METHOD FOR MANUFACTURING HIGHLY PURIFIED LACTOFERRIN AND LACTOPEROXIDASE FROM MILK, COLOSTRUM AND ACID OR SWEET WHEY

Abstract

A method for manufacturing a fraction comprising the proteins lactoferrin and/or lactoperoxidase from a source containing at least one of these proteins wherein the source is selected from the group consisting of milk, colostrum, acid or sweet whey, by means of a chromatographic separation process with a monolithic column having cation exchanger properties, wherein in the separation process a pH gradient or a combined pH and salt gradient elution is employed after loading the source to the column.

Further disclosed is a composition of matter comprising lactoferrin having a C value of >60% and A value of >1% or lactoperoxidase.

Claims

1. A method for manufacturing a fraction comprising the proteins lactoferrin and/or lactoperoxidase from a source containing at least one of these proteins wherein the source is selected from the group consisting of milk, colostrum, acid or sweet whey, by means of a chromatographic separation process with a monolithic column having strong cation exchanger properties, wherein in the separation process a pH gradient or a combined pH and salt gradient elution is employed after loading the source to the column.

2. The method of claim 1, wherein the pH gradient starts in a pH range of about 4.0 to about <pH 8.0.

3. The method of claim 1, wherein the pH gradient terminates in a range of about pH 8 to pH<13.

4. The method of claim 1, wherein the source is filtered prior to loading of the source to the column.

5. The method of claim 1, wherein a fraction A is collected which elutes at a pH range of about pH 8 to about pH<11, in particular about pH 8.9 to about pH 10 or about pH 6.6 to about pH 7.5 at a higher conductivity in the range about 5 to 55 mS/cm.

6. The method of claim 1, wherein a fraction B is collected which elutes at a pH range of about pH >10.4 to about 12, in particular about pH >11 to about 11.7, or about pH 9.6 to about pH 10.7 at a higher conductivity in the range about 5 to 55 mS/cm.

7. The method of the claim 1, wherein the chromatographic separation process comprises the steps of (i) Adjusting the pH value of the source to a value lower than pH 7, in particular lower than pH 6.5; (ii) Contacting the source of step (i) with a monolithic column having strong cation exchanger properties; followed by (iii) Flowing a pH gradient buffer through the column thereby increasing the pH value; and (iv) Collecting a fraction A which elutes at a pH range of about pH 8 to about pH<11, in particular about pH 8.0 to about pH 10, preferably at a pH range of about pH 8.2 to about pH 10, more preferably about pH 8.9 to about pH 10, or about pH 6.6 to about pH 7.5 at a higher conductivity in the range about 5 to 55 mS/cm and typically comprises lactoperoxidase; and/or (v) Collecting a fraction B which elutes at a pH range of about pH >10 to about pH 12.0, preferably about pH >10.4 to about 12, more preferably about pH >11.0 to about pH 12.0, in particular about pH >11 to about 11.7, or about pH 9.6 to about pH 10.7 at a higher conductivity about 5 to 55 mS/cm and typically comprises lactoferrin; (vi) Optionally further processing the fractions A and/or B, in particular by treatment for neutralising, concentrating, preservation and the like.

8. The method of claim 7, wherein the source is filtered prior to step (ii) or wherein the monolithic column is prior to step (ii) equilibrated with an equilibration buffer having a pH value of about pH<7, in particular about pH<6.5.

9. The method of claim 1, wherein the monolithic column having strong cation exchanger properties is selected from the group consisting of a —SO.sub.3H modified monolithic column, —COOH modified monolithic column, —OSO.sub.3H modified monolithic column or —OPO.sub.3H modified monolithic column.

10. The method of claim 1, wherein the salt gradient is performed by concentration of salts, in particular the salt gradient corresponds to a conductivity in a range of about 5 mS/cm to about 55 mS/cm.

11. The method of claim 1, wherein prior to step (iii) or (iv) the column is flushed with the equilibration buffer of claim 8.

12. The method according to claim 1, wherein the lactoferrin and lactoperoxidase containing fractions are dried, in particular by spray drying.

13. The method according to claim 1, wherein the purity of lactoferrin is >90% and the purity of lactoperoxidase is >50%, in particular wherein the lactoferrin C value is >50 and the lactoferrin A value is >1.

14. The method according to claim 1, wherein the column is sanitised by flushing the column with a buffer of about pH >12 after step (iv) or (v).

15. A composition of matter comprising lactoferrin having a C value of >60% and A value of >1%.

16. The composition of matter according to claim 15 wherein the C value is 70% or more and the A value is 2% or more.

17. A composition of matter comprising lactoferrin or lactoperoxidase obtainable by a method according to claim 1.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0059] FIG. 1. Chromatograph of LF elution peak, composed from LF elution subpeaks. The phenomenon is a consequence of small differences in LF isoelectric point (IEP) due to its iron content. Voswinkel et al. [32] showed, that decrease in iron content consequently lowers IEP of LF to a small degree.

[0060] FIG. 2. Chromatographs of (A) acid whey and (B) commercial LF products and LF.

