PROTEASE COMPOSITION

FIELD OF THE INVENTION

This invention relates generally to a composition comprising the cysteine protease ananain, a reducing agent and a buffer, to methods of stabilizing ananain while retaining protease activity, and methods of activating ananain zymogen for proteolysis.

BACKGROUND TO THE INVENTION
Ananain

Ananain (EC 3.4.22.31) is a plant cysteine protease in the papain superfamily of cysteine proteases. Ananain has a broad specificity for peptide bonds and catalyzes the hydrolysis of various proteins. Ananain is found in bromelain extract, a proteolytic extract obtained from the stems of pineapple (Ananus comosus). Ananain differs from other papain superfamily proteases in comprising a unique combination of acidic amino acids. Ananain is also considered to be an enzyme of considerable commercial importance due to strong proteolytic activity (Yongqing et al. 2019).

As a therapeutic, ananain-rich bromelain fractions have demonstrated utility as immunosuppressants, acting to inhibit the production of various cytokines including interleukin-2 (IL-2), IL-4, IL-6, tumor necrosis factor (TNF) and gamma interferon (INFγ) (WO2017/031299). Ananain-rich bromelain fractions have also been used as debriding agents (Orgill et al. 1996) and been suggested to block ERK-2 phosphorylation and the MAP kinase cascade, and CD4+ T cell proliferation (US 9,9663,777). Similar to other proteolytic enzymes (e.g., serine, aspartic and metalloproteases), the use of ananain as a therapeutic requires the prevention of unwanted protein degradation (including unwanted auto-proteolysis or self-degradation (Verma et al. 2016)).

Naturally occurring ananain from natural extracts of pineapple is produced by fractionation of bromelain extract and is known to be unstable, autolysing to constituent peptides and amino acids. In addition, the ananain comprised in fractions of bromelain extract lacks purity (Matagne et al. 2017). In contrast, recombinant ananain produced by in vitro expression in an appropriate host cell can be obtained and formulated into compositions of high purity. Such compositions suffer from an acknowledged lack of stability when formulated for physiological use; i.e., the active form of the enzyme is auto-proteolytic and undergoes a marked loss of activity over a relatively short time (i.e., a few hours). Additionally, freeze drying or alternate forms of formulating pure ananain result in irreversible denaturation of the enzyme such that when reconstituted, the enzyme preparation has lost significant activity.

The expression of an ananain zymogen (pro-ananain) overcomes the inherent instability of the enzyme, however such zymogen forms require activation under conditions of high ionic strength 50 mM, low pH (pH 4.0), and high temperature (55° C.) for 30-60 minutes. These conditions are inconsistent with in vivo use (Carter et al. 2000).

Accordingly, there is an unmet need in the art for compositions comprising pure and stable pro-ananain (i.e., separated from other proteases and/or other constituents), methods of making such compositions and methods of activating pro-ananain under physiologically compatible conditions to obtain mature ananain for various uses as described above.

It is an object of the invention to provide a composition of pure and stable pro-ananain (i.e., separated from other proteases and/or other active constituents), methods of making such compositions and methods of activating pro-ananain to obtain mature ananain that can go at least some way towards addressing this unmet need and/or to at least provide the public with a useful choice.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

SUMMARY OF THE INVENTION

In one aspect the invention relates to a composition comprising:

(a) recombinant pro-ananain
(b) a pharmaceutically acceptable buffer
(c) a pharmaceutically acceptable reducing agent, and
(d) sodium chloride (NaCl),
- wherein the concentration of the buffer is about 5 to about 30 mM,
- wherein the concentration of the reducing agent is about 10 to about 30 mM,
- wherein the concentration of the NaCl is about 140 to about 160 mM and,
- wherein the pH of the composition is about 5.0 to about 6.0.

In another aspect the invention relates to a method of making a composition comprising recombinant pro-ananain, the method comprising combining:

(a) recombinant pro-ananain with,
(b) a pharmaceutically acceptable buffer
(c) a pharmaceutically acceptable reducing agent, and
(d) sodium chloride (NaCl),
- wherein the concentration of the buffer is about 5 to about 30 mM,
- wherein the concentration of the reducing agent is about 10 to about 30 mM,
- wherein the concentration of the NaCl is about 140 to about 160 mM and,
- wherein the pH of the composition is about 5.0 to about 6.0.

In another aspect the invention relates to a method of providing a recombinant active ananain composition, the method comprising heating an aqueous composition of the invention to about 30° C. to about 44° C. for at least 5 min.

In another aspect the invention relates to method of providing a recombinant active ananain composition, the method comprising reconstituting a dry composition of the invention to form an aqueous composition and heating the aqueous composition to at least 30° C.

In another aspect the invention relates to an in vivo method of activating bromelain comprising (a) reconstituting a dry composition comprising at least 0.1% recombinant pro-ananain and > about 5% recombinant pro-bromelain and a physiological buffer in water to form an aqueous composition comprising about 4 to about 20 mM buffer, and (b), heating the reconstituted composition in vivo to about 37° C., wherein the reconstituted composition comprises a pH of about 5.0 to about 7.5.

In another aspect the invention relates to a method of activating bromelain comprising (a) reconstituting a dry composition comprising at least 0.1% recombinant pro-ananain and about >5% recombinant pro-bromelain and a physiological buffer in water to form an aqueous composition comprising about 4 to about 20 mM buffer, and (b), heating the aqueous composition in vitro to between about 30° C. and about 40° C. for about 5 to about 20 mins, wherein the reconstituted composition comprises a pH of between about 5 and about 7.5.

Other aspects of the invention may become apparent from the following description which is given by way of example only and with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

The invention will now be described by way of example only and with reference to the drawings in which:

FIG. 1 - The gene and protein sequences of pro-ananain. Shown in FIG. 1 are the primary amino acid (SEQ ID NO: 1) and nucleic acid (SEQ ID NO: 2) sequences of recombinant pro-ananain used in the work disclosed herein.

FIG. 2 - Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of pro-ananain activation in phosphate citrate buffer at varied pH values. SDS-PAGE was applied to visualize active ananain resulting from the activation process at different pH values in phosphate-citrate buffer. Pro-ananain and active ananain are displayed as a 36 kDa and a 24 kDa band, respectively, in a 12.5% SDS-gel. Lane 1, molecule weight markers. Lanes 2-9, incubation at various pH values.

FIG. 3 - Activity assay of pro-ananain activation in phosphate citrate buffer at varied pH values. Graphic representation of the activity of mature ananain resulting from the activation process at different pH values in phosphate-citrate buffer. PLQ is the fluorescent tripeptidyl substrate: MeOC—GGG—PLQ—GG—DPA—KK—NH₂. Cleavage at the core PLQ tripeptide tethered between the fluorescent MeOC donor group and the DPA quencher moiety releases the fluorescence of the MeOC donor group, which is detected by using a POLARstar fluorescent plate reader (BMG Labtech, Offenburg, Germany) with excitation and emission wavelengths of 320 nm and 420 nm, respectively. The initial rate of proteolysis of substrate is measured as the slope of the linear portion of the progressive curve and is converted to the unit of µg PLQ molecules per min.

FIG. 4 - SDS-PAGE of pro-ananain activation in varied concentrations of phosphate citrate buffer. SDS-PAGE was applied to visualize active ananain resulting from the activation process in different concentrations of phosphate-citrate buffer. Pro-ananain and active ananain are displayed as a 36 kDa and a 24 kDa band, respectively, in a 12.5% SDS-gel. Lane 1, molecule weight markers. Lanes 2 -17, incubation in various concentrations of phosphate citrate buffer.

