AU1313401A

AU1313401A - Method of obtaining immunoglobulins from colostrum and dairy sources

Info

Publication number: AU1313401A
Application number: AU13134/01A
Authority: AU
Inventors: Peter Hobman; Paul Signal
Original assignee: Fonterra Cooperative Group Ltd
Current assignee: Fonterra Cooperative Group Ltd
Priority date: 1999-10-26
Filing date: 2000-10-26
Publication date: 2001-05-08
Also published as: WO2001030168A1

Description

WO 01/30168 PCT/NZOO/00211 METHOD OF OBTAINING IMMUNOGLOBULINS FROM COLOSTRUM AND DAIRY SOURCES FIELD OF THE INVENTION 5 This invention relates to a method of obtaining immunoglobulin containing products from a dairy source and/or colostrum, in whole form, or as whey, permeate or serum, and products obtained from the method. 10 BACKGROUND TO THE INVENTION Immunoglobulins, growth factors and other biologically active components from milk and colostrum (e.g. lactoferrin, anti-inflamamatory factors, immune enhancing substances) are sought-after materials. Such 15 biologically active components can be sourced from milk, milk serum, colostrum or colostrum serums, but the concentration of such components is generally much higher in colostrum than milk. It will be appreciated, however, that the concentration of bioactive components in milk (or colostrum) may be elevated, for example by immunisation or 20 hyperimmunisation of animals. The separation and purification of biologically active materials from milk and/or colostrum has significant commercial advantages over the sourcing of such active materials from blood. Blood serum antibodies are 25 difficult to collect and it is therefore difficult to produce commercial quantities for use in medications. Furthermore, secretory immunoglobulins (eg, IgA) seem to be more stable against proteases and acidic conditions than serum immunoglobulins.

WO 01/30168 PCT/NZOO/00211 2 United States patent No. US 5,747,031 describes a process for isolating immunoglobulins from whey involving the sequential precipitation of lipids and non-immunoglobulin proteins through a sequence of admixtures of cationic polymer and fatty acid in series, followed by 5 isolation of immunoglobulins from the supernatant of the mixture. This process results in a large volume of waste material which is unsuitable for food use and difficult to dispose of. Hahn et al ("Bovine whey fractionation based on cation-exchange 10 chromatography", Journal of Chromatography A, 795, (1998), 277-287) compares the use of a number of cation ion-exchange resins for suitability in purifying immunoglobulins from whey. The starting material for their investigation was dilute acid casein whey or colostrum whey. The conditions used for preparation of the feed material were not 15 representative of what is available for whey production on a commercial scale. Furthermore, the problem of hydraulic capacities normally associated with whey production is compounded by the dilution of the whey. Also the purity of the eluted IgG in this study was low because the pH of loading was set to maximise the yield of bound IgG, therefore 20 retaining large quantities of beta-lactoglobulin and other proteins which then co-elute with the IgG. The present inventors are not aware of any other commercial process or processes which enable production of commercially useful 25 quantities of immunoglobulin, an immunoglobulin-rich fraction, or other biologically active components, from colostrum and/or a dairy source. It is an object of the present invention to provide a method of obtaining products containing immunoglobulins from a dairy and/or 30 colostrum source which reduces or overcomes the above-mentioned problems, or which at least provides the public with a useful alternative. Other objects of the invention may become apparent from the following description which is given by way of example only.

WO 01/30168 PCT/NZOO/00211 3 SUMMARY OF THE INVENTION According to one aspect of the present invention there is provided 5 a method of producing products containing immunoglobulin(s) from a dairy and/or colostrum source by ion-exchange, the method including the steps of: - taking a feedstock from a dairy source and/or colostrum, 10 - adjusting the pH of the feedstock to a pH in the range 4.5 to 6.5, - subjecting the feedstock to ion-exchange using a cation exchanger to produce a high purity immunoglobulin fraction and a non-bound fraction containing commercially useful 15 amounts of immunoglobulin(s). Preferably, the immunoglobulin(s) may be igG. Preferably, in the non-bound fraction at least 1 % of the total 20 protein content may be immunoglobulin(s). Preferably , the feedstock may be cheese or casein whey, skim milk, whole milk, colostrum, colostrum whey or colostrum serum, hyperimmunised milk or colostrum, or a reconstituted form of such 25 streams. Preferably, the feedstock may be derived from colostrum. Preferably, the feedstock may be a microfiltered colostrum serum. 30 In a further preferred form the pH may be adjusted to be in the range substantially 5.4 to 5.6.