[0061] FIG. 3. Scheme of LF isolation from acid whey using one 8 L monolith column, CIMmultus™ SO3; BIA Separations and basic information on the mass balance of the process.

DETAILED DESCRIPTION OF THE INVENTION

[0062] Monolithic columns with strong cation exchanger groups CIMmultus™ SO3—strong CEX from BIA Separations were tested in different elution modes to isolate LF and LPO from filtered whey or milk separately. Loading of LF/LPO was performed at natural pH of whey, while the two target proteins were eluted separately by (I) stepwise increase of the pH, (II) conductivity steps, (III) linear conductivity gradient and (IV) linear pH gradient.

[0063] In the case of step elution modes, performed for comparison, the purity of LF achieved was about 95%, while in the case of conductivity gradient the purity achieved was slightly higher than 95%. Surprisingly, it has been found that according to the invention an elution with an increasing pH gradient, in particular with a linear increasing pH gradient, the protein purity achieved was over 98%. The purity was proven by HPLC, SDS-page analysis, Bioanalyzer and SEC chromatography. The method of the invention provides for these results by a single chromatographic step and on a production scale. By the way, according to Laurell's method [26] used for purity calculation in EP 0253395, the calculated purity for the product of the invention which is obtainable by the method of the invention is >118%. It seems that the established method becomes outdated and is not suitable any more for the correct determination of LF purity.

[0064] In the case of linear pH gradient elution, it was noticed that LF elution was composed of several smaller sub-peaks (FIG. 1). The latter suggests that LF was also separated on the level of protein iron content, from lower to higher. The reason for this was already discussed and proven by others [32].

[0065] Concentrated LF water dispersion was dried by spray drying or lyophilizer, and unsaturated iron-binding capacity (UIBC) for LF was than measured. According to Iron Colorimetric Assay Kit provided by NRL Pharma Inc. Japan (detailed principle was published by Ito et al. [33]), LF obtained by the method of the invention had an iron saturation level (already bound iron—A value) between 2% to 4.9%. Its potential for iron binding (iron binding capacity—C value), also called unsaturated iron-binding capacity (UIBC), was above 70%. The result ranks at the top in comparison to products present on the market, whose A and C values rank between 4.6-11.7% and 34.4-52.1%, respectively (see Table 1). At the same time their total bioactivity (C value+A value) is usually lower than in comparison to the product of the invention.

TABLE-US-00001 TABLE 1 Bioactivity of commercially available LF samples and LF of the invention (Arhel d.o.o.) Samples of LF C value [%] A value [%] (A + C)* [%] Supps Planet 34.4 4.6 39.0 Lactoferrin Life Extension 48.6**(31.1-vz.) 11.7**(7.5-vz.) 60.3 (Lactoferrin caps-Bioferrin; 95% Apolactoferrin) Ingredia 52.1 7.7 59.8 Nutritional (Prodiet Lactoferrin, >95%) NRL Pharma 49.2 9.0 58.2 LF of the 70.0-74.0 2.0-4.9 74.9 (avg.) invention *The total bioactivity (A + C) is expressed by summing the C and A values, which gives the percentage of active protein in the sample. **Value is recalculated according to the quantity of pure LF in the sample, which is 64%.

[0066] The method of the invention provides a method for manufacturing a fraction comprising the proteins lactoferrin or lactoperoxidase from a source containing at least one of these proteins, wherein the source is selected from the group consisting of milk, colostrum, acid or sweet whey, by means of a chromatographic separation process with a monolithic column having strong cation exchanger properties, in particular a —SO.sub.3H modified monolithic column wherein in the separation process a pH gradient elution is employed after loading the source to the column. A monolithic column is commonly a chromatographic separation equipment comprising a hollow body wherein a porous solid material is contained which is a polymerisate of monomers. Pores of the material are formed e.g. during the polymerisation process (U.S. Pat. Nos. 4,923,610, 4,952,344, 4,889,623).

[0067] Suitable devices are described in the prior art, for example EP 1058844, EP 777725 and are commercially available. The monolithic chromatography material used herein is modified with —SO.sub.3H moieties which are exposed i. a. at the surfaces of the porous material. The surface modification with —SO.sub.3H groups provides the material with so called strong cation exchanging properties (CAX). The skilled person knows that other materials, e. g. classified as weak cation exchanger. In contrast to this, for other purposes anion exchanger (AEX) can be used.

[0068] A pH gradient chromatography is conducted by increasing the pH from a starting value to an end point. It can be designed in an almost linear shape but also a different course is possible as long as the result of the invention, i. e. the products lactoferrin and/or lactoperoxidase of the invention are obtained. The skilled person knows how to perform the gradient chromatography as such. An optimisation of the conditions of the cationic gradient chromatography—based on the explicit and implict disclosure of the invention-lies within skilled person's routine and is not related with undue burden of experimentation.

[0069] In order to establish conditions for a reproducible processing it can be advantageous to equilibrate the monolithic column prior to the actual separation by pH gradient. In this case the column is flushed with an equilibration buffer. Typically, the column is flushed with a volume of the equilibration buffer equivalent to 8-12× dead volumes of the column. The selection of the starting pH value of the chromatography is to some extent influenced by the pH value of the source containing LF and/or LPO. For example, if acid whey is the source for LF and/or LPO, the pH value for equilibration can be pH 4.6 whereas if sweet whey is used, the pH value can be higher, i. e. pH 5.0 to pH 6.5.