FIG. 5 - Activity assay of pro-ananain activation in varied concentrations of phosphate citrate buffer. Graphic representation of the activity of mature ananain resulting from the activation process in different concentrations of phosphate-citrate buffer. PLQ is the fluorescent tripeptidyl substrate: MeOC-GGG-PLQ-GG-DPA-KK-NH₂. Cleavage at the core PLQ tripeptide tethered between the fluorescent MeOC donor group and the DPA quencher moiety releases the fluorescence of the MeOC donor group, which is detected by using a POLARstar fluorescent plate reader (BMG Labtech, Offenburg, Germany) with excitation and emission wavelengths of 320 nm and 420 nm, respectively. The initial rate of proteolysis of substrate is measured as the slope of the linear portion of the progressive curve and is converted to the unit of µg PLQ molecules per min.

FIG. 6 - SDS-PAGE of pro-ananain activation in varied concentrations of L-Cysteine (L-Cys) in phosphate citrate buffer. SDS-PAGE was applied to visualize active ananain resulting from the activation process in different concentrations of L-Cys in phosphate citrate buffer. Pro-ananain and active ananain are displayed as a 36 kDa and a 24 kDa band, respectively, in a 12.5% SDS-gel. Lane 1, molecule weight markers. Lanes 2 - 13, incubation in various concentrations of L-Cys.

FIG. 7 - Activity assay of pro-ananain activation in varied concentrations of L-Cys in phosphate citrate buffer. Graphic representation of the activity of mature ananain resulting from the activation process in different concentrations of L-Cys in phosphate citrate buffer. PLQ is the fluorescent tripeptidyl substrate: MeOC—GGG—PLQ—GG—DPA—KK—NH₂. Cleavage at the core PLQ tripeptide tethered between the fluorescent MeOC donor group and the DPA quencher moiety releases the fluorescence of the MeOC donor group, which is detected by using a POLARstar fluorescent plate reader (BMG Labtech, Offenburg, Germany) with excitation and emission wavelengths of 320 nm and 420 nm, respectively. The initial rate of proteolysis of substrate is measured as the slope of the linear portion of the progressive curve and is converted to the unit of µg PLQ molecules per min.

FIG. 8 - SDS-PAGE of pro-ananain activation at varied temperatures in phosphate citrate buffer. SDS-PAGE was applied to visualize active ananain resulting from the activation process at different temperatures between 0 and 46° C. in phosphate citrate buffer. Pro-ananain and active ananain are displayed as a 36 kDa and a 24 kDa band, respectively, in a 12.5% SDS-gel. Lane 1, molecule weight markers. Lanes 2 - 15, incubation at different temperatures.

FIG. 9 - Activity assay of pro-ananain activation at varied temperatures in phosphate citrate buffer. Graphic representation of the activity of mature ananain resulting from the activation process at different temperatures between 0 and 50° C. in phosphate citrate buffer. PLQ is the fluorescent tripeptidyl substrate: MeOC—GGG—PLQ—GG—OPA—KK—NH₂. Cleavage at the core PLQ tripeptide tethered between the fluorescent MeOC donor group and the DPA quencher moiety releases the fluorescence of the MeOC donor group, which is detected by using a POLARstar fluorescent plate reader (BMG Labtech, Offenburg, Germany) with excitation and emission wavelengths of 320 nm and 420 nm, respectively. The initial rate of proteolysis of substrate is measured as the slope of the linear portion of the progressive curve and is converted to the unit of µg PLQ molecules per min.

FIG. 10 - SDS-PAGE of pro-ananain activation in sodium acetate buffer at varied pH values. SDS-PAGE was applied to visualize active ananain resulting from the activation process at different pH values in sodium acetate buffer. Pro-ananain and active ananain are displayed as a 36 kDa and a 24 kDa band, respectively, in a 12.5% SDS-gel. Lane 1, molecule weight markers. Lanes 2-9, incubation at various pH values.

FIG. 11 - Activity assay of pro-ananain activation in sodium acetate buffer at varied pH values. Graphic representation of the activity of mature ananain resulting from the activation process at different pH values in sodium acetate buffer. PLQ is the fluorescent tripeptidyl substrate: MeOC—GGG—PLQ—GG—DPA—KK—NH₂. Cleavage at the core PLQ tripeptide tethered between the fluorescent MeOC donor group and the DPA quencher moiety releases the fluorescence of the MeOC donor group, which is detected by using a POLARstar fluorescent plate reader (BMG Labtech, Offenburg, Germany) with excitation and emission wavelengths of 320 nm and 420 nm, respectively. The initial rate of proteolysis of substrate is measured as the slope of the linear portion of the progressive curve and is converted to the unit of µg PLQ molecules per min.

FIG. 12 - SDS-PAGE of pro-ananain activation in varied concentrations of sodium acetate buffer. SDS-PAGE was applied to visualize active ananain resulting from the activation process in different concentrations of sodium acetate buffer. Pro-ananain and active ananain are displayed as a 36 kDa and a 24 kDa band, respectively, in a 12.5% SDS-gel. Lane 1, molecule weight markers. Lanes 2 -10, incubation in various concentrations of sodium acetate buffer.

FIG. 13 - Activity assay of pro-ananain activation in varied concentrations of sodium acetate buffer. Graphic representation of the activity of mature ananain resulting from the activation process in different concentrations of sodium acetate buffer. PLQ is the fluorescent tripeptidyl substrate: MeOC—GGG—PLQ—GG—DPA—KK—NH₂. Cleavage at the core PLQ tripeptide tethered between the fluorescent MeOC donor group and the DPA quencher moiety releases the fluorescence of the MeOC donor group, which is detected by using a POLARstar fluorescent plate reader (BMG Labtech, Offenburg, Germany) with excitation and emission wavelengths of 320 nm and 420 nm, respectively. The initial rate of proteolysis of substrate is measured as the slope of the linear portion of the progressive curve and is converted to the unit of µg PLQ molecules per min.

FIG. 14 - SDS-PAGE of pro-ananain activation in varied concentrations of L-Cys in sodium acetate buffer. SDS-PAGE was applied to visualize active ananain resulting from the activation process in different concentrations of L-Cys in sodium acetate buffer. Pro-ananain and active ananain are displayed as a 36 kDa and a 24 kDa band, respectively, in a 12.5% SDS-gel. Lane 1, molecule weight markers. Lanes 2 - 10, incubation in various concentrations of L-Cys.

FIG. 15 - Activity assay of pro-ananain activation in varied concentrations of reducing agent in sodium acetate buffer. Graphic representation of the activity of mature ananain resulting from the activation process at varied concentrations of reducing agent: L-Cys or ascorbic acid in sodium acetate buffer. PLQ is the fluorescent tripeptidyl substrate: MeOC—GGG—PLQ—GG—DPA—KK—NH₂. Cleavage at the core PLQ tripeptide tethered between the fluorescent MeOC donor group and the DPA quencher moiety releases the fluorescence of the MeOC donor group, which is detected by using a POLARstar fluorescent plate reader (BMG Labtech, Offenburg, Germany) with excitation and emission wavelengths of 320 nm and 420 nm, respectively. The initial rate of proteolysis of substrate is measured as the slope of the linear portion of the progressive curve and is converted to the unit of µg PLQ molecules per min.

FIG. 16 - SDS-PAGE of pro-ananain activation at varied temperatures in sodium acetate buffer. SDS-PAGE was applied to visualize active ananain resulting from the activation process at different temperatures between 0 and 50° C. in sodium acetate buffer. Pro-ananain and active ananain are displayed as a 36 kDa and a 24 kDa band, respectively, in a 12.5% SDS-gel. Lane 1, molecule weight markers. Lanes 2 - 18, incubation at various temperatures.

