CA1210329A - Production of purified porcine immunoglobulins - Google Patents

Abstract

THE ABSTRACT OF THE DISCLOSURE

A method for the continuous production of a purified immunoglobulin preparation wherein the antibodies are substantially enriched by means of a selective two-step ammonium sulfate fractionation procedure employing centrifugation and ion depletion processes. The purified immunoglobulins are subsequently commingled with condensed skim milk and spray dried. The resulting product for example is employed in the formulation of milk replacers for artificial rearing of neonatal pigs to provide the passive immunity to disease normally provided by sows' colostrum and later milk.

Description

~2~03~9 ACKGROUND OF TH~ INV~TIO~

The present invention relates to a method Eor the continuous production of a purified immunoglobulin preparation.
The immunoglobulin can be prepared from either porcine or bovine blood collected for example at ~bat~oirs from inspecte~ animals. The preparation from porcine blood has application în the rearing of neonatal pigs while that from bovine blood has potential use in the rearing of calves. The following description is directed primarily to the porcine application.
The neonatal piglet is born without the ability to fight disease and is dependent upon colos~ral and la~er milk from the sow to provide immunoglobulins which confer passive immunity to disease for the first 2 to 3 weeks of life. The piglet's endogenous immune system begins to function and produce antibodies in response to environmental stimuli at approximately 2 weeks of age. On an average, 1.5 - 2.0 pig ~ar litter of 8 - lO are lost b~tween birth and weaning at 3 -5 weeks of age as the result of a variety of sow and piglet rQlatad factor6, including the inability of the piglet to obtain sufficient immunoglobulins from ~he sows' clostrum.
Problems at lactation, extra large litters, within litte~
competition, poor nursing sows and sow death resulting in partial starvation and weakness leading to chillin~ and crushing which account ~or n!any of these losses. Clearly, a , milk replacer imparting the required passive disease immunity would allow piglets to be raised from birth independent of the sow and thereby reduce piglet mortality that is mo~ pronounced during the first ~ - ~ days of life (Van der ~Iyae, H. 1972, Proc. Br. Soc. Animal Prod. 33 - 36).
The availability of a milk ba~ed immunoglobulin enriched product which can be used in milk replacer formulation ~ill allow for the rearing of neonatal pigs in a practical environmen~ ~eparate from the dam and allow for populating of new swine units by hy~terectomy with pigs that are Specific Pathogen Free (SPF). Traditionally artificial rearing and derivation o~ SPF pigs wa~ only possible under sterile conditions impractical at the farm level. This system of rai~ing SPF pig~ required specialized and expensive aquipment.
Artificial rearing of neonatal pigs in a non-isolated or l~commercial~' environment has met with little success as the piglet is deprived of ~ow's colostrum and later milk required to provide ~he immunoglobulins which confer pas~ive immunity to disease-cau6ing and other organiæms encountered in a

2~ non-isolated en~ironment. A more practical approach would be to surgicall~ remove the piglets from the sow by either caesarean 6ection or hy~terectomy and rear them ~rom birth in a 'l~ommercial" tnon-isolated) environment providing disease resi~tance from birth by supplementing the milk replacer with porcine immunoglobulins. The weaker pigs in a naturally farrowed litter could be selected soon after birth and reared ~ 1Lat32~

ar~ificially away from the æow. Those piglets contributing most to the early post-na~al mortality could thereby be saved.
Scoot and Coworkers ~J. of Animal Science, 1972, Vol.
35, Pages 1201 - 1205) obtained a survival ra~e of 75% or more when immunoglobulins were ~ed ~or 10 days postpartum to three lots of pigs and this did not differ significantly from control piglets nursing from their dam. Corresponding survival rates of the negative control pigs was 0.33 and 25%. Similar results were ob~ained by ~cCallum, Ellio~ and owen in 197~ ~Can. J.
Animal Sci., Vol. 57, Pages 151 - 158).
The immunoglobulins requir~d to confer passive disease immunity are also present in porcine blood, adequa~e quantities of which are available in meat packing plants. Curren~ly such porcine blood is processed to produce blood meal. Isola~ion a~d concentration o~ these immunoglobulins from porcine ~lood has been previ3usly accomplished by cumbersome methods in quantitiefi sufficient to demonstrate that the immunoglobulins 80 derived can be added to ~he diet of neonatal pigs removed from the sow at birth and confer upon them the passive immunity to di~ease which will allow them to survive in a commercial environme~t, thereby ensuring ~he succes~ of artificial rearing.
The mechanism by which immu~oglobulins can be removed from porcine blood has been a subject of investigation for many years. Methodology employed to-date has been essentially a batch multi-step process which i5 slow, cumbersome, low yielding and not readily adaptabla to commercial production.