[0070] It can be advantageous to filter the source prior to loading of the source to the column. Generally, filter means used in the diary industries can be employed. Particularly useful are ceramic TFF filters, spiral-wound membranes or other continuous filtration technologies. After loading the source on the monolithic column, in principle, the pH gradient chromatography can start. It may be useful, however, that prior to starting the pH gradient chromatography, a further flushing of the column with a pH value around the equilibration conditions can be employed to remove impurities. This supports the separation of the proteins to be manufactured, because proteins or other contaminants which elute at that pH value do not pollute the separation of LF und/or LPO.

[0071] The pH gradient starts with a pH value typically in a pH range of 4.0 to <pH 8.0, preferably in a pH range of 4.0 to 7.5, preferably in a pH range of about 4.0 to about <pH 7, in particular in a pH range of about 4.5 to about <pH 6.5. The pH gradient terminates typically in a range of about pH 8 to pH<13, preferably in a pH range of pH 8 to pH 12, in particular pH 8 to pH<12. FIG. 1 depicts a typical course of a pH gradient chromatography.

[0072] It should be noted that also the ionic strength of the elution buffer can have some influence on the separation. The ionic strength can also follow an increasing gradient during the chromatographic separation overlaying the necessary pH gradient. The necessary pH gradient for use in combination with the salt gradient mentioned before starts with a pH value typically in a pH range of 4.0 to <pH 8.0, preferably in a pH range of 4.0 to 7.5, preferably in a pH range of 4.0 to <pH 7, in particular in a pH range of 4.5 to <pH 6.5. The pH gradient used in combination with the salt gradient terminates typically in a range of pH 8 to pH<13, preferably in a pH range of pH 8 to pH 12, in particular in a pH range of pH 8 to pH<12.

[0073] For example LPO is eluting at a pH range of about pH 8 to about pH<11 if the ionic strength is equivalent to a conductivity of about 15 mS/cm, but at lower pH in the range of about pH 6.6 to about pH 7.5 if the conductivity increases from 4 to 55 mS/cm, preferably from 5 to 55 mS/cm. The elution of LF follows a similar regime. If the conductivity is medium but stays constant, LF elutes in a range of about pH 10.7 to about pH 11.7, if the conductivity increases from 4 to 55 mS/cm, preferably from 5 to 55 mS/cm, LF is eluting at lower pH in the range of about pH 9.6 to about pH 10.7. The ionic strength, i. e. conductivity can be adjusted by adding suitable salts. Using suitable salts also the pH value of the elution buffer may be adjusted. A fraction A eluting at a pH range of about pH 8 to about pH<11, preferably at a pH range of pH 8.0 to pH 10.0, preferably at a pH range of pH 8.2 to pH 10.0, in particular about pH 8.9 to about pH 10, or about pH 6.6 to about pH 7.5 at higher conductivity about 5 to 55 mS/cm is collected. This fraction contains typically lactoperoxidase. A fraction B eluting at a pH range of >10 to pH 12.0, preferably at a pH range of pH >10.4 to pH 12, preferably about pH >11 to about 12, in particular about pH >11 to about 11.7, or about pH 9.6 to about pH 10.7 (higher conductivity, about 5 to 55 mS/cm) is collected. This fraction contains typically lactoferrin. In the following a typical performance of the method of the invention is described. The pH ranges where LF and LPO are eluting correspond to a medium conductivity of about 15 mS/cm. The chromatographic separation process comprises the steps of

(i) Adjusting the pH value of the source to a value lower than pH 7, in particular lower than pH 6.5;
(ii) Contacting the source of step (i) with a monolithic column having strong cationic properties, in particular a —SO.sub.3 modified monolithic column; followed by
(iii) Flowing a gradient buffer through the column thereby increasing the pH value; and
(iv) Collecting a fraction A which elutes at a pH range of about pH 8 to about pH<11, preferably at a pH range of pH 8.0 to pH 10.0, preferably at a pH range of pH 8.2 to pH 10.0, in particular about pH 8.9 to about pH 10; and/or
(v) Collecting a fraction B which elutes at a pH range of >10 to pH 12.0, preferably about pH >10.4 to about 12, preferably at a pH range of >11 to pH 12, in particular about pH >11 to about 11.7;
(vi) Optionally further processing the fractions A and/or B, in particular by treatment for neutralising, concentrating, preservation and the like.

[0074] In order to remove unwanted material, the source containing the lactoferrin and/or lactoperoxidase is filtered prior to step (ii) through a ceramic filter.

[0075] The monolithic column is equilibrated prior to step (ii) with an equilibration buffer having a pH value of about pH<7, in particular about pH<6. The column is flushed with the equilibration buffer prior to step (iii) or (iv).

[0076] After collecting the lactoferrin and lactoperoxidase containing fractions these are further processed by spray drying.