FIG. 17 - Activity assay of pro-ananain activation at varied temperatures in sodium acetate buffer. Graphic representation of the activity of mature ananain resulting from the activation process at different temperatures between 22 and 50° C. in sodium acetate buffer. PLQ is the fluorescent tripeptidyl substrate: MeOC—GGG—PLQ—GG—DPA—KK—NH₂. Cleavage at the core PLQ tripeptide tethered between the fluorescent MeOC donor group and the DPA quencher moiety releases the fluorescence of the MeOC donor group, which is detected by using a POLARstar fluorescent plate reader (BMG Labtech, Offenburg, Germany) with excitation and emission wavelengths of 320 nm and 420 nm, respectively. The initial rate of proteolysis of substrate is measured as the slope of the linear portion of the progressive curve and is converted to the unit of µg PLQ molecules per min.

FIG. 18 - SDS-PAGE of pro-ananain activation in 2-(N-morpholino)ethanesulfonic acid (MES) buffer at varied pH values. SDS-PAGE was applied to visualize active ananain resulting from the activation process at different pH values in MES buffer. Pro-ananain and active ananain are displayed as a 36 kDa and a 24 kDa band, respectively, in a 12.5% SDS-gel. Lane 1, molecule weight markers. Lanes 2 - 8, incubation at various pH values.

FIG. 19 - Activity assay of pro-ananain activation in MES buffer at varied pH values. Graphic representation of the activity of mature ananain resulting from the activation process at different pH values in MES buffer. PLQ is the fluorescent tripeptidyl substrate: MeOC—GGG—PLQ—GG—DPA—KK—NH₂. Cleavage at the core PLQ tripeptide tethered between the fluorescent MeOC donor group and the DPA quencher moiety releases the fluorescence of the MeOC donor group, which is detected by using a POLARstar fluorescent plate reader (BMG Labtech, Offenburg, Germany) with excitation and emission wavelengths of 320 nm and 420 nm, respectively. The initial rate of proteolysis of substrate is measured as the slope of the linear portion of the progressive curve and is converted to the unit of µg PLQ molecules per min.

FIG. 20 - The SDS-PAGE of pro-ananain activation in varied concentrations of MES buffer. SDS-PAGE was applied to visualize active ananain resulting from the activation process in different concentrations of MES buffer. Pro-ananain and active ananain are displayed as a 36 kDa and a 24 kDa band, respectively, in a 12.5% SDS-gel. Lane 1, molecule weight markers. Lanes 2 - 15, incubation in various concentrations of MES buffer.

FIG. 21 - Activity assay of pro-ananain activation in varied concentrations of MES. Graphic representation of the activity of mature ananain resulting from the activation process in different concentrations of MES buffer. PLQ is the fluorescent tripeptidyl substrate: MeOC—GGG—PLQ—GG—DPA—KK—NH₂. Cleavage at the core PLQ tripeptide tethered between the fluorescent MeOC donor group and the DPA quencher moiety releases the fluorescence of the MeOC donor group, which is detected by using a POLARstar fluorescent plate reader (BMG Labtech, Offenburg, Germany) with excitation and emission wavelengths of 320 nm and 420 nm, respectively. The initial rate of proteolysis of substrate is measured as the slope of the linear portion of the progressive curve and is converted to the unit of µg PLQ molecules per min.

FIG. 22 - SDS-PAGE of pro-ananain activation in varied concentrations of L-Cys in MES buffer. SDS-PAGE was applied to visualize active ananain resulting from the activation process in different concentrations of L-Cys in MES buffer. Pro-ananain and active ananain are displayed as a 36 kDa and a 24 kDa band, respectively, in a 12.5% SDS-gel. Lane 1, molecule weight markers. Lanes 2 - 9, incubation at various concentrations of L-Cys in MES buffer.

FIG. 23 - Activity assay of pro-ananain activation in varied concentrations of L-Cys in an MES buffer. Graphic representation of the activity of mature ananain resulting from the activation process in different concentrations of L-Cys in MES buffer. PLQ is the fluorescent tripeptidyl substrate: MeOC—GGG—PLQ—GG—DPA—KK—NH₂. Cleavage at the core PLQ tripeptide tethered between the fluorescent MeOC donor group and the DPA quencher moiety releases the fluorescence of the MeOC donor group, which is detected by using a POLARstar fluorescent plate reader (BMG Labtech, Offenburg, Germany) with excitation and emission wavelengths of 320 nm and 420 nm, respectively. The initial rate of proteolysis of substrate is measured as the slope of the linear portion of the progressive curve and is converted to the unit of µg PLQ molecules per min.

FIG. 24 - SDS-PAGE of pro-ananain activation at varied temperatures in MES buffer. SDS-PAGE was applied to visualize active ananain resulting from the activation process at different temperatures between 22 and 50° C. in MES buffer. Pro-ananain and active ananain are displayed as a 36 kDa and a 24 kDa band, respectively, in a 12.5% SDS-gel. Lane 1, molecule weight markers. Lanes 2 - 18, incubation at various temperatures.

FIG. 25 - Activity assay of pro-ananain activation at varied temperatures in MES buffer. Graphic representation of the activity of mature ananain resulting from the activation process at different temperatures between 0 and 50° C. in MES buffer. PLQ is the fluorescent tripeptidyl substrate: MeOC—GGG—PLQ—GG—DPA—KK—NH₂. Cleavage at the core PLQ tripeptide tethered between the fluorescent MeOC donor group and the DPA quencher moiety releases the fluorescence of the MeOC donor group, which is detected by using a POLARstar fluorescent plate reader (BMG Labtech, Offenburg, Germany) with excitation and emission wavelengths of 320 nm and 420 nm, respectively. The initial rate of proteolysis of substrate is measured as the slope of the linear portion of the progressive curve and is converted to the unit of µg PLQ molecules per min.

FIG. 26 - SDS-PAGE of pro-ananain activation in sodium citrate buffer at varied pH values. SDS-PAGE was applied to visualize active ananain resulting from the activation process at different pH values in sodium citrate buffer. Pro-ananain and active ananain are displayed as a 36 kDa and a 24 kDa band, respectively, in a 12.5% SDS-gel. Lane 1, molecule weight markers. Lanes 2 - 9, incubation at various pH values.

FIG. 27 - Activity assay of pro-ananain activation in sodium citrate buffer at varied pH values. Graphic representation of the activity of mature ananain resulting from the activation process at different pH values in sodium citrate buffer. PLQ is the fluorescent tripeptidyl substrate: MeOC—GGG—PLQ—GG—DPA—KK—NH₂. Cleavage at the core PLQ tripeptide tethered between the fluorescent MeOC donor group and the DPA quencher moiety releases the fluorescence of the MeOC donor group, which is detected by using a POLARstar fluorescent plate reader (BMG Labtech, Offenburg, Germany) with excitation and emission wavelengths of 320 nm and 420 nm, respectively. The initial rate of proteolysis of substrate is measured as the slope of the linear portion of the progressive curve and is converted to the unit of µg PLQ molecules per min.

FIG. 28 - SDS-PAGE of pro-ananain activation in varied concentrations of sodium citrate buffer. SDS-PAGE was applied to visualize active ananain resulting from the activation process at different pH values in phosphate-citrate buffer. Pro-ananain and active ananain are displayed as a 36 kDa and a 24 kDa band, respectively, in a 12.5% SDS-gel. Lane 1, molecule weight markers. Lanes 2 - 10, incubation in various concentrations of sodium citrate buffer.

FIG. 29 - Activity assay of pro-ananain activation in varied concentrations of sodium citrate buffer. Graphic representation of the activity of mature ananain resulting from the activation process in different concentrations of sodium citrate buffer. PLQ is the fluorescent tripeptidyl substrate: MeOC—GGG—PLQ—GG—DPA—KK—NH₂. Cleavage at the core PLQ tripeptide tethered between the fluorescent MeOC donor group and the DPA quencher moiety releases the fluorescence of the MeOC donor group, which is detected by using a POLARstar fluorescent plate reader (BMG Labtech, Offenburg, Germany) with excitation and emission wavelengths of 320 nm and 420 nm, respectively. The initial rate of proteolysis of substrate is measured as the slope of the linear portion of the progressive curve and is converted to the unit of µg PLQ molecules per min.

