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WRITTEN PRELIMINARY DOCTORAL EXAMINATION FOR STEVEN BRANDON

Food Microbiology - Susan Barefoot - May 24, 2004

Due Friday, May 28, 2004, by 4:30 PM

You are chief scientist and manager of a project team whose task will be to filter-sterilize

an IgA-enriched liquid whey supplement. Avoiding the use of thermal sterilization processes is essential to the retention of biological activity in the finished product. The

pH of the supplement typically is 6.0-6.5. Product consumers will include infants,

children and immune-suppressed individuals.

The IgA content of the whey supplement product is achieved by concentrating and

diafiltering raw materials using composite metallic membrane ultrafilters. Your objective is to explore the feasibility of using composite membrane filters to sterilize the product. As we discussed in your office, I will interpret this to indicate that I should use Scepter

membranes.

Discuss the feasibility of producing a commercially sterile product using a composite

metallic membrane ultrafiltration system. What requirements must the composite

metallic membrane ultrafiltration system meet to be suitable for producing a sterile

product? What alternative filtration systems (or other cold sterilization methods) might

be employed? What other questions should be considered in establishing the feasibility of the system?

Discuss the regulatory, economic, scientific (including engineering and safety) and

practical issues to be considered in developing the sterilization process. Conclude your

discussion by providing recommendations for the process to your company.

Background Information

Scepter® Membranes The type of membrane system proposed for the current project is the Scepter® 0.1-µm

rated microfiltration membrane.a This membrane consists of a thin layer of sintered of

300-nm titanium dioxide particles applied across the inner surface and extending into

the pores of sintered, Type 316L stainless steel porous tubes. The resulting porous

matrix forms the 0.1-µm rated Scepter microfiltration (MF) membrane (Figure 1, below).b Sections of porous stainless steel tubes are welded to one another and to tube

sheets at each end of the resulting tubes to which is also welded a Type 316L stainless

steel permeate shell. The resulting membrane module is thus constructed entirely of

stainless steel and the sintered titanium dioxide coating without the need to depend on

epoxy or elastomeric seals to separate the filtered permeate stream from the unfiltered retentate.

® Scepter is a registered trademark of Graver Technologies, Inc., Newark DE.

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Figure 1. 20,000X SEM photograph of Scepter 0.1-µm rated Scepter membrane.

Conventional Scepter modules, such as those used in corn-wet milling and raw cane

sugar processing plants (see Figure 2, below), are not 3-A certified, i.e. they are not manufactured to standards established by the 3-A Sanitary Standards Committee, which

develops sanitary design standards for dairy and food processing, packaging and

handling equipment and systems.c

Figure 2. A large module, used in the corn wet-milling industry.d

The lack of compliance with 3-A sanitary standards of the conventional module design

lies in the way the solid (non-porous) tubes, which are welded to each end of the porous

tubes, are sealed into the permeate side of the tube sheets. In conventional (non 3-A

sanitary) modules, these tubes are inserted into the tube sheet hole and then

mechanically expanded, or “rolled” to force the outside of the tube to press against the inside of the tube sheet hole. This technique does not ensure that the space between

the outside of the tube and the inside of the tube sheet hole is completely filled.

Scott Wittwer, Scepter Product Manager, Graver Technologies, reports that “We have

never tried to get 3A approval, but I have the specifications and know who to contact. I

think we could get by with minimal change in our current tube and module designs, but it would be costly. No one in the dairy industry is knocking our door down to have us

get 3A certification and, until they do, we are not going to spend the time and money to

seek approval.” He goes on to point out that the welds between tube segments could

also be of concern, stating “Another issue is the porous to porous and porous to solid

tube welds. I think the current orbital welding technology helps us with that hurdle.”e In

recent years, Graver has manufactured a small number of test modules, using 6-mm diameter tubes, for use in pharmaceutical applications (see Figure 3, below). I do not

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know whether the issues, discussed above, were addressed in producing these test modules.

Figure 3. Test module for pharmaceutical applications.

Scepter membrane modules are able to operate at extremes of temperature (to 177°C)

and pressure (to 69 bar) and are able to withstand exposure to harsh chemicals (pH 0-

14) provided they are compatible with Type 316L stainless steel.f This means that Scepter modules, unlike polymeric membrane modules, may be thermally sterilized by

exposure to steam at 121°C (2.0 bar)g for 30 minutes to achieve commercial sterility

prior to introduction of the food product.