[0077] The obtained lactoferrin or lactoperoxidase is of high purity. The purity of lactoferrin is >98% and the purity of lactoperoxidase is >78%. Furthermore, the lactoferrin C value is ≥60% and the lactoferrin A value is ≥1%. Preferably, the lactoferrin C value is >70% and the lactoferrin A value is >2%. Preferably, the lactoferrin C value is ≥70.0%, preferably between 70.0% to 80.0%, more preferably between 70.0% and 77.0%. Preferably, the lactoferrin A value is preferably between 1.0% and 7.0%, preferably between 2.0% and 7.0%, preferably between 2.0% and 5.0%, more preferably between 2% and 4%, preferably ≥2.0% and/or preferably <3.9%,

[0078] According to another embodiment of the invention the monolithic column can be sanitised by flushing with a buffer of about pH >13, typically after step (iv) or (v).

[0079] A sanitising step is performed by flushing column with deionized water (10-15 CVs), 1M NaOH (4-10 CVs) with contact time of 1 to 3 h and again flushing with water (>30 CVs). This step may be executed every 8-10 chromatographic runs.

[0080] The uniqueness of the novel approach according to the method of the invention is also in the facts that it (I) does not require any chemical modification of the source (e.g. whey) from which the protein is isolated, but merely pre filtration using standard filtration techniques, (II) in contrast to other processes (e.g. EP2421894 A1 [29]) uses inexpensive chemicals (buffers) in low amounts, (III) provides separate highly pure fractions of LPO and LF, (IV) enables the production of LF with pre-defined share of already bound Fe and (V) results in high protein recovery, which is higher than 85%, including desalting/concentrating of protein elution dispersion and drying production steps.

[0081] All references cited herein are incorporated by reference to the full extent to which the incorporation is not inconsistent with the express teachings herein.

[0082] The invention is further explained and illustrated in and by the following non-limiting examples.

EXAMPLES

[0083] Analytics:

[0084] HPLC Analytics:

[0085] To determine LF and LPO in samples, a chromatographic system equipped with MWD multi-wavelength detector and conductivity monitor with pH measuring kit (PATfix™, BIA Separations d.o.o., Ajdovscina, Slovenia) was employed. Chromatographic separations were run on CIMac™ S03-0.1 (Pores 1.3 μm, BIA Separations d.o.o., Ajdovscina, Slovenia) analytical column using two sodium phosphate monobasic buffer solutions (25 mM, pH=7.5) in conductivity gradient mode which increased from 3.5 to 152 mS/cm. The elution of analysed proteins were monitored by MWD detector at 226 nm. Detailed information about the chromatographic method is presented in Table 2, while chromatograms of some analysed samples are shown in FIGS. 1 and 2. All samples were prior analysis filtered through CHROMAFIL® A-45/25, 0.45 μm, cellulose mixed esters filters.

TABLE-US-00002 TABLE 2 Chromatographic system and method used for sample analysis. Chromatographic system: PATfix ™ HPLC system Chromatographic column: CIMac ™ SO3, 100 μL (BIA Separations d.o.o., Ajdovscina, Slovenia) Injection volume: 15 μL Detection: 226 nm B (%) A (%) (c(NaH.sub.2PO.sub.4 × 2H.sub.2O) = Time (c(NaH.sub.2PO.sub.4 × 2H.sub.2O) = 25 mM, c(NaCl) = Flow (min) 25 mM, pH = 7.5) 2M, pH = 7.5) (mL/min) 0   100  0 2.6 0.2 100  0 2.6  1.24  0 100 2.6  1.85  0 100 2.6  1.87 100  0 2.6 3.0 100  0 2.6

[0086] Determination of C- and A-Values:

[0087] To determine C- and A-values of dry LF samples we used Iron Colorimetric Assay Kit provided by NRL Pharma Inc., Japan (detailed principle was published by Ito et al. [33]), while to determine A-value for dry and liquid samples we used the same approach as described in literature [33,34] in combination with HPLC method.

[0088] Determination of C Values:

[0089] To determine the C value of LF, LF was saturated by a known excess amount of iron. The remaining iron was coloured by a chelating reagent (maximum absorbance 760 nm). The remaining iron is quantified spectrophotometrically at 760 nm. A serial dilution of the iron complex is prepared and a calibration curve is determined spectrophotometrically at 760 nm. The bound iron is calculated by subtracting the remaining iron from the added iron. The C value is indicated in relative terms, wherein the calculated theoretical iron binding capacity of LF (each molecule of LF can bind 2 iron atoms) is set as 100%.