FIG. 30 - SDS-PAGE of pro-ananain activation in varied concentrations of L-Cys in sodium citrate buffer. SDS-PAGE was applied to visualize active ananain resulting from the activation process in different concentrations of L-Cys in sodium citrate buffer. Pro-ananain and active ananain are displayed as a 36 kDa and a 24 kDa band, respectively, in a 12.5% SDS-gel. Lane 1, molecule weight markers. Lanes 2 - 10, incubation in various concentrations of L-Cys.

FIG. 31 - Activity assay of pro-ananain activation in varied concentrations of L-Cys in sodium citrate buffer. Graphic representation of the activity of mature ananain resulting from the activation process in different concentrations of L-Cys in sodium citrate buffer. PLQ is the fluorescent tripeptidyl substrate: MeOC—GGG—PLQ—GG—DPA—KK—NH₂. Cleavage at the core PLQ tripeptide tethered between the fluorescent MeOC donor group and the DPA quencher moiety releases the fluorescence of the MeOC donor group, which is detected by using a POLARstar fluorescent plate reader (BMG Labtech, Offenburg, Germany) with excitation and emission wavelengths of 320 nm and 420 nm, respectively. The initial rate of proteolysis of substrate is measured as the slope of the linear portion of the progressive curve and is converted to the unit of µg PLQ molecules per min.

FIG. 32 - SDS-PAGE of pro-ananain activation at varied temperatures in sodium citrate buffer. SDS-PAGE was applied to visualize active ananain resulting from the activation process at different temperatures between 24 and 50° C. in sodium citrate buffer. Pro-ananain and active ananain are displayed as a 36 kDa and a 24 kDa band, respectively, in a 12.5% SDS-gel. Lane 1, molecule weight markers. Lanes 2 - 10, incubation at different temperatures.

FIG. 33 - Activity assay of pro-ananain activation at varied temperatures in sodium citrate buffer. Graphic representation of the activity of mature ananain resulting from the activation process at different temperatures between 24 and 50° C. in sodium citrate buffer. PLQ is the fluorescent tripeptidyl substrate: MeOC—GGG—PLQ—GG—DPA—KK—NH₂. Cleavage at the core PLQ tripeptide tethered between the fluorescent MeOC donor group and the DPA quencher moiety releases the fluorescence of the MeOC donor group, which is detected by using a POLARstar fluorescent plate reader (BMG Labtech, Offenburg, Germany) with excitation and emission wavelengths of 320 nm and 420 nm, respectively. The initial rate of proteolysis of substrate is measured as the slope of the linear portion of the progressive curve and is converted to the unit of µg PLQ molecules per min.

FIG. 34 - SDS-PAGE of pro-ananain activation in phosphate buffered saline (PBS) buffer at varied pH values. SDS-PAGE was applied to visualize active ananain resulting from the activation process at different pH values in PBS buffer. Pro-ananain and active ananain are displayed as a 36 kDa and a 24 kDa band, respectively, in a 12.5% SDS-gel. Lane 1, molecule weight markers. Lanes 2-9, incubation at various pH values.

FIG. 35 - Activity assay of pro-ananain activation in PBS buffer at varied pH values. Graphic representation of the activity of mature ananain resulting from the activation process at different pH values in PBS buffer. PLQ is the fluorescent tripeptidyl substrate: MeOC—GGG—PLQ—GG—DPA—KK—NHz. Cleavage at the core PLQ tripeptide tethered between the fluorescent MeOC donor group and the DPA quencher moiety releases the fluorescence of the MeOC donor group, which is detected by using a POLARstar fluorescent plate reader (BMG Labtech, Offenburg, Germany) with excitation and emission wavelengths of 320 nm and 420 nm, respectively. The initial rate of proteolysis of substrate is measured as the slope of the linear portion of the progressive curve and is converted to the unit of µg PLQ molecules per min.

FIG. 36 - SDS-PAGE of pro-ananain activation in varied concentrations of L-Cys in PBS buffer. SDS-PAGE was applied to visualize active ananain resulting from the activation process in different concentrations of L-Cys in PBS buffer. Pro-ananain and active ananain are displayed as a 36 kDa and a 24 kDa band, respectively, in a 12.5% SDS-gel. Lane 1, molecule weight markers. Lanes 2 - 10, incubation in various concentrations of L-Cys.

FIG. 37 - Activity assay of pro-ananain activation in varied concentration of L-Cys or ascorbic acid in PBS buffer. Graphic representation of the activity of mature ananain resulting from the activation process at varied concentrations of reducing agent: L-Cys or ascorbic acid in PBS buffer. PLQ is the fluorescent tripeptidyl substrate: MeOC—GGG—PLQ—GG—DPA—KK—NH₂. Cleavage at the core PLQ tripeptide tethered between the fluorescent MeOC donor group and the DPA quencher moiety releases the fluorescence of the MeOC donor group, which is detected by using a POLARstar fluorescent plate reader (BMG Labtech, Offenburg, Germany) with excitation and emission wavelengths of 320 nm and 420 nm, respectively. The initial rate of proteolysis of substrate is measured as the slope of the linear portion of the progressive curve and is converted to the unit of µg PLQ molecules per min.

FIG. 38 - SDS-PAGE of pro-ananain activation at varied temperatures in PBS buffer. SDS-PAGE was applied to visualize active ananain resulting from the activation process at different temperatures between 22 and 50° C. in PBS buffer. Pro-ananain and active ananain are displayed as a 36 kDa and a 24 kDa band, respectively, in a 12.5% SDS-gel. Lane 1, molecule weight markers. Lanes 2 - 18, incubation at various temperatures.

FIG. 39 - Activity assay of pro-ananain activation at varied temperatures in PBS buffer. Graphic representation of the activity of mature ananain resulting from the activation process at different temperatures between 22 and 50° C. in PBS buffer. PLQ is the fluorescent tripeptidyl substrate: MeOC—GGG—PLQ—GG—DPA—KK—NH₂. Cleavage at the core PLQ tripeptide tethered between the fluorescent MeOC donor group and the DPA quencher moiety releases the fluorescence of the MeOC donor group, which is detected by using a POLARstar fluorescent plate reader (BMG Labtech, Offenburg, Germany) with excitation and emission wavelengths of 320 nm and 420 nm, respectively. The initial rate of proteolysis of substrate is measured as the slope of the linear portion of the progressive curve and is converted to the unit of µg PLQ molecules per min.

FIG. 40 - Activity assay of water reconstituted pro-ananain formulations. Five lyophilized pro-ananain formulations were resuspended with 0.5 ml water pre-warmed at 37° C. The solutions were incubated at 37° C. for 120 min for a time course examination of active ananain. PLQ is the fluorescent tripeptidyl substrate: MeOC—GGG—PLQ—GG—DPA—KK—NH₂. Cleavage at the core PLQ tripeptide tethered between the fluorescent MeOC donor group and the DPA quencher moiety releases the fluorescence of the MeOC donor group, which is detected by using a POLARstar fluorescent plate reader (BMG Labtech, Offenburg, Germany) with excitation and emission wavelengths of 320 nm and 420 nm, respectively. The initial rate of proteolysis of substrate is measured as the slope of the linear portion of the progressive curve and is converted to the unit of µg PLQ molecules per min.