Commercial sterility of equipment and containers used for aseptic processing and packaging of food means the condition achieved by application of heat, chemical

sterilant(s), or other appropriate treatment that renders the equipment and containers

free of viable microorganisms having public health significance, as well as microorganisms of non-health significance, capable of reproducing in the food under normal non-

refrigerated conditions of storage and distribution.h

Commercial sterility implies that the inactivation of all microorganisms that endanger

public health to a very low probability of survival. For canned foods, the critical organism

is Clostridium botulinum. The 12-decimal (12D) concept is a minimum process for inactivation of C. botulinum in canned foods is accepted in principle by regulatory agencies

and the food industry. However, its interpretation has changed from a literal 12 decimal

reduction to what is now generally accepted as a probability of survival of 10-12. i

Since the stainless steel of the porous tubular supports, used by Scepter technology has

relatively high thermal conductivity (13.4 W/m•K),j heat is distributed quickly and

uniformly throughout the porous tube structure, reaching points in the structure, such as

“dead-end” pores that might be difficult to reach by chemical sanitizers (required by polymeric membranes), such as hydrogen peroxidek, whose transport depends on

molecular diffusion, which may not be effective against all microorganisms and which

may be consumed in chemical reactions before all microorganisms are destroyed.

Competing inorganic membrane technologies such as CeraMeml, CarboSep and KeraSepm

and Membraloxn are also able to be thermally sterilized. However, with each of these membranes, great care must be taken to avoid overly rapid heating and cooling rates to

avoid cracking the membrane elements due to mechanical stresses induced by thermal

expansion and contraction. This has the effect of extending the time required to

accomplish sterilization which wastes online processing time. More significantly, the risk

of such cracking increases the potential for contamination of the filtered product, or

permeate, stream by passage of microbes from the retentate via the crack. Such a

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possibility would necessitate verification of membrane integrity following each sterilization procedure, creating additional down time and added cost of operation.

In the early 1990’s, the predecessor to the current 0.1-µm rated Scepter MF membrane

was referred to as “altered substrates” because the original intention was to modify the

previously existing porous Type 316L stainless steel tubes to have a smaller pore size

thereby providing a superior support or substrate for coatings applied to the inner

surface of the tubes from a recirculating solution or suspension – a formed-in-place (FIP) membrane. In 1993, Barefoot, et al., reported results of microbial challenge tests

conducted on this membrane, referred to as “DS-type altered substrates” in the paper.

That paper concluded that “Microfiltration of whey on the FIP metallic membrane system

reduced lactococci by 5-7 logs, but permitted passage of 66 to 77% of the IgG.”o

The original version of the sintered titanium dioxide membrane were formed using Whitaker, Clark and Daniels, Inc. (now Mineral & Pigment Solutions, Inc.) No. 3330 food-

grade titanium dioxide pigmentp, which is in the anatase crystalline form. In 1992,

production switched to using DuPont Ti-Pure R-101, which is in the rutile crystalline

form. In the sintering furnace, the anatase converts to rutile, which is 5% more dense

than anatase.q This resulted in contraction of the particles causing cracks to form in the sintered titania membrane, thereby increasing the probability of passage of

microorganisms. I believe this manufacturing change took place after the tests reported

in 1993. So, it is possible that membranes produced after 1992 would have higher

microbial rejection than those reported on in the 1993 paper.

A full-scale system, in operation in a fructose refinery in Nebraska since late 1995

filtering 55% fructose high fructose corn syrup (55 HFCS) downstream of the final evaporator, typically achieves about 7 LRV overall.r It should be noted, however, that

this assessment is based on rejection of yeast, mold and mold spores, as well as

bacteria.

Since the mid-1990s, there has been an on-going effort to produce a smaller pore size

membrane. In September 2003, Scott Wittwer informed me that they had produced a

stable 0.02-µm rated Scepter ultrafiltration (UF) membrane that was ready for testing.

With help from Drs. Ron Thomas and Scott Whiteside, Dr. Charles Morr and I obtained

two of these new UF membrane modules for a test program conducted in the DuPont

Packaging Lab in Newman Hall during the Fall 2003 semester. I assisted Dr. Morr in his

investigation of using diafiltration of skim milk to produce a reduced-lactose skim milk

product. Unlike lactose-free milk products currently on the market, which have been treated with lactase to convert lactose to glucose and galactose, the new product would

contain very little sugar, a plus for those suffering with diabetes – not to mention being

more marketable to fans of the low-carbohydrate diet.