[0090] Calculation of the C Value:

[00001]$\begin{array}{cc}\mathrm{theoretical}\mathrm{iron}\mathrm{binding}\mathrm{capacity}\left(\mathrm{LF}\right)\left[\frac{\mu g}{\mathrm{dl}}\right]=\frac{\mathrm{amount}\mathrm{of}\mathrm{LF}}{\mathrm{volume}\mathrm{of}\mathrm{sample}}\times \frac{M\left(\mathrm{Fe}\right)}{M\left(\mathrm{LF}\right)}\times \frac{1000\mu l\times 100000\mu l}{1\mathrm{mg}\times 1\mathrm{dl}}& \left(1\right)\end{array}$

[0091] M(Fe): molecular weight of iron; M(LF): molecular weight of LF

[00002]$\begin{array}{cc}\mathrm{iron}\mathrm{bound}\mathrm{by}\mathrm{LF}\left[\frac{\mu g}{dl}\right]=c\left({\mathrm{Fe}}_{\mathrm{Start}}\right)-c\left({\mathrm{Fe}}_{\mathrm{End}}\right)& \left(2\right)\end{array}$

[0092] c(Fe.sub.Start): starting concentration of iron; c(Fe.sub.End): concentration of remaining iron

[00003]$\begin{array}{cc}C\mathrm{value}\left[\%\right]=\frac{\mathrm{iron}\mathrm{bound}\mathrm{by}\mathrm{LF}}{\mathrm{theoretical}\mathrm{iron}\mathrm{binding}\mathrm{capacity}\mathrm{LF}}\times 100& \left(3\right)\end{array}$

[0093] Determination of a Values:

[0094] To determine the A value of LF, LF was denaturated by a denaturating reagent and the released iron was coloured by a chelating agent (maximum absorbance 760 nm). The released iron is quantified spectrophotometrically at 760 nm. A serial dilution of the iron complex is prepared and a calibration curve is determined spectrophotometrically at 760 nm. The already bound iron (A value) is indicated in relative terms, wherein 2 iron atoms bound to one LF molecule is defined as 100%.

[0095] Calculation of the a Value:

[00004]$\begin{array}{cc}\left(\mathrm{LF}\right)\left[\frac{\mu g}{dl}\right]=\frac{\mathrm{amount}\mathrm{of}\mathrm{LF}}{\mathrm{volume}\mathrm{of}\mathrm{sample}}\times \frac{M\left(\mathrm{Fe}\right)}{M\left(\mathrm{LF}\right)}\times \frac{1000\mu l\times 100000\mu l}{1\mathrm{mg}\times 1\mathrm{dl}}& \left(1\right)\end{array}$

[0096] M(Fe): molecular weight of iron; M(LF): molecular weight of LF

[00005]$\begin{array}{cc}A\mathrm{value}\left[\%\right]=\frac{\mathrm{iron}\mathrm{released}\mathrm{from}\mathrm{LF}}{\mathrm{theoretical}\mathrm{iron}\mathrm{binding}\mathrm{capacity}\mathrm{LF}}\times 100& \left(4\right)\end{array}$

[0097] Scheme of A Pilot System

[0098] FIG. 3 depicts a flow sheet about the principles in the technology for LP/LPO isolation and approximate values on the mass balance of the process on a scale of one chromatographic run. Before LF/LPO isolation, 1,100 L of acid whey is filtered using ceramic TFF filter system with a pore diameter lower than 0.8 μm. Filtered whey (1,000 L) is then pumped trough an equilibrated chromatographic column. The column-bound LF and LPO are after that eluted by a combination of different buffer solutions. Eluted fractions are then concentrated and, if necessary, desalted. After drying of concentrated protein, 60 to 90 g of dry LF product are obtained. Negligibly changed flow trough whey and whey slurry can be, separately or mixed, further used or processed downstream.

Example 1

[0099] A monolith column, 80 mL CIMmultus™ SO3; BIA Separations was before loading equilibrated using 800 mL of buffer solution A (sodium phosphate or citrate buffer: 5-50 mM, pH =4.6). After the equilibration, filtered acid whey was pumped through the column until the column capacity for LF and LPO were reached. The saturation of the column capacity was verified by analysing flow-through samples on the outflow side of the column by the HPLC method described in the Analytics section. The volume of whey pumped through the column at a flow rate of 0.24 L/min was usually 10 to 20 L, which was mainly depending on LF/LPO concentration in processed whey. The column was then flushed with buffer solution A. Separation of LF and LPO was triggered by flushing the column using two buffer solutions (mixture of sodium citrate, phosphate, TRIS and carbonate buffers, 4 to 50 mM, pH=4.6 and 12.0) in linear pH gradient mode. The pH was gradually linearly changed in a range from 4.6 to 12.0. The pH ranges for LPO/LF elution were 8.9-10 and 11-11.7, respectively. The results of the separation procedure were two, chromatographically very well separated elution fractions of LPO and LF, which were further easily processed separately. In the following steps, fractions of the isolated proteins were neutralised to pH=6 using a small amount of appropriate acid solution, concentrated using TFF membrane with a pore size from 1 to 50 kDa and spray dried. Final LF and LPO purities were >98% and >70%, respectively. LF C- and A-values were determined to be 71% and 3.4%, respectively.