FIG. 41 - The proteolytic activity of ananain against human plasma proteins. SDS-PAGE was applied to visualize the proteolytic activity of ananain with human plasma proteins. Lane 1, molecule weight markers; Lane 2 - ana alone - ananain only. A(-): Albumin-partially depleted human plasma, A(+): Albumin-partially depleted human plasma + ananain; B(-):Albumin-fully depleted human plasma, B(+) Albumin-fully depleted human plasma + ananain; C(-): Human plasma, C(+): Human plasma + ananain; D(-): Human serum, D(+) Human serum + ananain. The reaction was prepared in PBS (pH7.4), incubated at 37° C. for 15 min. Active ananain from the phosphate citrate pro-ananain formulation maintained its proteolytic activity against human plasma proteins at a > 1:100 ratio within 15 min. The clear reduction of albumin in sample A, C and D in present of ananain indicates that albumin is a protein substrate for ananain. The cleavage results in fragments of varied molecular weights, including 1, 2, 3 and 4 as marked with arrows, which are not presented in sample B, confirming that the source of these fragments is albumin. Besides albumin, high molecular weight human proteins or complexes, marked as bands 5, 6 and 7 are also highly likely protein substrates for ananain.

FIG. 42 - The amino acid (SEQ ID NO: 3) and nucleic acid (SEQ ID NO: 4) sequences of pro-stem bromelain (pro-SB) used herein.

FIG. 43 - SDS-PAGE showing cleavage of pro-SB by active ananain. 40 µg of pro-SB was mixed with 1.6 µg of active ananain, in a final volume of 160 µl of AMT buffer, pH 5.0, with 12 mM L-Cys. The solutions were incubated at room temperature (RT) and at every designated time point of 0, 1, 2, 3, 5, 10 and 15 min (lanes 2-8), a 20 µl of solution was subjected to SDS-PAGE analysis, as described previously. As control, a sample with 5 µg of pro-SB (lane 9) and another with 0.2 µg of active ananain (lane 10) in 20 µl of AMT buffer with 12 mM L-Cys was respectively incubated at RT for 120 min. Lane 1 = molecular weight markers.

FIG. 44 - SDS-PAGE showing cleavage of pro-SB by immobilized ananain. 2 mg of active ananain was immobilized in a 5 ml HiTrap™ NHS-Activated HP affinity column (GE Health, IL, USA). A 5 mg of pro-SB was resuspended in 5 ml AMT buffer, pH 5.0, with 12 mM L-Cys. The pro-SB was circulated through the ananain-immobilized column at RT for overnight. The collected activated SB was subject to SDS-PAGE analysis and was confirmed to be fully cleaved. Lane 1 = molecular weight markers.

FIG. 45 - Proteolytic activity of activated stem bromelain against PRR substrate The proteolytic activity of activated stem bromelain was assayed using the fluorescent tripeptidyl substrate, PRR, in the form of MeOC—GGG—PRR—GG—OPA—KK—NH₂ (Mimotopes, Clayton, Australia). Following the activation process, 200 nM of activated SB was mixed with a final concentration of 25 µM of PRR in a final volume of 100 µl AAB, Cleavage at the core PRR tripeptide releases the fluorescence of the MeOC donor group, which is detected by using a POLARstar fluorescent plate reader, as described previously. Solid circles line = proteolysis due to active stem bromelain. Open circles = proteolysis due to pro-SB.

FIG. 46 - The amino acid (SEQ ID NO: 5) and nucleic acid (SEQ ID NO: 6) sequences of pro-fruit bromelain (pro-FB) used herein.

FIG. 47 - SDS-PAGE showing cleavage of pro-FB by immobilized ananain. 2 mg of active ananain was immobilized in a 5 ml HiTrap™ NHS-Activated HP affinity column (GE Health, IL, USA). A 5 mg of pro-FB was resuspended in 5 ml AMT buffer, pH 5.0, with 12 mM L-Cys. The pro-FB was circulated through the ananain-immobilized column at RT for overnight. The collected activated FB was subject to SDS-PAGE analysis and was confirmed to be fully cleaved. Lane 1 = molecular weight markers.

FIG. 48 - – Proteolytic activity of activated fruit bromelain against FVR-AMC-substrate. Activity of activated fruit bromelain was measured using a fluorescent tripeptidyl substrate, FVR-7-amino-4-methylcoumarin (FVR-AMC). A 10 nM of activated FB was mixed with a final concentration of 50 µM of FVR-AMC in a final volume of 100 µl AAB buffer, pH 4.0. Cleavage of FVR-AMC releases the fluorescent AMC group, which is detected by using a POLARstar fluorescent plate reader, using excitation and emission wavelengths of 360 and 460 nm, respectively. Solid circles = proteolysis due to active fruit bromelain. Open circles = proteolysis due to pro-FB.

DETAILED DESCRIPTION OF THE INVENTION
Definitions

The following definitions are presented to better define the present invention and as a guide for those of ordinary skill in the art in the practice of the present invention.

Unless otherwise specified, all technical and scientific terms used herein are to be understood as having the same meanings as is understood by one of ordinary skill in the relevant art to which this disclosure pertains.

It is also believed that practice of the present invention can be performed using standard cell biology, microbiological, molecular biology, pharmacology and biochemistry protocols and procedures as known in the art, and as described, for example in numerous commonly available reference materials relevant in the art to which this disclosure pertains.

Examples of definitions of common terms in microbiology, molecular biology and biochemistry can be found in Methods for General and Molecular Microbiology, 3rd Edition, C. A. Reddy, et al. (eds.), ASM Press, (2008); Encyclopedia of Microbiology, 2nd ed., Joshua Lederburg, (ed.), Academic Press, (2000); Microbiology By Cliffs Notes, I. Edward Alcamo, Wiley, (1996); Dictionary of Microbiology and Molecular Biology, Singleton et al. (2d ed.) (1994); Biology of Microorganisms 11^th ed., Brock et al., Pearson Prentice Hall, (2006); Genes IX, Benjamin Lewin, Jones & Bartlett Publishing, (2007); The Encyclopedia of Molecular Biology, Kendrew et al. (eds.), Blackwell Science Ltd., (1994) and Molecular Biology and Biotechnology: a Comprehensive Desk Reference, Robert A. Meyers (ed.), VCH Publishers, Inc., (1995).

The term “pro-ananain” as used herein means the non-proteolytic zymogen form of ananain. In some embodiments, pro-ananain comprises the amino acid sequence of SEQ ID NO: 1.

The terms “ananain” and “mature ananain” are used interchangeably herein and mean the proteolytic, activated ananain enzyme having the ability to catalyse proteolysis.

The term “pro-bromelain” as used herein means the non-proteolytic zymogen form of bromelain. In some embodiments, pro-bromelain is pro-stem bromelain. In some embodiments, pro-stem bromelain comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, pro-bromelain is pro-fruit bromelain. In some embodiments, pro-fruit bromelain comprises the amino acid sequence of SEQ ID NO: 5.

The terms “bromelain” and “mature bromelain” are used interchangeably herein and mean the proteolytic, activated bromelain enzyme having the ability to catalyse proteolysis.

The terms “recombinant pro-ananaiii” and “recombinant pro-bromelain” refer to polypeptides that are expressed in vitro from a polynucleotide sequence that is removed from sequences that surround it in its natural context and/or is recombined with sequences that are not present in its natural context, A “recombinant” pro-ananain or pro-bromelain polypeptide sequence is produced by translation from a “recombinant” polynucleotide sequence.

The term “a stable composition” as used herein means a composition, preferably a dry composition comprising pro-ananain or pro-bromelain or both that can be reconstituted as an aqueous composition comprising active ananain or active bromelain or both. In one embodiment a stable composition is a lyophilized composition as described herein, comprising a pharmaceutically acceptable buffer, NaCl, a pharmaceutically acceptable reducing agent and recombinant pro-ananain or recombinant pro-bromelain or both.

The term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, “about 100” means from 90 to 110 and “about six” means from 5.4 to 6.6.

The term “comprising” as used in this specification means “consisting at least in part of”. When interpreting statements in this specification that include that term, the features, prefaced by that term in each statement, all need to be present but other features can also be present. Related terms such as “comprise” and “comprised” are to be interpreted in the same manner.

The term “consisting essentially of” as used herein means the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

The term “consisting of” as used herein means the specified materials or steps of the claimed invention, excluding any element, step, or ingredient not specified in the claim.