In our first test with the Scepter UF membrane, we ran two UF membrane and two MF

membranes simultaneously. The permeate from both MF modules was markedly turbid

(probably indicating the passage of phospholipids, according to Dr. Morr) in contrast to the UF membranes which produced transparent, yellow-colored (due to the riboflavin)

permeates. Dr. Morr determined that “the diafiltered UF permeate fractions contained

nearly half of the skim milk whey proteins, but no casein.s His recommendation is to

repeat the test with a tighter (about 10 kDa MWCO) UF membrane to retain all of the

whey proteins. By contrast, Dr. Morr concluded that the MF membrane was

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unacceptable for his application due to its high degree of passage of whey protein.t Even though Dr. Morr did not attempt to differentiate between the immunoglobulins and the

smaller whey proteins, his observation of relatively high passage of whey proteins tends

to support the results reported in the 1993 paper.

The Scepter UF membrane could be considered as a possible alternative to the MF

membrane. To the best of my knowledge no bacterial challenge test has yet been

conducted on this membrane. The factor which is likely to favor the MF membrane over the UF is that the MF membrane is its ability likely to pass more of the valuable IgA and

other immunoglobulins, while rejecting microorganisms.

Sterile Filtration “There are many organizations involved in setting standards for sterilizing filters, among them ASTM, U.S. Pharmacopoeia, FDA, DIN (Germany) and Health Industry

Manufacturers Association (HIMA). Most agree that a 0.2-µm membrane used for

sterilizing purposes should retain Brevundimonas diminuta (formerly called Pseudomonas

diminutau) at a level of at least 107 cfu/cm² of membrane area.”v However, increasing

concerns about passage of organisms smaller than B. diminuta (e.g. P. Picketti, P. cepacia, Leptospria, Acholeplama, Mycoplasma)w, have led to the wider use of 0.1-µm

membrane for use in sterile filtration applications. It has been demonstrated that a

number of organisms that can penetrate 0.2/0.22-µm rated membranes, including

Acidovorax (formerly Pseudomonas) avenae citrulli, Comamonas (formerly

Pseudomonas) acidovorans, Hydrogenophaga (formerly Pseudomonas) pseudoflava, and Janthinobacterium lividum, are retained by 0.1-µm rated filters.x For this reason

consideration of use of the 0.1-µm rated Scepter membrane is appropriate for this

application.

Immunoglobulins – IgA Immunoglobulins are any of a set of serum glycoproteins which have the ability to bind

other molecules with a high degree of specificity. Molecules which are foreign or non-self

are called antigens; and when an antigen is introduced into a vertebrate, the

immunoglobulin induced is called an antibody. The critical property of this antibody is

that it will combine specifically with the inducing antigen.y There are five classes of

immunoglobulins: IgG, IgA, IgM, IgD and IgE. Of these five, the most abundant in human serum are IgG, IgA and IgM. All immunoglobulins have a basic structure

composed of four polypeptide chains connected to each other by disulfide bridges (see

Figure 4, below). Each light chain usually consists of about 220 amino acids and has a

mass of approximately 25 kDa. Each heavy chain consists of about 440 amino acids and

has a mass of about 50 to 70 kDa. The heavy chains are structurally distinct for each immunoglobulin class or subclass.z

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Figure 4. Basic structure of immunoglobulins showing light and heavy chains. aa

IgG (see Figure 5, below) is the major immunoglobulin of human serum, accounting for

80% of the immunoglobulins.bb IgG neutralizes toxins, opsonizes bacteria, and activates

complement, maternal antibodies. IgG has a molecular mass of 146 kDa.cc Opsonization

is the process in which microorganisms or other particles are coated by antibody and/or

complement, thereby being prepared for recognition and ingestion by phagocytic cells.dd

Figure 5. Basic structure of human Immunoglobulin G. ee

IgA accounts for 15% of the immunoglobulin in human serum. Some IgA is present n the

serum as a monomer of two heavy and two light chains. Most IgA, however, occurs in

the serum as a polymerized dimer held together by a J chain. IgA has special features that are associated with secretory mucosal surfaces. IgA, when transported from the

mucosa-associated lymphoid tissue to mucosal surfaces, acquires a protein termed the

secretory component. Secretory IgA (sIgA, see Figure 6, below) is the primary

immunoglobulin of the secretory immune system, which is found in the gastrointestinal

tract, upper and lower respiratory tracts and genitourinary system. In these body areas, sIgA plays a major roll in protecting the surface tissues from infectious microorganisms

by the formation of an immune barrier. For example, in breast milk sIgA helps protect

nursing newborns.ff IgA levels are considerable in the sero-mucous secretion such as

saliva, tears, nasal fluids, sweat, colostrum and secretions of the lungs and

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gastrointestinal tract. It is believed that IgA plays a major role in protecting the exposed epithelium.gg