WO 01/30168 PCT/NZOO/00211 4 Preferably, at least 7% of the total protein content of the non bound fraction may be immunoglobulin(s). Preferably, IgG. In a further preferred form the method may further include 5 adjusting the pH of the ion-exchange medium to be substantially the same as that of the feedstock, prior to ion-exchange. According to a further aspect of the present invention there is provided a high purity immunoglobulin fraction derived by an ion-exchange 10 method as herein described from a feedstock of a dairy source and/or colostrum. According to a further aspect of the present invention there is provided a non-bound fraction containing a commercially useful amount of 15 immunoglobulin(s) derived by an ion-exchange method as herein described from a feedstock of a dairy source and/or colostrum. According to a further aspect of the present invention there is provided a dietary or nutritional supplement including a high purity fraction 20 and/or a non-bound fraction as herein described. 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 figures and examples. 25 BRIEF DESCRIPTION OF THE FIGURES Figure 1: Is a flow diagram showing a process for the production of an eluate fraction rich in immunoglobulin from colostrum or 30 whole milk, including the method of the invention, in one preferred form. Figure 2: Shows the influence of load pH on the yield purity and IgG production from the process of the present invention using a WO 01/30168 PCT/NZOO/00211 5 micro-filtered colostrum permeate (MFCP) as the feed material. DETAILED DESCRIPTION OF THE INVENTION 5 In broad terms the method of the invention involves feeding a selected feed material prepared from cheese or casein whey, directly from skim milk, whole milk, colostrum whey (either cheese or casein), colostrum, colostrum serum, hyperimmunised milk or colostrum, or a 10 reconstituted form of such streams adjusting the pH of that selected material, so that beta-lactoglobulin and alpha-lactalbumin and a proportion of IgG do not bind strongly, and applying the material to a selected ion exchange media. A low yield/high purity immunoglobulin-rich fraction is achieved, together with a non-bound fraction which includes commercially 15 useful amounts of immunoglobulin. The non-bound fraction contains a high protein content and so remains commercially useful rather than being a waste product. For example, it may include at least 1 % immunoglobulin. In a preferred form, 20 using a colostrum feed material the non-bound fraction may contain at least 7% IgG. The non-bound fraction may be further processed (e.g. by evaporation or spray drying) to produce a colostrum product for use as an ingredient in health foods, supplements and the like. 25 The feed material may be prepared from cheese or casein whey, or directly from skim milk, whole milk, colostrum whey, colostrum or colostrum serum, hyperimmunised milk or colostrum, or a reconstituted form of such streams. In the case of using cheese or casein whey, the whey is preferably de-lipidised using microfiltration, thermo-calcic 30 precipitation or fat precipitation. In the case of using skim-milk or whole milk, the whey can be prepared directly via microfiltration. A preferred colostrum-based feed material may be prepared according to the microfiltration process described in the patent WO 01/30168 PCT/NZOO/00211 6 specification accompanying international patent application number PTC/NZO0/001 20. This preferred colostrum serum may contain at least 80% protein of which at least 20% may comprise immunoglobulins. 5 Where the feed material is a whey it may be pre-concentrated through the suitable use of ultrafiltration to produce a whey protein retentate. This has two benefits, the first being that the volume of liquid to be handled is reduced by the concentration factor employed in ultrafiltration, and the second being that if the retentate is then diluted the 10 conductivity of the feed material will effectively have been lowered. A lower conductivity can assist in the ion exchange step. The upper part of Figure 1 shows, in broad terms, the preparation of the feed material. It will be appreciated that the feed material may 15 include hyper-immune colostrum or milk. The pH of the feed material is adjusted to be in the range pH 4.5 to pH 6.5. The optimal pH will depend on the ion exchange resin used, the conductivity of the feed material, the source of the feed material (whether 20 milk-based, colostrum-based or whey-based) and the yield and degree of separation required. The pH may be adjusted with any acid, but preferably a strong acid such as hydrochloric acid or sulphuric acid. The preferred pH for a colostrum-based feed material was 25 investigated and the results are shown below, under the heading "pH Evaluation". An optimum pH may be in the range 5.4 to 5.6. The ion-exchange process may involve a stirred bed, fluidised bed, expanded bed or fixed bed in axial or radial flow mode. In one 30 embodiment, it may employ a packed-bed arrangement (either axial flow or radial flow) in conjunction with non-swelling ion-exchange media, or alternatively a stirred-bed arrangement in conjunction with non-swelling or swelling ion-exchange media. A cation ion-exchange media may WO 01/30168 PCT/NZOO/00211 7 preferably be selected, for example MacroPrep High S Cation Exchange Support, SP Sepharose big beads or Sepra-Prep S media. The ion-exchange medium is preferably pre-equilibrated to the pH 5 of the feed material prior to loading of the feed material. Pre-equilibration may be carried out with a slightly buffered organic acid system such as a solution of acetic acid and sodium acetate. After equilibration the medium is then preferably washed with water to remove the pre-equilibration buffer. 10 The feed material is then applied to the ion-exchange medium. The flowrate and volume of feed material will be dependent on the ion exchange medium employed and the properties of the feed material. The non-bound fraction is collected, the ion-exchange medium is washed to 15 remove residual feed material, and an eluent buffer is applied to the ion exchange medium to remove the immunoglobulin-rich fraction. The eluent buffer may be eluted using a salt solution having a conductivity greater than that of the feed material and less than 1.OM NaCl. Alternatively, a buffered system may be employed using a weak acid system such as 20 salt/phosphoric acid/sodium phosphate adjusted to a pH greater than that of the feed material. The elution and washing cycle of the ion-exchange medium is shown in Figure 1. 25 The immunoglobulin-rich eluate may be further purified (as shown in Figure 1) to remove salt either using a de-salting column or by nanofiltration or ultrafiltration. Ultrafiltration may be the preferred option. During this step the immunoglobulins are concentrated, and preferably 30 diafiltration water may be added to lower the salt content of the final product.