It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

DESCRIPTION

The core event in the activation of cysteine proteases is the cleavage at the peptide bond between the inhibitory sequence and the protease domain. Cleavage can be mediated by either the active site of a protease molecule itself (and subsequently toward other pro-enzyme molecules of the same kind known as ‘auto-activation’) or by a protease having a different identity (‘trans-activation’). Protease activation also involves interfering with the molecular interactions between the inhibitory sequence and the protease domain. Such interactions usually involve strong hydrogen bonding, charge interaction and Van der Waal force among residues of both structures. Accordingly, the buffer conditions (type, concentration and pH value) of a protease solution are important as they affect the strength of such inhibitory interactions. Moreover, it is generally believed that a reducing agent is critical for the proteolytic activity of cysteine proteases, either for its contribution on preventing oxidation of the active Cys residue, and/or by affecting the overall secondary structure of the protein. What the inventors have surprisingly found is that for cysteine proteases undergoing auto-activation, such as pro-ananain, careful selection of the types of buffers and reducing agents and accurate determining the concentrations of the components, has enabled them to identify physiologically relevant conditions for the activation of pro-ananain, including the activation of pro-ananain that has been stabilized in a dry powder.

In this project, a number of physiologically relevant buffers (phosphate citrate, sodium acetate, MES, sodium citrate and PBS) and reducing agents (L-Cys and ascorbic acid) were selected for developing the activation formula of pro-ananain comprised in the compositions described herein. In order to maximise the applicability of the final formula for humans and animals, the concentrations of the buffer and reducing agent were brought to the minimal range, yet the activation of pro-ananain achieves the maximal range at physiological temperatures of humans. Further, the established pro-ananain formula substantially encompasses the physiological pH range of humans from weak acidic (pH 5) to neutral (pH 7.5). Moreover, the inventors have surprisingly found that a composition as described herein, comprising an activation formula of pro-ananain, can be lyophilized to provide a stable composition comprising pro-ananain. That stable composition can then be reconstituted with water to provide a composition as described herein, comprising active ananain. The inventors believe that the provision of a lyophilized form of a pro-ananain formula as described herein is a cost-effective means of delivering pure and stable pro-ananain with a long shelf-life. Following reconstitution in purified water, the water-reconstituted formula provides an easy and effective way to rapidly generate active ananain having sustained proteolytic activity. In addition, the inventors have surprisingly found that the trans-activation of pro-stem bromelain and pro-fruit bromelain can be mediated by active ananain and used this knowledge to develop an activation formula comprising both pro-stem and pro-fruit bromelain enzymes.

Based on the surprising findings detailed herein the inventors believe that formulations of pure, stable ananain and/or ananain + bromelain can be provided for various research and therapeutic purposes, particularly at physiologically relevant conditions and using pharmaceutically acceptable constituents.

Accordingly, in one aspect the invention relates to a composition comprising:

(a) recombinant pro-ananain
(b) a pharmaceutically acceptable buffer
(c) a pharmaceutically acceptable reducing agent, and
(d) sodium chloride (NaCl),
- wherein the concentration of the buffer is about 5 to about 30 mM,
- wherein the concentration of the reducing agent is about 10 to about 30 mM,
- wherein the concentration of the NaCl is about 140 to about 160 mM and,
- wherein the pH of the composition is about 5.0 to about 6.0.

In one embodiment the recombinant pro-anariain comprises a polypeptide comprising at least 70% amino acid identity to (SEQ ID NO: 1).

In one embodiment the recombinant pro-ananain comprises at least 75%, preferably 80%, 85%, 90%, 95%, preferably 99% amino acid identity to (SEQ ID NO: 1).

In one embodiment the recombinant pro-ananain comprises (SEQ ID NO: 1). In one embodiment the recombinant pro-ananain consists or consists essentially of (SEQ ID NO: 1).

In one embodiment the composition is an aqueous composition.

In one embodiment the composition comprises about 0.001 mg/mL to about 10 mg/mL recombinant pro-ananain. In one embodiment the composition comprises about 1 mg/mL recombinant pro-ananain.

In one embodiment the composition comprises about 0.001 mM to about 1 mM recombinant pro-ananain. In one embodiment the composition comprises about 0.027 mM pro-ananain.

In one embodiment the buffer is selected from the group consisting of phosphate-citrate, sodium acetate, sodium citrate and 2-(N-morpholino)ethanesulfonic acid (MES) buffers.

In one embodiment the buffer comprises citric acid.

In one embodiment the buffer comprises sodium hydrogen phosphate.

In one embodiment the buffer is phosphate-citrate.

In one embodiment the composition comprises about 5 mM to about 20 mM phosphate-citrate buffer.

In one embodiment the composition comprises about 5.5 mM to about 17 mM phosphate-citrate buffer.

In one embodiment the composition comprises about 6 mM to about 15 mM phosphate-citrate buffer.

In one embodiment the composition comprises about 6.5 mM to about 13 mM phosphate-citrate buffer.

In one embodiment the composition comprises about 7 mM to about 11 mM phosphate-citrate buffer.

In one embodiment the composition comprises about 7.5 mM to about 10 mM phosphate-citrate buffer.

In one embodiment the composition comprises about 8 mM to about 9 mM phosphate-citrate buffer.

In one embodiment the composition comprises about 8 mM phosphate-citrate buffer.

In one embodiment the composition comprises 5 mM to 20 mM phosphate-citrate buffer.

In one embodiment the composition comprises 5.5 mM to 17 mM phosphate-citrate buffer.

In one embodiment the composition comprises 6 mM to 15 mM phosphate-citrate buffer.

In one embodiment the composition comprises 6.5 mM to 13 mM phosphate-citrate buffer.

In one embodiment the composition comprises 7 mM to 11 mM phosphate-citrate buffer.

In one embodiment the composition comprises 7.5 mM to 10 mM phosphate-citrate buffer.

In one embodiment the composition comprises 8 mM to 9 mM phosphate-citrate buffer.

In one embodiment the composition comprises 8 mM phosphate-citrate buffer.

In one embodiment the buffer comprises an acid salt.

In one embodiment the acid salt is sodium citrate.

In one embodiment the composition comprises about 5 mM to about 20 mM sodium citrate buffer.

In one embodiment the composition comprises about 5.5 mM to about 17 mM sodium citrate buffer. In one embodiment the composition comprises about 6 mM to about 15 mM sodium citrate buffer.

In one embodiment the composition comprises about 6.5 mM to about 13 mM sodium citrate buffer. In one embodiment the composition comprises about 7 mM to about 11 mM sodium citrate buffer.

In one embodiment the composition comprises about 7.5 mM to about 10 mM sodium citrate buffer. In one embodiment the composition comprises about 8 mM to about 9 mM sodium citrate buffer.

In one embodiment the composition comprises about 8 mM sodium citrate buffer.

In one embodiment the composition comprises 5 mM to 20 mM sodium citrate buffer.

In one embodiment the composition comprises 5.5 mM to 17 mM sodium citrate buffer.

In one embodiment the composition comprises 6 mM to 15 mM sodium citrate buffer.

In one embodiment the composition comprises 6.5 mM to 13 mM sodium citrate buffer.

In one embodiment the composition comprises 7 mM to 11 mM sodium citrate buffer.

In one embodiment the composition comprises 7.5 mM to 10 mM sodium citrate buffer.

In one embodiment the composition comprises 8 mM to 9 mM sodium citrate buffer.

In one embodiment the composition comprises 8 mM sodium citrate buffer.

In one embodiment the buffer is an acetate buffer.

In one embodiment the acetate buffer is sodium acetate.

In one embodiment the composition comprises about 3 mM to about 18 mM sodium acetate buffer.

In one embodiment the composition comprises about 3.5 mM to about 16 mM sodium acetate buffer.