- ~L2~L~3;~9t Newson ~Canadian Patent .~o. 1,046,407, 1979) describes a batch process for ~he production of a product whi~h contains only 20% immunoglobulin. This means that 75 - 90 g of the product must be fed to meet the suggested firs~ day requirement o~ 15 g of immunoglobulin: this quantity of material alone already exceed6 the daily dry matter requirement of a young pig (67.5 g) and leaves no room for the addition of skim milk powder (SMP) fat, minerals or vitamins. Casein, a normal constituent of SMP (25%), is essential for clo~ formation in the stomachs of young pigs.
~ further method0 belonging to the prior art ~Eibl. M.
Canadian Patent No. 1,112,166, 1981), describes the preparation o~ a high purity immunoglobulin ( 80% pure); however, the method of preparation is extremely long and involved and would not be economically ~easible.

SUM~A%Y OF THE INV~NTION

According to the present invention there is provided a method of continuous production of purified porcine or bovine immunoglobulins. ~n anti-coagulant is first added to collected porcine or bovine blood to preserve it and the cellular ~ractions are centrifugally removed. Ammonium sulfate is then added to the remaining plasma to obtain a saturation of approximately 20% to 30% to induce fibrin precipitation. The precipitated fibrin i6 removed by subjecting the plasma to .
..

32~

continuous cen~rifugation. The ammonium sulfate sa~uration of the plasma is then increased ~o a range of approximately 35% to 50~ and the product i~ subjec~ed to further centrifugation in order to remove, in the effluent, partially deproteina~ed immunoglobulins and albumins dissolved ~herein. The continuously discharged sludge from the centrifugation step, containing the immunoglobulins in the concen~rated form is collected, and water is added to redissolve the immunoglobulins. The resultant immunoglobulin solution is subjected to an ion depletion process to remove a major portion of the ammonium sulfate. The purified immunoglobulin product i~ then fiubjected to appropriate concentration and treatment for torage ~eg. freeze-drying), or is blended with suitable protein sources to provide an appropriate daily intake of immunoglobulins for the intended animal. For example, the immunoglobulin product intended for piglets derived from porcine blood may be blended with condensed skim milk to give a solids ratio of about 1:3 (immunoglobulins/skim milk solids), and the resulting mixture spray dried to yield a pig-milk ~ replacer component which can be reconstituted and fed as requirQd .
The method according to the present invention provides a product which is unexpectedly high in purity and is extremely e~ective for example, when derived from porcine blood, in the formulation of milk replacer~ for artificial rearing of neonatal pigs to provide the necessary passive immunity to Z~03~

disease otherwise provided by sow~' colos~rum and later milk.

BRIE:F DESCRIPTION OF THE: DRAWINGS

These and other object~ and advantages of the inven~ion will become apparen~ upon reading the following detailed description and upon referring to the drawings in which:
FIGURE 1 is a flow diagram for ~he preparation of a globulin fortifiad milk replacer supplement according to the proces6 of the present invention;
FIGURE 2 is a flow diagram for the preparation of purified porcine immunoglobulins according to a specific exampla; and FIGURE 3 i~ a flow diagram for the preparation of bovine immunoglobulin6 according to a specific example.
While the invention will be described in conjunction with axample embodiment~, it will be understood that it is not intended to limit the invention to such embodimen~s. On the contrary, it is intended to cover all alternatives, modifications and Qquivalent6 as may ~e included within the ~pirit and scQpe of the invention a~ de~ined by the appended claims. For example, while the invcntion will be described in conjunctîon with specific proces~es for preparing porcine immunoglobulin6, it is intended that the invention al60 extend to bovine immunoglobulins prepared from collected bovine blood.

