. The corresponding chromatogram is shown in FIG. 3 discussed herein above. Example 2 thus demonstrates the depletion of ACT from a C1-INH preparation obtained by a process involving multiple steps and comprising already a very low concentration of ACT by using AEX chromatography.

[0095] Example 3

Starting Material

[0096] Product obtained in the Berinert process, a highly purified C1-INH concentrate obtained by hydrophobic interaction chromatography (HIC) similar to the one described by Feussner et al. (doi: 10.1111/trf.12678) (batch No. 20181219-HW) stored at −20° C. was used. This material was dialyzed against 10 mM Tris, 32 mM AS pH 7.2 overnight at 4° C. and then subjected to anion exchange chromatography experiments comparing elution buffers 1 with different salt concentrations as represented in the following table b-1.

TABLE-US-00003 TABLE b-1 Experiments using different buffer 1 conductivity exper- buffer/ (25(±0.5)° C. column iment solution composition mS/cm load 1-6 equilibration 10 mM Tris, 32 mM buffer AS pH 7.2 1-6 wash buffer 10 mM Tris, 30 mM NaCl pH 7.2 1 elution buffer 1 10 mM Tris, 170 mM NaCl pH 7.2 18.7 c 2 10 mM Tris, 175 mM NaCl pH 7.2 18.9 c 3 10 mM Tris, 180 mM NaCl pH 7.2 19.2 b 4 10 mM Tris, 185 mM NaCl pH 7.2 19.8 b 5 10 mM Tris, 190 mM NaCl pH 7.2 20.2 c 6 10 mM Tris, 195 mM NaCl pH 7.2 20.7 a 1-6 elution buffer 2 10 mM Tris, 1M NaCl pH 7.2 1-6 regeneration 2M NaCl solution

Substances and Equipment

Substances:

[0097] De-ionised Water obtained via Milli-Q.

Equipment:

[0098]

TABLE-US-00004 Chromatography Device ÄKTA avant 25 Software Unicorn 6.4 pH meter Knick conductivity meter Knick weighing balance Sartorius magnetic stirrer Thermo Scientific column GE Healthcare, resin: Q HP, Lot No. 10270237 column dimensions inner diameter 0.77 cm, bed height 10 cm, column volume 4.7 mL

TABLE-US-00005 TABLE b-2 column loads a b c pH 7.18 7.16 7.15 conductivity 9.18 mS/cm 8.95 8.97 (22.1° C.) (21.7° C.) (22.9° C.) OD.sup.1 0.1026 0.0885 0.0911 (0.283 mg/mL) (0.244 mg/mL) (0.251 mg/mL) Volume.sup.2 205.6 mL 241 mL 234 mL total protein.sup.3 58.1 mg 58.8 mg 58.7 mg BC.sup.4 12.4 mg/mL 12.5 mg/mL 12.5 mg/mL .sup.11 OD = 2.76 mg/mL protein; .sup.2volume = volume used for loading onto column, also termed “column load” .sup.3total protein = total protein amount loaded [00001]${ }^4\mathrm{BC}=\mathrm{Binding}\mathrm{Capacity}=\frac{\mathrm{total}\mathrm{protein}\left(\mathrm{mg}\right)}{\mathrm{column}\mathrm{volume}\left(\mathrm{mL}\right)}$

TABLE-US-00006 TABLE b-3 ÄKTA program Pump A1 and Buffer equilibration buffer Pump A2 wash buffer Pump A3, A4, A6 different elution buffer 1 Pump B1 elution buffer 2 Pump A5 2M NaCl Pump S1 column load

TABLE-US-00007 TABLE b-4 Example of ÄKTA program Step Volume Inlet Flow rate (cm/h; mL/min) Outlet Equilibration   5 CV A1 150; 1.2 Waste Sample Application 241 mL S1 130; 1.0 Waste Washing   5 CV A2 130; 1.0 Waste Elution 1   5 CV A3 150; 1.2 Outlet 2 Elution 2   5 CV B1 150; 1.2 Outlet 3 Regeneration   5 CV A5 130; 1.0 Waste

[0099] Calculation of yields is based on volume of the respective column load a-c (see table b-1 above) and on the volumes of eluate 1 and 2 (23.5 mL, respectively).

Analytics

[0100] Samples from “column load” (L), “eluate 1” (E1) and “eluate 2” (E2) were analyzed by SDS-PAGE. Corresponding SDS-PAGE gels are represented in FIG. 4. Samples from “Column load” and “Eluate 2” were additionally tested for C1-INH activity in quality control laboratories.

[0101] Results of the comparison are summarised in the graph represented in FIG. 5, showing yields of C1-INH recovered in the respective eluate 2 in comparison to purity in %. In accordance therewith, eluent buffer 1 with NaCl concentration in the range of 175-190 mM NaCl, preferably 175-185 mM NaCl, most preferably 180 mM NaCl is best suited for maximum depletion of ACT without essential loss of C1-INH from C1-INH preparations. This corresponds to conductivities of eluent buffer 1 from 18.7 to 20.2 mS/cm at 25° C., preferably from 18.9 to 19.8 mS/cm at 25° C., most preferably of 19.2 mS/cm at 25° C. Eluent buffer 2 needs to be high enough to elute all C1-INH still bound to the anionic exchange chromatography columns and needs to be higher than 21.6. mS/cm at 25° C. One example for an eluent buffer 2 is a buffer composition of 10 mM Tris, 1M NaCl, pH 7.2

[0102] FIG. 6 shows that using an eluent buffer 1 of 170 mM NaCL (18.7 mS/cm at 25° C.) there is already some reduction of ACT but still 49% of the starting amount of ACT in the final product whereas using an eluent buffer 1 of 175 mM NaCl (18.9 mS/cm at 25° C.) there is only 17% of the starting amount of ACT in the final product. This illustrates how important the fine tuning of the ionic strength/conductivity of the eluent buffer 1 needs to be to allow a maximum yield of C1-INH and a minimum yield of ACT.

[0103] The present invention has been described above by making reference to specific examples. These examples are by no means intended to restrict the present invention, but to illustrate the way in which the present invention works.