Example 2

[0100] A monolith column, 80 mL CIMmultus™ SO3; BIA Separations was equilibrated before loading by using buffer solution B (sodium phosphate or citrate buffer: 5-50 mM, pH=5.0 to 6.5, as the pH of sweet whey). After that, sweet whey was allowed to flow through the column until the column capacity for LF and LPO reached. The saturation of the column capacity was verified by analysing flow-through samples on the outflow side of the column by HPLC method described in Analytics section. The volume of whey pumped through the column at a flow rate of 0.24 L/min was usually 10 to 40 L, which was mainly depending on LF/LPO concentration in processed whey. The column was then flushed with a buffer solution B. Separation of LF and LPO was triggered by flushing the column using two buffer solutions (mixture of sodium citrate, phosphate, TRIS and carbonate buffers, 4 to 50 mM, pH=5.0 and 12.0) in linear pH gradient mode. The pH is gradually linearly changed in a range from 5.0 to 12.0. The pH ranges for LPO/LF elution were 8.9-10 and 11-11.7, respectively. The results of the procedure mentioned above were two, chromatographically very well separated fractions of LPO and LF, which were further easily processed separately. In the following steps, fractions of the isolated proteins were neutralised to pH=6 using a small amount of appropriate acid solution, concentrated using TFF membrane with a pore size from 1 to 50 kDa and spray dried. Final LF/LPO purity was >98% or >70%. LF C- and A-values were determined to be 70.2% and 3.9%, respectively.

Example 3

[0101] A monolith column, 8 L CIMmultus™ SO3—Strong CEX; Bia Separations, was before loading equilibrated by 40 to 80 L of buffer solution C (sodium phosphate or citrate buffer: 5-50 mM with addition of NaCl, pH=4.6 and conductivity of 15 mS/cm). After that, acid whey was allowed to flow through the column until the column capacity for LF and LPO were reached. The saturation of the column capacity was verified by analysing flow-through samples on the outflow side of the column by HPLC method described in Analytics section. The volume of whey pumped through the column at a flow rate of 8 L/min was usually 1000 to 2000 L, which was mainly depending on LF/LPO concentration in processed whey. The column was then flushed with buffer solution C. Separation of LF and LPO was conducted by flushing the column using two buffer solutions (mixture of sodium citrate, phosphate, TRIS and carbonate, 4 to 50 mM with addition of NaCl, pH=4.6 and 12.0) in linear pH gradient mode. The pH was gradually linearly changed in a range from 4.6 to 12.0, while conductivity (15 mS/cm) stayed constant trough whole linear pH gradient. The pH ranges for LPO/LF elution were 8.2-9.3 and 10.7-11.2, respectively. The results of the procedure mentioned above were two, the chromatographically very well separated elution fractions of LPO and LF, which were further easily processed separately. In the following steps, the fractions of collected proteins were first neutralised to pH=6 using a small amount of appropriate acid solution, desalted and concentrated by using TFF membrane with a pore size from 1 to 50 kDa and dried by spray drying. Final LF/LPO purity was >98% or >75%. LF C- and A-values were determined to be 74.2% and 2.5%, respectively.

Example 4

[0102] A monolith column, 8 L CIMmultus™ SO3—Strong CEX; Bia Separations, was before loading equilibrated by using buffer solution C (sodium phosphate or citrate buffer: 5-50 mM, pH=5.0 to 6.5, as the pH of sweet whey). After that, acid whey was allowed to flow through the column until the column capacities for LF and LPO were reached. The saturation of the column capacity was verified by analysing flow through samples on outflow site of the column by HPLC method described in the Analytics section. The volume of whey pumped through the column at a flow rate of 8 L/min was usually 1000 to 2000 L, which was mainly depending on LF/LPO concentration in processed whey. Proteins with IEP <7.5 were initially eluted by pH step elution mode using a buffer solution C with pH=7.5 and then separation of LF and LPO was performed by flushing the column in linear pH gradient mode using same buffers as in Example 2. The pH was gradually linearly changed in a range from 7.5 to 12.0. The pH ranges for LPO/LF elution are 8.9-10 and 11-11.7, respectively. Separately collected elution fractions of LPO and LF were then neutralised to pH=6 using a small amount of appropriate acid solution, concentrated by using TFF membrane with a pore size from 1 to 50 kDa and dried by spray drying. Final LF and LPO purities were >98% and >75%, respectively. LF C- and A-values were determined to be 72.6% and 2.8%, respectively.

Example 5

[0103] A monolith column, 8 L CIMmultus™ SO3—Strong CEX; Bia Separations, was before loading equilibrated by using buffer solution C (sodium phosphate or citrate buffer: 5-50 mM with addition of NaCl, pH=4.6 and conductivity of 15 mS/cm). After that, acid whey was allowed to flow through the column until the column capacities for LF and LPO were reached. The saturation of the column capacity was verified by analysing flow through samples on the outflow side of the column by HPLC method described in the Analytics section. The volume of the whey pumped through the column at a flow rate of 8 L/min was usually 1000 to 2000 L, which was mainly depending on LF/LPO concentration in processed whey. The column was then flushed with buffer solution C. Proteins with IEP <7.5 were initially eluted by pH step elution mode using a buffer solution C with pH=7.5 and then separation of LF and LPO was performed by flushing the column in linear pH gradient mode using same buffers as in Example 3. The pH was gradually linearly changed in a range from 7.5 to 12.0, while the conductivity (15 mS/cm) stayed constant through the whole pH gradient. The pH ranges for LPO/LF elution were 8.2-9.3 and 10.7-11.2, respectively. Separately collected elution fractions of LPO and LF were then neutralised to pH=6 using a small amount of appropriate acid solution, concentrated by using TFF membrane with a pore size from 1 to 50 kDa and dried by spray drying technique. Final LF and LPO purities were >98% and >80%, respectively. LF C- and A-values were determined to be 71.6% and 3.2%, respectively.