~2~32~

Purified immunoglobulins according to the present inven~ion are prepared as outlined in the flow diagram of FIGURE 1. Blood is collected Erom a large number of pigs at a government inspected slaughter-house and preserved by the addition of an anti-coagulant, e.g., sodium citrate, to prevent clotting. The cellular frac~ions (erythrocytes, leukocytes and ~latelets) are removed centrifugally and account for about a 40% volume reduction, leaving about 60~ plasma. The plasma is chilled and ammonium sulfate added to obtain approximately 20%
- 30% saturation to induce fibrin precipita~ion. The ammonium sulfate can be added in a batch system or portion metered in-stream on a continuous flow basis to obtain the desired saturation. The precipitated fibrin is removed by subjec~ing the product to continuous cen~rifugation. Subsequently, saturation of ~he ammonium sulfate is increased from the range of 20% - 30% to a range of 35% to 50%. The resulting product containfi mainly aggregated immunoglobulins which are subjected to an enrichment centrifugation procedure. The albumins, being soluble at thifi stage, are voided in the ef1uent stream while ~he immunoglobulins are concentrated and continuously discharged as a sludge. The sludge is then reconstituted with water being added at a rate of approximately 5 ~/kg of material, ~ollowing which the redissolved immunoglobulins are subjected to a suitable ion depletion process to remove the ~lZ~32 ,. ~

ammonium sulfa~e te.g., ion exchange, ultrafiltration or elec~odialysis~.
Upon reduction o~ ammonium sulfate levels, the purified immunoglobulin~ may, for example, be fur~her concentrated by way o~ low temperature e~aporation, reverse osmosis or dewatering and freeze-dried for storage for subsequent use. Alternatively, the purified immunoglobulins may be blended immediately with suitable protein sour~es in a ratio appropriate for the daily intake of the animals for which they are intended. The protein sources might be one or more of the following skim milk, whey powder, lactalbumin, casein, caseinate~ and milk co-precipitates. Alternatively, protein products derived from protein sources such as ~oy meal, fish meal and canola meal might be used. To prepare a porcine immunoglobulin product particularly suited for piglets, the puri~ied immunoglobulins, after the reduc~ion of ammonium sulfate levels, are blended with condensed skim milk ~o give a solids ra~io of 1:3 ~immunoglobulins/skim milk solids). The resulting mixture is then spray dried to yield a pig-milk replacer component which can be blended with other required constituents, such as protein, fat, minerals, vitamins, reconstituted and fed upon demand. Optionally, addition of fat~ mineral~ and vitamin~ prior to or after drying gives a complete milk replacer for piglets.

~12~l~3;~

E~AMPLE 1 FIGURE 2 summarizes informa~ion collected on a major Pilot Plant ex~eriment in which 990 1 of plasma (approx. 1016 Kg at S.G. of 1.026) were processed to eventually yield 19.1 Kg of immunoglobulin of 60% purity. In ~his experiment, 254 1 of saturated ammonium sulfate were added to 990 1 of plasma tG
obtain 25% saturation. The precipitate (mainly fibrin) was con~inuously removed by means of a Westfalia (Trade Mark) clarifier (Model SA7-06) operating at 8380 rpm, a flow of 830 0 l/hr and a produc~ temperature of 6C. This yielded 195 Kg of fibrin and 1017 1 of defibrinated serum (approx. 1093 Kg at S.G. of 1.075). To this 463.8 1 of saturated ammonium sulfate were added to bring sa~urat~on to 45% and effect p~ecipitation of the immunoglobulin fraction. This material was subsequently subjected to continuous flow centrifugation using a Pfaudler (Trade Mark) decanting separator (Model ZlL) operating a~ 5650 rpm with a scroll to bowl differential of +10 rpm and a 140 mm regulatîng ring to give a 4.6 mm dry beach. During this operation, 98.7 Kg of sludge were recovered and subsequently ~ diluted wi~h wa~er at a rate of a~proximately 5 l/kg of sludge, in preparation for electrodialysis. This proce~s removed a ma~or por~ion of the ammonium sulfate previously added.
Electrodialysis was achieved by passing the reconstituted immunoglobulin through a "Stack-Pack" (Trade 25 ~ark~ unit manufactured by Ionics IncThis machine was ~2~3%~