IgA, in its monomeric form, has a molecular mass of 160 kDa, while sIgA has a mass of

360-400 kDa.hh Computer generated models of the structure of IgA, indicate that the

antigen-binding sites of the molecule are separated by 23 nm.ii

Figure 6. Basic structure of human sIgA. jj

As with the other classes of antibodies, deficiency of IgA can occur transiently, for example in infants when maternal IgA levels wane when breast feeding is discontinued, or

permanently, as in the not uncommon occurrence of patients with congenital IgA deficiency. Secretory IgA is known to have a beneficial protective effect on the intestinal

mucosa in infants. Stoliar et al., Lancet 1976; i:1258; Williams & Gibbons, Science 1972; 177:697. Eibl et al. (NEJM 1988; 319:1) found that oral feeding of an IgA-IgG

preparation minimized the risk of infants not fed breast milk of contracting necrotizing enterocolitis. The immunoglobulin preparation was made from human serum, Cohn's

Fraction II, by ion exchange chromatography. The preparations contained anywhere from

66% to 85% IgA, 15% to 34% IgG and 0.1% to 2% IgM.kk

Another recent paper found that sIgA may play a role in the in vitro neutralization of

HIV-2.ll

The idea of obtaining immunoglobulin rich extracts from bovine milk, whey or colostrum

using MF is not new. GalaGen, Inc. has patented a microfiltration technique for obtaining an immunoglobulin extract from bovine milk.

A method of microfiltering milk serum, colostrum or colostral serum has been

discovered which substantially reduces the bioburden in the product while providing high immunoglobulin yields. The method makes use of charged depth filters to provide

consistent bioburden control, resulting in whey products fortified with

immunoglobulins. Fortified whey products may be prepared using the microfiltration

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process of the invention. Microfiltration is performed before ultrafiltration to reduce bioburden and eliminate the need for multiple sterile filtrations after ultrafiltration. The

invention also provides a method of preparing whey for microfiltration by adjusting the pH of the whey to about 4.5 to 5.0 and ultrafiltering the whey to obtain a whey-

containing ultrafilter concentrate that can be consistently microfiltered without fouling of the microfilter membrane or depth filter.mm

GalaGen claims bovine colostrum preparations have repeatedly demonstrated their

effectiveness, particularly for use in prevention of diarrheal diseases and against oral

pathogens. Specific antibodies from the milk and colostrum of immunized cows appear efficacious in the treatment of diverse pathogens including bacteria and their toxins,

viruses, protozoal parasites and fungi.nn They make extensive claims for a range of

specific health issues, including several which directly affect the target consumers in the

current project.

The following section summarizes twenty years of scientific research on bovine colostrum ingredients. Many human disease conditions result from numerous harmful

organisms. Bovine colostrum preparations have been researched as a treatment for many illnesses.

Infantile Diarrhea

Formula-fed infants are more prone to a variety of infections. One reason for this may

be that commercial bovine milk derived infant formula lacks the active antibodies and other immune components naturally present in human breast milk. More than 15

articles and abstracts (from 1979 to the present) have been published discussing use of specific immunoglobulin products in the prevention and treatment of two major

causes of infantile diarrhea, E. coli and rotavirus. Use of these specific immunoglobulin products in multiple clinical studies successfully treated or prevented infantile diarrhea

in most, but not all studies. Where positive, they had the effect of reducing or preventing colonization, disease and mortality.

Oral Health Streptococcus mutans is the principal agent responsible for the formation of dental

caries (cavities). Recent studies have documented maternal-child transmission as the primary mode of infection; children colonized later in life appear to develop fewer

cavities. Several studies, including GalaGen research, have documented use of a mouth rinse containing bovine antibodies to reduce S. mutans colonization in adult

volunteers. In the treatment groups, fewer and smaller colonies of S. mutans were recovered, which suggests that the antibodies interfered with the metabolic growth of

existing colonies. Inhibition of glucosyltransferase (involved in adherence) by bovine

immunoglobulins has been reported as well. Specific antibodies can also be directed at the organisms implicated in periodontal (gum) disease.