Example 6

[0104] A monolith column, 8 L CIMmultus™ SO3—Strong CEX; Bia Separations, was before loading equilibrated by using buffer solution C (sodium phosphate or citrate buffer: 5-50 mM, pH=4.6). After that, acid whey was allowed to flow through the column until the column capacities for LF and LPO were reached. The saturation of the column capacity was verified by analysing flow through samples on the outflow side of the column by the HPLC method described in the Analytics section. The volume of the whey pumped through the column at a flow rate of 8 L/min was usually 1000 to 2000 L, which was mainly depending on LF/LPO concentration in processed whey. The column was then flushed with buffer solution C. Proteins with IEP <7.5 were initially eluted by pH step elution mode using a buffer solution C with pH=7.5 and then separation of LF and LPO was conducted by flushing the column using two buffer solutions (mixture of sodium citrate, phosphate, TRIS and carbonate, 4 to 50 mM with addition of NaCl to achieve appropriate conductivity, pH=4.6 and 12.0). The pH was gradually changed in a range from 7.0 to 12.0, while the gradient of conductivity increased from 4 to 55 mS/cm. The pH ranges for LPO/LF elution were 6.6-7.5 and 9.6-10.7, respectively. Separately collected elution fractions of LPO and LF were then neutralised to pH=6 using a small amount of appropriate acid solution, desalted and concentrated by using TFF membrane with a pore size from 1 to 50 kDa and dried by spray drying technique. Final LF and LPO purities were >98% or >85%. LF C- and A-values were determined to be 76.1% and 2.0%, respectively.