WO 01/30168 PCT/NZOO/00211 8 The concentrated product may optionally be evaporated to remove water and either spray-dried or freeze-dried. In order to avoid heat damage to the immunoglobulins, freeze-drying is preferred. 5 Although not shown in Figure 1, the non-bound fraction may also be further processed in substantially the same manner as the eluate fraction to produce a concentrated, dried, end-product. pH Evaluation 10 Spray dried skim colostrum (IMMULAC

TM

) was microfiltered on 0.1 micron ceramic membranes to produce a microfiltered colostrum permeate (MFCP). 15 MFCP was pH adjusted with 10% sulphuric acid and run through a 50ml ion exchange column packed with Sepra-Prep S media, at a flow rate of 0.5 column volumes per minute. The process involved: Column Start 20 Step Action Fluid Volume Time 1 Equilibrate Equilibration Buffer 2 0 2 Wash Water 2 4 3 Load MFCP 10 8 25 4 Wash Water 1.5 28 5 Elute Buffer 3 31 6 Wash Water 1.5 37 7 Regen Caustic/Salt 2 40 8 Wash Water 2 44 30 9 End 48 The MFCP was loaded at 3 different pH's - 5.90, 5.44 and 5.03. From the 500ml of MFCP loaded each cycle, 550ml of non-bound 35 fraction and 200ml of eluate fraction were collected. These were analysed WO 01/30168 PCT/NZOO/00211 9 for protein content using reverse phase HPLC (RP-HPLC) and Protein G HPLC. The results are shown in Figure 2 and summarised below. 5 Feed IgG Purity: the IgG/Protein content of the MFCP was 48.9%. Eluate Fraction Purity: the purity of the IgG eluate fraction dropped as the load pH of MFCP lowered. This may be 10 accounted for by more beta-lactoglobulin and alpha-lactalbumin being adsorbed to the resin at lower pH's. The optimal operating condition appeared to be around the middle condition (load pH 5.44) where the IgG/Protein purity 15 was 88%. Eluate Fraction Yield: yield of IgG increased from 15.6% at the high load pH to 36.5% at the low load pH. At the mid-point the IgG yield was 27.5%. The low 20 yield has the advantage that the non-bound fraction remains relatively high in immunoglobulins. Non-bound Fraction Purity: this remained relatively constant at 25 between 42 and 45% IgG/Protein. The curve is relatively flat as when more IgG is adsorbed, so is more other protein.