Candida albicans is a normal resident of the oral cavity (and gastrointestinal tract). In

subjects on certain antibiotics and in immuno-compromised patients, Candida overgrowth can cause severe health problems. The local infection in the oral cavity can

rapidly migrate into systemic infections, resulting in a high percentage of mortality. In a recently completed trial in bone marrow transplant patients, prophylactic

administration of anti-C. albicans bovine antibodies (supplied by GalaGen) as a

mouthrinse appeared to reduce colonization in a majority of patients with initial high colony counts in saliva samples. Thus, antibodies from GalaGen's bovine colostrum

have demonstrated activity against Candida albicans in humans. This evidence is relevant in other applications, as Candida albicans is the organism that causes yeast

infections, a health condition common in women.

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Diarrhea and Food Poisoning

In GalaGen research, colonization by other GI pathogens also appears to be negatively impacted by dietary antibodies. In normal adult volunteers fed live Shigella flexneri

bacteria, bovine immunoglobulins provided complete protection from illness and a marked reduction in diarrheal symptoms. Shigella can be particularly virulent, because

as few as 10 live organisms can cause major intestinal diarrheal symptoms. Shigella is

one of the organisms that commonly cause traveler's diarrhea, as well as food poisoning in the United States.

Cryptosporidium parvum is a protozoal parasite for which no approved efficacious

treatment is available. Specific bovine immunoglobulins (some supplied by GalaGen) have been reported to improve diarrheal symptoms and reduce oocyst shedding in

individual case studies and in clinical trials in severely immuno-compromised (AIDS) adult and pediatric patients. In most cases, patients had suffered debilitating diarrhea

for months to years and had failed all other available experimental therapeutics.

Cryptosporidium parvum also causes outbreaks of food poisoning.

Ulcers/Gastritis Long before medical science realized that stomach ulcers could be caused by bacterial

infection, milk and other dairy products were promoted for the relief of ulcer symptoms. It now appears that bovine colostral antibodies directed against H. pylori

may actively prevent adherence of the organism and inhibit colonization. Additionally, other bactericidal agents may be present in bovine colostrum and milk preparations

that impact H. pylori. In several studies in children and adults with chronic gastritis,

symptoms were improved, inflammation decreased, and colonization was reduced in response to oral antibody feeding; two additional studies were less conclusive.oo

Another company, Proliant, Inc., is marketing a line of dietary supplements called ImmunoLin, that they claim contain 40 - 45% bovine IgG.pp Their web site lists peer-

reviewed papers on relevant research including one that indicates that a large fraction of

ingested bovine immunoglobulin resists digestion in the adult human intestine.qq This

suggests that some of the bovine immunoglobulins, including IgA, survive to be

absorbed into the blood stream.

Discussion of Feasibility The feasibility of any microfiltration application may be considered in two parts: technical feasibility and economic feasibility. An application may be considered technically feasible

if the desired separation can be achieved. Economic feasibility requires that the

separation be accomplished at sufficiently low cost and the resulting separation is

sufficiently valuable so that the capital investment required to for testing, validation,

system design, construction and installation can be recovered in a time period, generally not exceeding 3 years (in applications with which I have experience).

Technical Feasibility There are several factors which must be met to make this application feasible.

1. membrane flux must be sufficient so that the resulting system size is reasonable,

2. enough IgA must pass through the membrane and 3. the final product must be essentially free of microorganisms.

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Flux

Flux must be determined by pilot testing. Of particular interest are how flux varies over

time and with concentration factor (volume reduction factor) and how flux responds to

the input parameters: crossflow velocity and transmembrane pressure. Both temperature and pH must be held at levels to minimize denaturing of the valuable IgA

and other immunoglobulins, hence these factors are not variables to be explored during

pilot testing.

IgA Passage

Passage of IgA must be maximized for the application to be considered feasible. The

passage of IgG reported in the 1993 paper (66 – 77% passage) offers some

encouragement.rr Dr. Morr’s observation last fall that the 0.1-µm rated Scepter MF

membrane retained very little of the total whey protein content of skim milk is also encouraging, however, it must be noted that this observation includes passage of the

more plentiful, smaller components of whey protein (BSA, α-lactalbumin and β-

lactoglobulin). Unfortunately, rejection of IgA is likely to be substantially greater than

that of IgG, due to the larger molecular size of IgA, especially considering that most of

the IgA is present as a 360 - 450 kDa dimer.