equipped with 25 cell pairs and electrodialysis was continued until a conductivity of 6 m - mho was obtained: equivalent to a~out 0.4% ammonium sulfate on a wet basis. In the end, 480.6 Kg of demineralized product were obtained.
Prior ar~ shows th~t the immunoglobulins account for about 27% of the proteins in blood plasma (Hawk, P.B., et. al.
1954, Practical Physiological Chemistry, 13th Ed~, Chapter 22, page 459). Prior ar~ also shows that the solids of blood is 8.51~ (+.2%) of ~hich 6.13% ~*.2%) is total protein (Donnelly, E.B. 1978, Ir. J. Food Sci. ~ Technology, Vol. 2, pages 31 -33). This means that plasma, prior to fibrin removal, contains 27 x S.13 or 1.66% immunoglobulins. From the data in ~able 1 it can clearly be seen that the aforementioned continuous process is a powerful means of concantrating the immunoglobulins from 1.66% in the plasma fraction to 13.3~
pro~ein in the precipitated immunoglobulin fraction recovered from the decanting centrifuga. This is a 8-fold increase in concentration effected by a single salt system used at two discriminating levels of saturation to preferentially remove fibrin at the 20% ~ 30% level of ammonium sulfate saturation and subsequentl~ precipitate the immunoglobulins at increased 6atu~ation levels of 35~ to 50% ammonium sulfate. By optimizing the ammonium sulfate saturation levels to preferably, re pectively, 25% and 45% saturation, the quan~ity of protein precipitated can be maximized and the efficiency of ..

~ ~Z~3~

the process increased.
The recoveIy of immunoglobulin by the presen~ process in this example is approximately 78%. This is based on the 1016 Kg of starting plasma containing approximately 1.66%
immunoglobulin which repre6ents a mass of 16.8 Kg. As the process is followed through the steps illustra~ed in FIGURE 2, a yield of 98.7 Kg of precipitated immunoglobulin sludge is recovered and contains 13.3% immunoglobulin. The yield of immunoglobulin at this point is 0.133 x 98.7 Kg or 13.1 Kg.
This represents a recovery of 78%. Some addi~ional handling losses can be expected during ~he deionizing and spray dried s~ages. Losses of immunoglobulin, due to passage through ultrafiltration (UF) or electrodialy~is (ED) membranes, is negligible as the mean diameter of the solvated species is well above maximum size opening in the membrane material. Losses on ion exchanye resin~ can be higher than either ED ar UF.
Immunoglobulin prepared by the present process, as illustrated ;n Example 1, contain~ low levels of ammonium sulfate as can be seen from the data in Table 2: 2.07% by conductivity and 2.31% by dif~erence. The~e low levels of residual ammonium sulfate ensure the product will he non-toxic to the pig and will not cause palatability problems in a complete milk replacery.
Products prepared according to the present invention have been analyzed for yeastO mold, Streptococci, Salmonella, thermophilic spores, Staphylococci, Coliforms, E. coli and .

- ~2 03;2~9 psychrotophs. In addition, they have been also analyzed for pathogenic viruses infective ~o swine. The results, summarized in Table 3, indicate there are no microbiological hazards associated with production of pig-milk replacer by the present process.
An example of a complete milk replacer formulation for piglets incorporating porcine immunoglobulins~ prepared according to the present invention ,in skim milk powder is as follow6:

Inqredients Percenta~e w/w Porcine Immunoglobulins 14.3 (in skim milk powder) Skim Milk Powder 47.6 Casein 8.0 D.L. Methionine 0.3 Soybean oil 25.0 Vitamin Premix l.o Trace ~ineral Premix 1.0 Calcium Chloride 0.17 ~ Emulsi~ying Agent(~3 1.2 Lactose 1.430 100.~0%

.:

~2~329 BIOLOGI~AL ~NIMAL~ TEST

Thirty-six ~iglets in ~hree replicates wer~ remo~ed from their dams at birth and di~ided into ~wo groups of 18 (6 x

3) piglets each and r~ared artificially until 21 days of age.
One group served as a control and received only mil~ replacer while the second group of 18 received ~he milk replacer supplemented wi~h immunoglobulins prepared using the process of the present inven~ion. Immunoglobulins were fed at a level of 10 gm/kg body weigh~ on day 1 and 2 gm/kg body weight on days 2 - 10 inclusive. Thereafter unsupplemented milk replacer was fed. The trial period was 21 days with the following results:
Immuno~lobulins +
No. of pigs started 18 18 No. of pigs surviving to 21 days 7 14 % Mortality 61.1 22.2 Average daily gain (gm) 100 140 The level of survival amongs~ control pigs (38.9%) was unusually high although not unexpected gi~en the minimal ~o disease status of the experimental unit in which the test was carried out. However, the survival amongst pigs supplemented with immunoglobulin prepared by the processed process (7~.8%) was significantly greater and due to the presence of the immunoglobulin supplements.

~Z~03;i~

The presen~ inven~ion provides, for the first time, a continuous flow process which makes ~he production of immunoglobulin products feasible on a commercial (plant) SiZQ
operation.

Exam~le 2 Bovine immunoglobins were fractiona~ed in a similar manner as previously reported for porcine immunoglobins in Example l. Bovine blood was collacted, preserved and centrifuged to remove red blood cells yielding 513.27 g of plasma containing approximately l.66~ of immunoglobin which repre~ents a mass of 8.5 g immunoglobin. FIGURE 3 is a flow diagram illustating the process which was carried out on the resultant plasma. The yield of immunoglobin sludge wa~ 79.5 g containing 34.~ solids to give a solids yield o~ 27.3 g. This product was analyzed and ~ound to contain 38.9~ protein.
Electrophoresis revealed that this protein contained 62.6%
immunoglobin. Therefore the resulting yield of immunoglobin was (~7.3 x .38g x .626 -) 6.6 g. This represents a yield (6.6/B.5) of 77.6% equivalent to the 78% yield for porcine immunoglobin in Example l. Thus, it is apparent that aquivalent yields of bovine immunoglobulin can be achieved us1ng this process.

9L2'~ 3~9 In conclusion it is apparent that there has been provided in accordance with ~he invention a me~hod for the continuoUs produc~ion of a purified immunoglobulin prepara~ion that Pully satisfies the objects, aims and advantages set forth above. While the invention has been described in conjunction with a specific embodiments thereof, it is eviden~ that many alternatives, modifica~ions and variations will be apparent to those skilled in the art in light of ~he foregoi~g description. Accordingly, it is intended ~o embrace all such alternatives, modifications and variations as fall within the spirit and broad scope of the invention.

3:~9 Table 1: Analy~ical da$a for solids, ash and protein on various fractions during the preparation of im~unoglobulin enriched pig-milk replacer componment.

_ Wt Total Immunoglo~
__ Fraction (Kg~ Solids(%) Ash(X) Protein~%~ bulin(%) Plasma 1016 8.51 (+.2)a _ 6.13(+~2)a 1-~6b Serum 1093 5.85 .123.39 Ppt. Immuno- 9B.7 38.70 .6822.16 13.3 globul;nC
Reconst1tuted - 6.25 .115.33 Immunoglobulind Electrodialyzed 480.6 4.11 .033.98 Immunoglobulind Condensed - 36~2 2.98 13.63 Sk~m Milk Immunoglobulin - 10.71 .74 5.96 1.6 Skim Milk Mixtured Spray Dried - 94.65 5.98 48.76 14.6 Immunoglobulin Sk~m M~lk M~xtured.~

a Data from Donnelly, Ir~sh J. of Food Sci. & Technology, 1978.
~ol. 2, Pages 31 - 38.
Data from Hawk et. al. 1954. Practical Physiologiçal Chemistry, 13th Edition, Chap. 22. - Page 459.
c Average of tw~ values.
d Average of six values.
e Mixed 3 parts condensed skim milk wlth 1 part electrodialyzed immunoglobulins and spray dried.