WO 01/30168 PCT/NZOO/00211 10 Protein Profile: the protein profile by RP-HPLC showed a high IgG content, as well as significant peaks at 5.0 minutes and 6.6 minutes. These peaks may be related to other bioactive proteins such as lactoferrin, 5 lactoperoxidase, and growth factors. Production Rate: theoretical production rate of IgG in the eluate fraction was calculated by assuming a protein loading factor of 60g/L resin. This leads to the volume of 10 MFCP loaded per cycle, and hence the cycle time. Production rates were factored for IgG purity, so that overall IgG production was optimised. At the middle load pH (5.44) the production rate could be 27 g/L/hour. On a 50 L column the key factors were: 15 Per Cycle Per Hour Volume MFCP loaded 2235 2666 Cycle Time 117 minutes 20 Protein Production 3.0kg 1.5kg IgG Production 2.6kg 1.3kg Example 1 25 Spray dried skim colostrum (IMMULACTM) was reconstituted with demineralised water to 12% total solids. The solution was then heated to 50 0 C prior to microfiltration. The microfiltration plant was operated using a 0.1tm ceramic membrane, with a crossflow rate of 6-7m/sec. The 30 temperature was maintained at 50"C throughout the process. The permeate from microfiltration (MFCP) was collected. A 50ml radial flow column (RFC) (bed depth 3.28 cm) was packed with MacroPrep High S Cation Exchange Support. The RFC was pre- WO 01/30168 PCT/NZO0/00211 11 equilibrated with 2 column volumes (CV's) of a buffer solution containing 0.025 M sodium acetate and 0.05 M sodium chloride. The RFC was then washed with 2 CV's of water. Flowrate was 25 ml/min. 5 The MFCP was pH adjusted to pH 5.44 using 10% sulphuric acid. 10 CV's of pH-adjusted permeate was applied to the RFC at a flowrate of 0.5 CV/min. Following loading, the RFC was washed with 2 CV of water. 3 CV of eluant (0.5M sodium chloride, 0.05M monosodium 10 phosphate, pH 8.0) was applied to the RFC and the fraction collected. The feed material, fraction (eluate) and non-bound fraction were analysed for IgG using a Pharmacia HiTrap Protein G 1ml column, and were analysed for protein using a Reverse-Phase (RP) HPLC method 15 utilising a Pharmacia RPC Source 1ml column with a gradient elution with acetonitrile. The compositional results were as follows; MFCP Eluate Fraction Non-bound Fraction Protein (g/L) 8.774 3.359 6.566 IgG (g/L) 4.290 2.946 2.814 Volume (ml) 500 200 550 IgG/Protein (%) 48.9 87.7 42.9 IgG Yield (%) 27.5 72.2 20 Example 2 Spray dried skim colostrum (IMMACULAC