The issue of immunoglobulin rejection by MF membranes has been the subject of

research for at least 12 years. One important factor is to operate at low the

transmembrane pressure to minimize the formation of a self-rejecting protein layer on

the surface of the MF membrane. An approach that has reportedly been successful is to

pump the permeate in the permeate shell co-currently with the retentate. This has the

effect of approximately matching the pressure drop profile down the length of the MF module, so that at every point down the length, the transmembrane pressure can be

held to very low levels (< 1 bar).ss However, Pafylias, et al., reports higher flux with co-

current permeate MF operation, but no significant change in protein rejection using

ceramic MF membranes.tt

Diafiltration

Diafiltration (DF) is defined by Munir Cheryan as “the convective elimination of

permeable solutes by the addition of fresh solvent (water, or other) to the retentate.”uu

For food processing, continuous DF is preferable to batch DF, because it is desirable to keep the labile food product continuously advancing through the various unit operations,

rather than remaining stagnant. There are two approaches to continuous DF that have

been employed in the design of Scepter MF systems in recent years: 1-stage DF and 2-

stage counter-flow DF. In 1-stage DF (see Figure 7, below), the dilution water is mixed

with the retentate stream coming from an upstream MF concentration system (labeled “clarifier” in Figure 7). If the ratio of the volumetric flow of dilution water added to the

volumetric flow of retentate (less the volume insoluble solids in the retentate) is termed

variable “a,” the dilution factor of soluble solids by DF is equal to 1+ a.

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Figure 7. Schematic diagram of 1-Stage diafiltration system

In a 2-Stage Counter-Flow DF system (see Figure 8, below), dilution water is injected

into the second DF stage and the permeate stream from the second DF stage is mixed

with the incoming feed stream (labeled “clarifier retentate” in Figure 8). In this case the

dilution factor of soluble solids in the DF system is 1 + a + a². This approach has the

ability to achieve a higher degree of removal of soluble solids for a given amount of

water (diluent) flow and membrane area. It, does, however increase the complexity and capital cost of the DF system.

Figure 8. Schematic diagram of 2-Stage Counter-flow diafiltration system

Microbial Rejection – Engineering Issues

In view of the 5 to 7 LRV for lactococci by altered substrate, sintered titanium dioxide

membrane that reported in the 1993 paper, it appears that a single pass through the

membrane will not be sufficient to achieve commercial sterility. That means that either

the MF process must be repeated, at least once, or MF must be augmented by additional hurdles that effectively add to the LRV of the single MF step. The shortcomings of

repeated MF operations are increased cost of filtration and increased loss of the valuable

IgA and other immunoglobulins.

It should be noted that, while 0.1-µm rated microfiltration membranes are able to

remove most (if not all) bacteria (and larger microorganisms including yeast, mold and

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microscopic parasites) from a food stream, they should not be expected to remove contaminates smaller than the rated pore size of the membrane to any useful degree.

That means viruses, prions (including BSEvv), other proteins that induce allergic reaction

in some people and various toxic substances will not be removed from the process

stream by MF. Therefore, it is necessary to verify that the incoming feed stream is free

of these contaminates prior to MF.

It is desirable, especially considering the sensitivity of the target consumer, to monitor the quality of the raw milk supplied to the process. In addition to testing for the raw

milk for pathogenic bacteria, using, e.g., the Lumac ATP-bioluminescent test for raw milk

is reportedly a quick and effective technique,ww the source animals should be tested to

confirm that they are BSE-free. The animal feeds given to these animals should also be

monitored.

To minimize the rate of microbial growth and to minimize the risk of denaturing the

immunoglobulins, it is desirable to perform all unit operations at low temperature (<

10°C).

The raw milk will contain milk fats present in the form of lipid micelles which will be

rejected by the MF membrane and can contribute to membrane fouling. It is probably

best to centrifuge the raw milk upstream of MF to remove these lipid micelles. This operation also removes bovine mammary cells and some bacteria from the milk, thereby

contributing to the total LRV of the process. This would mean that the MF system is run

on skim milk. It might also be desirable to take measures to precipitate casein prior to

MF. The feed stream to MF would then be whey. This would reduce the solids content of

the stream to be processed by MF possibly resulting in higher flux and higher immunoglobulin passage. However, the precipitation step could also negatively impact

the stability of the immunoglobulins, which are sensitive to changes in pH.

Combining MF with High Hydrostatic Pressure (HPP) could achieve commercial sterility in

the product without unacceptable loss of IgA, either through membrane rejection or

thermal or chemical denaturing. This would need to be confirmed by pilot testing.

“Pressures between 300 and 600 MPa have shown to be an effective method to inactivate microorganisms including most infectious food-borne pathogens. In addition to

microbial destruction, it has been reported that HP improves rennet or acid coagulation

of milk without detrimental effects on important quality characteristics, such as taste,

flavor, vitamins, and nutrients.”xx The stability of IgA to HPP would need to be verifed.