In one embodiment the composition comprises about 4 mM to about 14 mM sodium acetate buffer.

In one embodiment the composition comprises about 4.5 mM to about 12 mM sodium acetate buffer.

In one embodiment the composition comprises about 5 mM to about 10 mM sodium acetate buffer.

In one embodiment the composition comprises about 5.5 mM to about 8 mM sodium acetate buffer.

In one embodiment the composition comprises about 6 mM to about 7 mM sodium acetate buffer.

In one embodiment the composition comprises about 6 mM sodium acetate buffer.

In one embodiment the composition comprises 3 mM to 18 mM sodium acetate buffer.

In one embodiment the composition comprises 3.5 mM to 16 mM sodium acetate buffer.

In one embodiment the composition comprises 4 mM to 14 mM sodium acetate buffer.

In one embodiment the composition comprises 4.5 mM to 12 mM sodium acetate buffer.

In one embodiment the composition comprises 5 mM to 10 mM sodium acetate buffer.

In one embodiment the composition comprises 5.5 mM to 8 mM sodium acetate buffer.

In one embodiment the composition comprises 6 mM to 7 mM sodium acetate buffer.

In one embodiment the composition comprises 6 mM sodium acetate buffer.

In one embodiment the buffer is 2-(N-morpholino)ethanesulfonic acid (MES).

In one embodiment the composition comprises about 5 mM to about 25 mM MES buffer.

In one embodiment the composition comprises about 7 mM to about 23 mM MES buffer.

In one embodiment the composition comprises about 9 mM to about 21 mM MES buffer.

In one embodiment the composition comprises about 11 mM to about 19 mM MES buffer.

In one embodiment the composition comprises about 13 mM to about 17 mM MES buffer.

In one embodiment the composition comprises about 14 mM to about 16 mM MES buffer.

In one embodiment the composition comprises about 15 mM MES buffer.

In one embodiment the composition comprises 5 mM to 25 mM MES buffer.

In one embodiment the composition comprises 7 mM to 23 mM MES buffer.

In one embodiment the composition comprises 9 mM to 21 mM MES buffer.

In one embodiment the composition comprises 11 mM to 19 mM MES buffer.

In one embodiment the composition comprises 13 mM to 17 mM MES buffer.

In one embodiment the composition comprises 14 mM to 16 mM MES buffer.

In one embodiment the composition comprises 15 mM MES buffer.

In one embodiment the buffer is phosphate buffered saline (PBS).

In one embodiment the reducing agent is L- cysteine (L-Cys).

In one embodiment the composition comprises about 8 mM phosphate citrate buffer and about 10 mM to about 30 mM L-Cys.

In one embodiment the composition comprises about 8 mM phosphate citrate buffer and about 11 mM to about 28 mM L-Cys.

In one embodiment the composition comprises about 8 mM phosphate citrate buffer and about 12 mM to about 26 mM L-Cys.

In one embodiment the composition comprises about 8 mM phosphate citrate buffer and about 13 mM to about 24 mM L-Cys.

In one embodiment the composition comprises about 8 mM phosphate citrate buffer and about 14 mM to about 22 mM L-Cys.

In one embodiment the composition comprises about 8 mM phosphate citrate buffer and about 15 mM to about 20 mM L-Cys.

In one embodiment the composition comprises about 8 mM phosphate citrate buffer and about 16 mM to about 18 mM L-Cys.

In one embodiment the composition comprises about 8 mM phosphate citrate buffer and about 17 mM L-Cys.

In one embodiment the composition comprises about 8 mM phosphate citrate buffer and about 16 mM L-Cys.

In one embodiment the composition comprises 8 mM phosphate citrate buffer and 10 mM to 30 mM L-Cys.

In one embodiment the composition comprises 8 mM phosphate citrate buffer and 11 mM to 28 mM L-Cys.

In one embodiment the composition comprises 8 mM phosphate citrate buffer and 12 mM to 26 mM L-Cys.

In one embodiment the composition comprises 8 mM phosphate citrate buffer and 13 mM to 24 mM L-Cys.

In one embodiment the composition comprises 8 mM phosphate citrate buffer and 14 mM to 22 mM L-Cys.

In one embodiment the composition comprises 8 mM phosphate citrate buffer and 15 mM to 20 mM L-Cys.

In one embodiment the composition comprises 8 mM phosphate citrate buffer and 16 mM to 18 mM L-Cys.

In one embodiment the composition comprises 8 mM phosphate citrate buffer and 17 mM L-Cys.

In one embodiment the composition comprises 8 mM phosphate citrate buffer and 16 mM L-Cys.

In one embodiment the composition comprises about 8 mM sodium citrate buffer and about 10 mM to about 30 mM L-Cys.

In one embodiment the composition comprises about 8 mM sodium citrate buffer and about 12 mM to about 29 mM L-Cys.

In one embodiment the composition comprises about 8 mM sodium citrate buffer and about 14 mM to about 28 mM L-Cys.

In one embodiment the composition comprises about 8 mM sodium citrate buffer and about 16 mM to about 27 mM L-Cys.

In one embodiment the composition comprises about 8 mM sodium citrate buffer and about 18 mM to about 26 mM L-Cys.

In one embodiment the composition comprises about 8 mM sodium citrate buffer and about 20 mM to about 24 mM L-Cys.

In one embodiment the composition comprises about 8 mM sodium citrate buffer and about 21 mM to about 23 mM L-Cys.

In one embodiment the composition comprises about 8 mM sodium citrate buffer and about 22 mM L-Cys.

In one embodiment the composition comprises 8 mM sodium citrate buffer and 10 mM to 30 mM L-Cys.

In one embodiment the composition comprises 8 mM sodium citrate buffer and 12 mM to 29 mM L-Cys.

In one embodiment the composition comprises 8 mM sodium citrate buffer and 14 mM to 28 mM L-Cys.

In one embodiment the composition comprises 8 mM sodium citrate buffer and 16 mM to 27 mM L-Cys.

In one embodiment the composition comprises 8 mM sodium citrate buffer and 18 mM to 26 mM L-Cys.

In one embodiment the composition comprises 8 mM sodium citrate buffer and 20 mM to 24 mM L-Cys.

In one embodiment the composition comprises 8 mM sodium citrate buffer and 21 mM to 23 mM L-Cys.

In one embodiment the composition comprises 8 mM sodium citrate buffer and 22 mM L-Cys. In one embodiment the composition comprises about 6 mM sodium acetate buffer and about 5 mM to about 19 mM L-Cys.

In one embodiment the composition comprises about 6 mM sodium acetate buffer and about 6 mM to about 18 mM L-Cys.

In one embodiment the composition comprises about 6 mM sodium acetate buffer and about 7 mM to about 17 mM L-Cys.

In one embodiment the composition comprises about 6 mM sodium acetate buffer and about 8 mM to about 16 mM L-Cys.

In one embodiment the composition comprises about 6 mM sodium acetate buffer and about 9 mM to about 15 mM L-Cys.

In one embodiment the composition comprises about 6 mM sodium acetate buffer and about 10 mM to about 14 mM L-Cys.

In one embodiment the composition comprises about 6 mM sodium acetate buffer and about 11 mM to about 13 mM L-Cys.

In one embodiment the composition comprises about 6 mM sodium acetate buffer and about 12 mM L-Cys.

In one embodiment the composition comprises 6 mM sodium acetate buffer and 5 mM to 19 mM L-Cys.

In one embodiment the composition comprises 6 mM sodium acetate buffer and 6 mM to 18 mM L-Cys.

In one embodiment the composition comprises 6 mM sodium acetate buffer and 7 mM to 17 mM L-Cys.

In one embodiment the composition comprises 6 mM sodium acetate buffer and 8 mM to 16 mM L-Cys.

In one embodiment the composition comprises 6 mM sodium acetate buffer and 9 mM to 15 mM L-Cys.