TM

) was reconstituted and 25 microfiltered as for Example 1. The MFCP was pH adjusted to 5.59 with sulfuric acid and 2 WO 01/30168 PCT/NZOO/00211 12 column volumes (CV) applied to a 9.4cm high by 2.6cm diameter packed bed column of SP Sepharose big beads (50ml bed volume). The column was washed with 2 CV of 0.05M acetate buffer and 2 CV of 0.05M acetate, 0.06M NaCl. The IgG rich fraction was then eluted with 2 CV of 5 2.85M NaCl. Each fraction was analysed for IgG as in Example 1 and purity was estimated from this assay by the ratio of the absorbance at 280nm of IgG to total absorbance at 280nm of all peaks. The analysis of these fractions is given below. MFCP Eluate Fraction Non-bound Fraction Protein (g/L) 7.85 - IgG (g/L) 2.10 0.592 0.662 IgG/Protein (%) * 18.0 69.1 7.1 10 * Purity, estimated from the assay used in Example 1 by the ratio of absorbance at 280nm of IgG to total absorbance at 280nm of all peaks. Thus, the method of the invention produces a high purity immunoglobulin fraction from colostrum and/or a dairy source, 15 appropriately pre-treated, by ion-exchange technology. The fraction is of high purity but low yield, with the result that the non-bound fraction retains sufficient immunoglobulin to be commercially valuable in its own right. The commercial application of these products may, for example, be in the area of health food supplements and other nutritional or dietary 20 products or supplements. Where in the foregoing description reference has been made to specific components or integers of the invention having known equivalents then such equivalents are herein incorporated as if incorporated as if 25 individually set forth. Although this invention has been described by way of example and with reference to possible embodiments thereof it is to be understood that modifications or improvements may be made thereto without 30 departing from the scope or spirit of the invention.

Claims

1. A method of producing products containing immunoglobulin(s) from a dairy and/or colostrum source by ion-exchange, the method including the steps of: 10 - taking a feedstock from a dairy source and/or colostrum; - adjusting the pH of the feedstock to a pH in the range 4.5 to 6.5; - subjecting the feedstock to ion-exchange using a cation exchanger to produce a high purity immunoglobulin fraction 15 and a non-bound fraction containing commercially useful amounts of immunoglobulin(s).

2. A method according to claim 1 wherein the immunoglobulin(s) is IgG. 20

3. A method according to claim 1 or claim 2 wherein at least 1 % of the total protein content of the non-bound fraction is immunoglobulin(s). 25

4. A method according to any one of claims 1-3 wherein the feedstock is cheese or casein whey, skim or whole milk, colostrum, colostrum whey, hyperimmunised milk or colostrum, colostrum serum, or a reconstituted form of such streams. 30

5. A method according to claim 4 wherein the feedstock is derived from colostrum.

6. A method according to claim 5 wherein the feedstock is a microfiltered colostrum serum. 35 WO 01/30168 PCT/NZOO/00211 14

7. A method according to any one of claims 1-6 wherein the pH is adjusted to be in the range substantially 5.4 to 5.6.

8. A method according to claim 7 wherein at least 7% of the total 5 protein content of the non-bound fraction is immunoglobulin.

9. A method according to any one of claims 1-8 further including adjusting the pH of the ion-exchange medium to be substantially the same as that of the feedstock, prior to ion-exchange. 10

10. A method according to any one of claims 1-9 wherein the high purity immunoglobulin fraction is further processed by optional de salination, concentration, evaporation and drying to produce an immunoglobulin-rich product. 15

11. A method according to any one of claims 1-9 wherein the non bound fraction is further processed by optional de-salination, concentration, evaporation and drying to produce an immunoglobulin-containing product. 20

12. A high purity immunoglobulin fraction derived from a feedstock of a dairy source and/or colostrum according to a method of any one of claims 1-9. 25

13. A non-bound fraction containing a commercially useful amount of immunoglobulin(s) derived from a feedstock of a dairy source and/or colostrum according to a method of any one of claims 1-9.

14. An immunoglobulin-rich product derived from a feedstock of a dairy 30 source and/or colostrum according to the method of claim 10.

15. An immunoglobulin-containing product derived from a feedstock of a dairy source and/or colostrum according to the method of claim 11. WO 01/30168 PCT/NZOO/00211 15

16. A dietary or nutritional supplement including a high purity immunoglobulin fraction according to claim 12. 5

17. A dietary or nutritional supplement including a non-bound fraction according to claim 13.

18. A dietary or nutritional supplement including an immunoglobulin rich product of claim 14 and/or an immunoglobulin-containing 10 product of claim 15.

19. A method of producing products containing immunoglobulin(s) from a dairy and/or colostrum source by ion-exchange substantially as herein described and with reference to the accompanying examples 15 and figures.

20. A high purity immunoglobulin fraction substantially as herein described and with reference to the accompanying figures and examples. 20

21. A non-bound fraction containing a commercially useful amount of immunoglobulin(s) substantially as herein described and with reference to the accompanying figures and examples.