Another possibility is the addition of a bacteriocin, e.g. nisin to the milk as an additional hurdle. The combination of HPP with nisin in processing goat cheese has been reported.

They found that “Combination of 500 MPa and nisin was the most effective treatment to

inactivate cheese indigenous microbiota.”yy However, since nisin is currently approved

by the FDA only for use in cheese, obtaining approval could prove costly. Furthermore,

the stability of Ig exposed to nisin and the safety of ingestion of nisin by infants, children and the immuno-deficient would have to be investigated.

The effectiveness of irradiation as a cold pasteurization method to control foodborne

diseases caused by pathogenic microorganisms and parasites, especially in food to be

consumed raw or partially processed, is established.zz A new paperaaa reports that

continuous-flow UVC irradiation is very effective, particularly against resistant pathogens

(e.g. parvoviruses and bacteria) at UVC doses preserving protein activity. They report

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that “for immunoglobulins the average recovered activity for two independent measurements exceeded 95% for anti-tetanus antibodies and 93% for anti-

pneumococcal antibodies when the UVC dosage was 1000 J/m². Neither aggregation nor

fragmentation of immunoglobulins was observed.” They conclude by saying “Over a

wide range of UVC dosages this new technology is effective against a wide range of

pathogens. It is notably effective against B19 (virus), Gram negative and Gram positive bacteria at UVC dosages preserving plasma protein activity. It is likely to be applicable to

the inactivation of emerging viruses.”

Microbial Rejection – Food Safety Issues

Due to the extreme susceptibility to illness caused by ingestion of pathogenic

microorganisms of members of the target consumer groups: the immuno-deficient,

infants (who are unable to consume breast milk) and children, achieving commercial

sterility in the final product in this application is essential. Persons with Acquired

Immunodeficiency Syndrome (AIDS) are susceptible to many types of infection including illness from foodborne pathogens. They are at higher risk than are otherwise healthy

individuals for severe illness or death.bbb

In the case of infant formula, strict regulations have been passed by Congress with the

intent of protecting this extremely vulnerable group. In 1980, Congress passed the

Infant Formula Act which amended the Federal Food, Drug, and Cosmetic Act to include

section 412 (21 U.S.C. 350a). FDA in turn adopted regulations implementing the act, including regulations on recall procedures, quality control procedures, labeling and

nutrient requirements. This was followed in 1986 by seven amendments.

The 1986 amendments:

(1) State that an infant formula is deemed to be adulterated unless it provides certain required nutrients, meets the quality factor requirements established by the Secretary

of Health and Human Services (the Secretary) (and, by delegation, FDA), and is manufactured in accordance with CGMP and quality control procedures established by

the Secretary; (2) require that the Secretary issue regulations establishing requirements for quality

factors and CGMP, including quality control procedures,

(3) require that infant formula manufacturers regularly audit their operations to ensure that those operations comply with CGMP and quality control procedure regulations;

(4) expand the circumstances in which manufacturers must make a submission to the agency (manufacturers must do so when making major changes that may affect

whether the formula is adulterated); (5) specify the nutrient quality control testing that must be done on each batch of infant

formula; (6) modify the infant formula recall requirements; and

(7) give the Secretary authority to establish requirements for retention of records, including records necessary to demonstrate compliance with CGMP and quality control

procedures.ccc

Several types of requirements are specific to infant formula. These include: • Current good manufacturing practices (CGMP) help ensure that all of the required

nutrients and other ingredients are included at designated levels in the formula, and that the formula is not contaminated with microorganisms or other materials that may be

harmful to infants. The designated levels of nutrients must be within ranges established by statute and regulation.

• Quality control procedures ensure that the infant formula contains the nutrients that

are necessary to support growth and development, at the appropriate levels, not only

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when it enters into commerce but throughout its shelf life. Under the authority of the act, FDA has promulgated regulations that specify quality control procedures for assuring

nutrient content of infant formulas, records and reports, and submission requirements. • Nutrient requirements. Section 412(i) of the act includes a table that lists nutrients

that every infant formula must contain. This section also establishes a minimum level for each of the listed nutrients and a maximum level for certain of those nutrients. FDA may

revise the list by regulation. Currently listed nutrient requirements for infant formulas are

found in 21 C.F.R. 107.100. • Quality factor. This term refers to the nutrient potency and biological effectiveness of a

formula, as formulated, e.g., formulas need to be adequate to support normal physical growth. Subsequent processing, ingredient interactions, and time should not reduce

biological effectiveness of a formula. Manufacturers need to make sure that unsafe nutrient levels or by-products are not created from ingredient addition or breakdown, or

interactions caused by processing or time.ddd

With these stringent regulations in mind, the proposed MF filter-sterilization of whey

must be validated in a process similar to that described for high hydrostatic pressure processes in a recent Food Technology article.eee Although the mechanism of sterilization

is different, the meticulous validation steps, described in this article, apply equally well

to filter sterilization as they do to high pressure processes.