In one embodiment the composition comprises 6 mM sodium acetate buffer and 10 mM to 14 mM L-Cys.

In one embodiment the composition comprises 6 mM sodium acetate buffer and 11 mM to 13 mM L-Cys.

In one embodiment the composition comprises 6 mM sodium acetate buffer and 12 mM L-Cys.

In one embodiment the composition comprises about 15 mM MES buffer and about 7 mM to about 21 mM L-Cys.

In one embodiment the composition comprises about 15 mM MES buffer and about 8 mM to about 20 mM L-Cys.

In one embodiment the composition comprises about 15 mM MES buffer and about 9 mM to about 19 mM L-Cys.

In one embodiment the composition comprises about 15 mM MES buffer and about 10 mM to about 18 mM L-Cys.

In one embodiment the composition comprises about 15 mM MES buffer and about 11 mM to about 17 mM L-Cys.

In one embodiment the composition comprises about 15 mM MES buffer and about 12 mM to about 16 mM L-Cys.

In one embodiment the composition comprises about 15 mM MES buffer and about 13 mM to about 15 mM L-Cys.

In one embodiment the composition comprises about 15 mM MES buffer and about 14 mM L-Cys.

In one embodiment the composition comprises 15 mM MES buffer and 7 mM to 21 mM L-Cys.

In one embodiment the composition comprises 15 mM MES buffer and 8 mM to 20 mM L-Cys.

In one embodiment the composition comprises 15 mM MES buffer and 9 mM to 19 mM L-Cys.

In one embodiment the composition comprises 15 mM MES buffer and 10 mM to 18 mM L-Cys.

In one embodiment the composition comprises 15 mM MES buffer and 11 mM to 17 mM L-Cys.

In one embodiment the composition comprises 15 mM MES buffer and 12 mM to 16 mM L-Cys.

In one embodiment the composition comprises 15 mM MES buffer and 13 mM to 15 mM L-Cys.

In one embodiment the composition comprises 15 mM MES buffer and 14 mM L-Cys.

In one embodiment the composition comprises PBS and about 8 mM to about 22 mM L-Cys.

In one embodiment the composition comprises PBS and about 9 mM to about 21 mM L-Cys.

In one embodiment the composition comprises PBS and about 10 mM to about 20 mM L-Cys.

In one embodiment the composition comprises PBS and about 11 mM to about 19 mM L-Cys.

In one embodiment the composition comprises PBS and about 12 mM to about 18 mM L-Cys.

In one embodiment the composition comprises PBS and about 13 mM to about 17 mM L-Cys.

In one embodiment the composition comprises PBS and about 14 mM to about 16 mM L-Cys.

In one embodiment the composition comprises PBS and about 15 mM L-Cys.

In one embodiment the composition comprises PBS and 8 mM to 22 mM L-Cys.

In one embodiment the composition comprises PBS and 9 mM to 21 mM L-Cys.

In one embodiment the composition comprises PBS and 10 mM to 20 mM L-Cys.

In one embodiment the composition comprises PBS and 11 mM to 19 mM L-Cys.

In one embodiment the composition comprises PBS and 12 mM to 18 mM L-Cys.

In one embodiment the composition comprises PBS and 13 mM to 17 mM L-Cys.

In one embodiment the composition comprises PBS and 14 mM to 16 mM L-Cys.

In one embodiment the composition comprises PBS and 15 mM L-Cys.

In one embodiment the composition comprises about 150 mM NaCl.

In one embodiment the composition comprises 140 mM to 160 mM NaCl.

In one embodiment the composition comprises 150 mM NaCl.

In one embodiment the composition has a pH of about 5.1 to about 5.9.

In one embodiment the composition has a pH of about 5.2 to about 5.8.

In one embodiment the composition has a pH of about 5.3 to about 5.7.

In one embodiment the composition has a pH of about 5.4 to about 5.6.

In one embodiment the composition has a pH of about 5.5.

In one embodiment the composition has a pH of 5.0 to 6.0

In one embodiment the composition has a pH of 5.1 to 5.9

In one embodiment the composition has a pH of 5.2 to 5.8.

In one embodiment the composition has a pH of 5.3 to 5.7.

In one embodiment the composition has a pH of 5.4 to 5.6

In one embodiment the composition has a pH of 5.5.

In one embodiment the composition is a dry composition.

In one embodiment the dry composition is a freeze-dried composition.

In one embodiment the dry composition is a lyophilized composition.

In one embodiment the dry composition is in the form of a powder.

In one embodiment the freeze-dried or lyophilized composition comprises about 0.001 to about 10 mg recombinant pro-ananain.

In one embodiment the composition further comprises (e) pro-bromelain. In one embodiment the pro-bromelain is at a ratio of pro-ananain:pro-bromelain of about 1:25 to about 1:50.

In one embodiment the pro-bromelain is recombinant pro-bromelain.

In one embodiment the pro-bromelain is stem pro-bromelain. In one embodiment the pro-bromelain is fruit pro-bromelain.

In one embodiment the composition consists essentially of (a), (b), (c) and (d) in any of the embodiments set out above. In one embodiment the composition consists essentially of (a), (b), (c), (d) and (e) in any of the embodiments set out above.

In one embodiment the composition consists of (a), (b), (c) and (d) in any of the embodiments set out above. In one embodiment the composition consists of (a), (b), (c), (d) and (e) in any of the embodiments set out above.

Methods

In another aspect the invention relates to a method of making a composition comprising recombinant pro-ananain, the method comprising combining:

(a) recombinant pro-ananain with
(b) a pharmaceutically acceptable buffer
(c) a pharmaceutically acceptable reducing agent, and
(d) sodium chloride (NaCI),
- wherein the concentration of the buffer is about 5 to about 30 mM,
- wherein the concentration of the reducing agent is about 10 to about 30 mM,
- wherein the concentration of the NaCI is about 140 to about 160 mM and,
- wherein the pH of the composition is about 5.0 to about 6.0.

In one embodiment the recombinant pro-ananain comprises a polypeptide comprising at least 70% amino acid identity to (SEQ ID NO: 1).

In one embodiment the recombinant pro-anariain comprises at least 75%, preferably 80%, 85%, 90%, 95%, preferably 99% amino acid identity to (SEQ ID NO: 1).

In one embodiment the recombinant pro-ananain comprises (SEQ ID NO: 1). In one embodiment the recombinant pro-ananain consists or consists essentially of (SEQ ID NO: 1).

In one embodiment the composition is an aqueous composition.

In one embodiment the composition comprises about 0.001 to about 10 mg/mL recombinant pro-ananain. In one embodiment the composition comprises about 1.0 mg/mL recombinant pro-ananain.

In one embodiment the composition comprises about 0.001 mM to about 1 mM pro-ananain. In one embodiment the composition comprises about 0.027 mM pro-anariain.

In one embodiment the buffer is selected from the group consisting of phosphate-citrate, sodium acetate, sodium citrate and 2-(N-morpholino)ethanesulfonic acid (MES) buffers.

In one embodiment the buffer comprises citric acid.

In one embodiment the buffer comprises sodium hydrogen phosphate.

In one embodiment the buffer is phosphate-citrate.

In one embodiment the composition comprises about 5 mM to about 20 mM phosphate-citrate buffer. In one embodiment the composition comprises about 5.5 mM to about 17 mM phosphate-citrate buffer.

In one embodiment the composition comprises about 6 mM to about 15 mM phosphate-citrate buffer.

In one embodiment the composition comprises about 6.5 mM to about 13 mM phosphate-citrate buffer.

In one embodiment the composition comprises about 7 mM to about 11 mM phosphate-citrate buffer. In one embodiment the composition comprises about 7.5 mM to about 10 mM phosphate-citrate buffer.

In one embodiment the composition comprises about 8 mM to about 9 mM phosphate-citrate buffer. In one embodiment the composition comprises about 8 mM phosphate-citrate buffer.