~ .

~2~32g ~ "

Table 2: Analysis of pig-milk replacer component prepared according to Example 1 (75% skim milk solids and 25% immunoglobul;ns) after spray drying.a Total (NH4)2S4 in Protein Total NPN NPN Contribution b Final Product (%) Nitrogen Nitrogen Nitrogen (N~4)2S04 Milk 8y Con- By (%) (%) ~%~ (%) ductivity Difference 7.64 8.22 .58 .49 .09 2.07 2.31 (48.76% protein) a Average of five values.
b NPN - non-protein nitrogen 3~

Table_3: Microbiological analysis of immunoglobulin enriched pig-milk replacer component . . . _ Organism Log10 Maximum Number Reported Total bacteria 4.97 Yeast 2.~3 Mold 1 74 Yeast ~ Mold 2.93 Streptococci 2.97 Psychrotrophs 3.86 Salmonella negative Thermophilic spores negat;ve Most probable number / g Staphlococc~ g.l Coliforms ~confirmed) 36 E. coli 3.6 Influenza virus negative TGE virus negative HEV virus negative

Claims (13)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method of continuous production of purified immunoglobulins comprising the sequential steps of:
(a) adding an anti-coagulant to collected blood to prevent the formation of fibrin;
(b) centrifugally removing from the blood the cellular fractions;

(c) adding ammonium sulfate to the remaining plasma to obtain a first stage ammonium sulfate saturation of approximately 20% to 30% to induce fibrin precipitation:

(d) removing the precipitated fibrin by subjecting the plasma to continuous centrifugation:

(e) increasing the ammonium sulfate saturation of the remaining plasma to a second stage ammonium sulfate range of approximately 35% to 50%:
(f) subjecting the plasma to further centrifugation to remove in the effluent partially deproteinated immunoglobulins and albumins:

(g) collecting the continuously discharged immunoglobulin - containing sludge from this centrifugation step and adding water to redissolve therein the immunoglobulins:

(h) subjecting the aqueous immunoglobulin solution to an ion depletion process to remove a major portion of the ammonium sulfate: and (i) recovering the purified immunoglobulin material.

2. A method according to claim 1 wherein the purified immunoglobulin material from step (h) is blended with a suitable protein source in a ratio appropriate for the daily intake of an animal to which it is to be fed.

3. A method according to claim 1 wherein the collected blood is porcine.

4. A method according to claim 2 wherein the purified aqueous immunoglobulin recovered from step (h) is blended with a protein source selected from the group consisting of skim milk, whey powder, lactalbumin, casein, caseinates and milk co-precipitates.

5. A method according to claim 3 wherein the purified aqueous immunoglobulin material obtained from step (h) is blended with condensed skim milk to give a solids ratio of about 1:3 (immunoglobulins/skim milk solid), and the resulting mixture is spray dried to yield a pig-milk replacer component which can be reconstituted and fed as required.

6. A method according to claim 5 wherein fat, protein, minerals and vitamins are added to the product prior to or after spray drying to give a complete milk replacement.

7. A method according to claim 5 or 6 wherein the anti-coagulant is sodium citrate.

8. A process according to claim 5 or 6 wherein ammonium sulfate is added to the plasma to obtain a saturation of 20% to 30% in a batch system or portion metered in-stream on a continuous flow basis.

9. A process according to claim 5 or 6 wherein ammonium sulfate is increased to a concentration in the range of 35% to 50% by addition of ammonium sulfate in a batch system or portion metering in-stream on a continuous flow basis.

10. A process according to claim 5 or 6 wherein the immunoglobulin solution is subjected to an ion depletion process selected from the group consisting of ion exchange, diafiltration, ultrafiltration or electrodialysis..

11. A process according to claim 5 wherein the ammonium sulfate concentration is increased to approximately 25% in the first stage of ammonium sulfate saturation.

12. A process according to claim 11 wherein the ammonium concentration is increased to approximately 45% in the second stage of ammonium sulfate saturation.

13. A method according to claim 1 wherein the collected blood is bovine.