REFERENCES

[0105] [1] Susana A. Gonzalez-Chavez, Sigifredo Arevalo-Gallegos, and QuintinRascon-Cruz. Lactoferrin: structure, function and applications. International Journal of Antimicrobial Agents, 33(4): 301.e1-301.e8, 2009. [0106] [2] Nicolas Urtasun, Maria Fernanda Baieli, Daniela BelenHirsch, Maria Camila MartinezCeron, Osvaldo Cascone, and Federico Javier Wolman. Lactoperoxidase purification from whey by using dye affinity chromatography. Food and Bioproducts Processing, 103:58-65, 2017. [0107] [3] Lin Chen, Chen Guo, Yueping Guan, and Huizhou Liu. Isolation of lactoferrin from acid whey by magnetic affinity separation. Separation and Purification Technology, 56(2):168-174, 2007. [0108] [4] Rhee-M. S. Imm J Y Noh, K. H. Separation of lactoferrin from model whey protein mixture by reverse micelles formed by cationic surfactant. Food Sci. Biotechnol., 14:131-136., 2005. [0109] [5] Shalan Alwan Al-Mashikhi, Eunice Li-Chan, and ShuryoNakai. Separation of immunoglobulins and lactoferrin from cheese whey by chelating chromatography. Journal of Dairy Science, 71(7):1747-1755, 1988. [0110] [6] S. Yoshida, Z. Wei, Y. Shinmura, and N. Fukunaga. Separation of lactoferrin-a and -b from bovine colostrum. Journal of Dairy Science, 83(10):2211-2215, 2000. [0111] [7] Jonatan Andersson and Bo Mattiasson. Simulated moving bed technology with a simplified approach for protein purification: Separation of lactoperoxidase and lactoferrin from whey protein concentrate. Journal of Chromatography A, 1107(1):88-95, 2006. [0112] [8] Rong Rong Lu, Shi Ying Xu, Zhang Wang, and Rui Jin Yang. Isolation of lactoferrin from bovine colostrum by ultrafiltration coupled with strong cation exchange chromatography on a production scale. Journal of Membrane Science, 297(1):152-161, 2007. [0113] [9] T. Uchida, S. Dosako, K. Sato, and H. Kawakami Sequential separation of lactoferrin, lactoperoxidase, and secretory component by sulfate-linked ion-exchange chromatography. Milchwissenschaft, 9(58):482-486, January 2003. [0114] [10] O. Cascone Mariano Grasselli. Separation of lactoferrin from bovine whey by dye affinity chromatography. Netherlands Milk and Dairy Journal, 4(50):551-561, January 1996. [0115] [11] Enrique Alvarez-Guerra and Angel Irabien. Extraction of lactoferrin with hydrophobic ionic liquids. Separation and Purification Technology, 98:432-440, 2012. [0116] [12] Vojtech Adam, OndrejZitka, Petr Dolezal, Ladislav Zeman, Ales Horna, Jaromir Hubalek, Jan Sileny, Sona Krizkova, Libuse Trnkova, and Rene Kizek. Lactoferrin isolation using monolithic column coupled with spectrometric or micro-amperometric detector. Sensors (Basel), 1(8):464-487, January 2008. [0117] [13] J. H. Nuijens and H. H. Van Veen. Isolation of lactoferrin from milk, Jan. 19 1999. U.S. Pat. No. 5,861,491. [0118] [14] Conan J. Fee and Amita Chand. Capture of lactoferrin and lactoperoxidase from raw whole milk by cation exchange chromatography. Separation and Purification Technology, 48(2):143-149, 2006. Separation and Purification in the Food Industry. [0119] [15] Chalore Teepakorn, Koffi Fiaty, and Catherine Charcosset. Comparison of membrane chromatography and monolith chromatography for lactoferrin and bovine serum albumin separation. Processes, 4(3), 2016. [0120] [16] P. Milavec Zmak, H. Podgornik, J. Jancar, A. Podgornik, and A. Strancar. Transfer of gradient chromatographic methods for protein separation to convective interaction media monolithic columns. Journal of Chromatography A, 1006(1):195-205, 2003. International Symposium on Preparative and Industrial Chromatography and Allied Techniques. [0121] [17] Shuichi Yamamoto and Ayako Kita. Theoretical background of short chromatographic layers: Optimization of gradient elution in short columns Journal of Chromatography A, 1065(1):45-50, 2005. 1st Monolith Summer School. [0122] [18] Bo Wang, Yakindra Prasad Timilsena, Ewan Blanch, and Benu Adhikari. Characteristics of bovine lactoferrin powders produced through spray and freeze drying processes. International Journal of Biological Macromolecules, 95:985-994, 2017. [0123] [19] K. Sato and S. I. Dousako. Process of separating, purifying and recovering milk proteins capable of binding iron, Mar. 27 1991. EP Patent App. EP19,900,117,442. [0124] [20] K. Sato, M. Shiba, A. Shigematsu, N. Teduka, and A. Tomizawa. Method for producing lactoferrin, Oct. 13 2004. EP Patent App. EP20,040,008,008. [0125] [21] G. Rowe, H. Aomari, and D. Petitclerc. New purification method of lactoferrin, November 16 2006. WO Patent App. PCT/CA2006/000,780. [0126] [22] H. Kawakami, M Tanimoto, and S. Dousako. Process for separating and purifying lactoferrin from milk using sulfate compound, Sep. 9 1992. EP Patent 0,348,508. [0127] [23] Z. Mozaffar, S. H. Ahmed, V. Saxena, and Q. R. Miranda. Sequential separation of whey, Aug. 1 2000. U.S. Pat. No. 6,096,870. [0128] [24] J. Souppe. Milk protein isolate and process for its preparation, Dec. 10 2013. U.S. Pat. No. 8,603,560. [0129] [25] Kussendrager K. D., Kivits M. G. C., and Verver A. B. Process for isolating lactoferrin and lactoperoxidase from milk and milk products, and products obtained by such process, Jun. 16 1998. CA Patent 2,128,111. [0130] [26] S. Okonogi, M. Tomita, T. Tomimura, Y. Tamura, and T. Mizota. A process for producing bovine lactoferrin in high purity, Jul. 5 2006. EP Patent 0,253,395. [0131] [27] K. D. Kussendrager, M. G. C. Kivits, and A. B. Verver. Process for isolating lactoferrin and lactoperoxidase from milk and milk products, and products obtained by such process, January 21 1997. U.S. Pat. No. 5,596,082. [0132] [28] Jean-Paul Perraudin. Method for production of lactoferrin, August 2015. U.S. Pat. No. 9,115.211 B2. [0133] [29] Kevin Neil Pearce Shaojiang Chen. Method of preparing low-iron lactoferrin, February 2012. EP2421894A1. [0134] [30] Hassan Mohamed Hassan Abdalla. Method for purifying lactoferrin, December 2014. U.S. Pat. No. 9,458,225B2. [0135] [31] Jan H. Nuijens Veen Harry H. Van. Isolation of lactoferrin from milk, August 1995. WO1995022258A2. [0136] [32] Linda Voswinkel, Thomas Vogel, and Ulrich Kulozik. Impact of the iron saturation of bovine lactoferrin on adsorption to a strong cation exchanger membrane. International Dairy Journal, 56:134-140, 2016. [0137] [33] Satoshi Ito, Katsuya Ikuta, Daisuke Kato, Kotoe Shibusa, Noriyasu Niizeki, Hiroki Tanaka, Lynda Addo, Yasumichi Told, Mayumi Hatayama, Junki Inamura, MotohiroShindo, Katsunori Sasaki, Naomi Iizuka, Mikihiro Fujiya, Yoshihiro Torimoto, and Yutaka Kohgo. Non-transferrin-bound iron assay system utilizing a conventional automated analyzer. ClinicaChimica Acta, 437:129-135, 2014. [0138] [34] Robert J. Hilton, Matthew C. Seare, N. David Andros, Zachary Kenealey, Catalina Matias Orozco, Michael Webb, and Richard K. Watt. Phosphate inhibits in vitro Fe3+ loading into transferrin by forming a soluble Fe(iii) phosphate complex: A potential non-transferrin bound iron species. Journal of Inorganic Biochemistry, 110:1-7, 2012.