HACCP Plan

A Hazard Analysis Critical Control Point (HACCPfff) plan must be developed for this

process. The most obvious hazard is passage of microorganisms into the final product.

The critical point is then to monitor the MF (and any other hurdle unit operations added

to the process) to verify that they are rejecting microbes as expected.

The FDA states that, “Validation should include microbiological challenges to simulate

‘worst case’ production conditions regarding the size of microorganisms in the material

to be filtered.”ggg The validation of sterilization-grade membranes is integral to ensuring

the efficient and safe use of microfiltration systems. Current validation methods, based

on standard plate counts, require 48 hours of culture for results to become available, which creates time delays within the manufacturing process and quality control (QC)

backlogs. Molecular DNA tests, e.g., PCR, and probe hybridization methods, are useful

and rapid for bacterial enumeration, but these methods do not necessarily confirm that

the organisms detected are viable, and they are expensive and technically demanding.

Griffiths, et al., reports on a comparison of four methods for the production of filter challenge test data, to the desired test sensitivity, within 24 hours using bioluminescent

and fluorescent recombinant strains of the test organism Brevundimonas diminuta.hhh

The following process flow chart lists indicates suggested unit operations and critical

control points that must be monitored to maintain food safety.

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Figure 9. Process flow chart and HACCP diagram for production of IgA concentrate from

bovine milk.

Economic Feasibility The economic feasibility of the process depends greatly on flux rate determined in pilot

testing. It also depends on the value of the resulting product and on the costs of

operation of the process, as well as the cost of monitoring to insure production of a safe

product. All of these factors would need to be evaluated by pilot testing.

Conclusions & Recommendations As an initial plan, I recommend that the process flow chart presented in Figure 9, above.

Raw milk should be obtained from suppliers who can confirm monitoring the health of

their dairy cattle of the feeds they consume. The raw milk should be transported

hygienically at low temperature, as is standard operating procedure in the US. The raw

milk should be tested for the presence of pathogenic organisms before delivery is

accepted. The raw milk should be chilled further, if necessary before the lipid micelles and cellular debris is removed by centrifugation. The centrate (skim milk) should then

be subjected to HPP to destroy most microorganisms present in the milk. Casein

precipitation would occur next, provided this can be accomplished without negatively

affecting the stability of the immunoglobulins. Microfiltration would then be used to

provide an additional 5 to 7 LRV hurdle and to remove dead cells killed in the preceding HPP operation. Ultrafiltration membranes could then be used to concentrate the

immunoglobulins. The new 0.02-µm rated Scepter ultrafiltration (UF) membrane might

be tight enough to reject immunoglobulins (especially the relatively large IgA molecules)

while passing water, riboflavin, lactose and other soluble solids. Diafiltration with the

same type of UF membrane and using sterile-filtered water can be used to further purify the immunoglobulin concentrate by washing out the soluble solids. A final treatment

with continuous flow UVC irradiation (which has been shown to cause minimal damage to

Dairy Cattle

Animal Feed

Raw Milk

Centrifuge

CCP = RPM

MF CCP = LRV

Chiller CCP = Temp.

HPP CCP = Press,

LRV

UVC Irradiation

DF (purification)

UF (Ig conc.)

Aseptic Packaging

Cold Storage

CCP = BSE Test

CCP = BSE Test

CCP = Pathogen test

CCP = LRV

CCP = Temp.

CCP = sterility

26 May 2004

16

Ig activity) should be employed to provide a final, broad-spectrum hurdle. UVC irradiation is able to destroy, not only pathogenic bacteria, but also viral contaminates.

Finally, aseptic packaging techniques should be employed to maintain the sterility of the

product, which should be stored and shipped at refrigerated temperatures to protect the

immunoglobulins and to maximize shelf life of the product.

The efficacy of all of the hurdle operations must be validated through extensive pilot

testing. The safety of the final product must be confirmed by a clinical study to win FDA

approval for its use in a new infant formula as detailed by Dr. Duane Brooks.iii Approval

for use of the IgA-enriched liquid whey supplement in products intended for the immuno-

deficient should be pursued simultaneously.

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