The manufacture and purification of lactoferrin (LF), a multifunctional iron-binding glycoprotein primarily found in milk, involves several carefully controlled steps to ensure purity, bioactivity, and safety. LF has important nutraceutical, anti-inflammatory, and antimicrobial properties. It plays an important role in iron metabolism. The protein is also strongly associated with hydroxyapatite in building synthetic bone structures and general bone building. In this post we cover the extraction and purification from milk and then look at how precision fermentation is being exploited as the next alternative commercial process.
LF is a 78 kDa iron-binding protein of the transferrin family with an isoelectric point around 9.0 It has two metal binding sites (Chandan et al., 2015). It has 651 amino acids and its principal role is to transport iron to the newly born calf after birth. Other roles include ion transport, antiviral and antifungal activity as well as wound healing and some anti-inflammatory activity. It forms two globular lobes, an N-lobe and a C-lobe and each lobe is connected via a single hinge formed by parts of the alpha helix (Gonzalez-Chavez et al., 2009).
The most striking physicochemical feature of lactoferrin is its extraordinarily high affinity for iron, which can increase the bioavailability of this metal in intestinal cells, stabilize reduced iron ions, and reduce the irritation of the intestines and stomach. Each lobe binds one ferric ion in coordination with a carbonate ion.
In 2024, it was reported that purified bovine lactoferrin cost anywhere between $700 and $2,000/kg.
The Raw Material: Milk
This protein is present in a concentration of 3.2 mg/L in milk. The level in milk which is produced by the mammary glands varies depending on the nutritional and functional status of the animal. The actual levels of LF vary between 0.1 to 0.3 mg/ml and is further increased to between 2 and 5 mg/ml in colostrum. If the bovine mammary gland is involuted, the level of LF can reach between 20 and 30 mg/ml which is 100 fold higher (Bishop et al., 1976). The occurrence of mastitis in cows also raises lactoferrin levels.
To help in the design of a purification process means understanding the protein of interest and the other proteins that serve as contaminants or hinder purification. Milk is complex in composition containing many proteins especially casein, lactoglobulin and albumin which are present in greater amounts than LF. LF is not only found in milk but also specific granules of polymorphonuclear leukocytes (Baggiolini et al., 1970) .
One of the main issues in its separation is the similarity in molecular weight to bovine serum albumin (BSA) which has a molecular weight of 66.5 kDa. There is also a similarity in both proteins preventing foam formation at their isoelectric points through reduction of surface tension.
Lactoferrin is a valuable protein for medical purposes. Peptides fragments derived from this protein called lactoferricins are effective against streptococci, Escherichia coli and can stop biofilm formation by oral pathogens (Dionysius & Milne, 1997).
This is an overview of the typical lactoferrin production process, especially when derived from cow’s milk (the most common source). Presently, about 20 to 30 tonnes are produced annually for the nutraceutical industry.
1. Raw Material Collection
-
Source: Usually cow milk or whey (a by-product of cheese production).
-
Goal: Obtain a rich and consistent starting material containing lactoferrin.
-
Considerations: Quality control for microbial safety, pH, protein content, and fat levels.
2. Pre-Treatment of Milk/Whey Solution/Colostrum
-
Defatting: Centrifugation removes cream/fat. Usually operated at 2000 x g.30 min. The defatted (skimmed) milk was diluted twice with deionized water.
-
Casein Removal: Acidification (2 N HCl) to pH 4.6 at room temperature or enzymatic treatment removes casein, leaving whey proteins. This is normally called acid whey.
-
Microfiltration: The precipitate of casein is removed by filtration. Further clarification of the liquid also removes bacteria. Casein is usually processed into cheese.
-
Precipitate Removal: The acid whey is usually neutralized to pH 6.8 with 2N NaOH which generates a precipitate that is then removed by centrifugation (10,000 x g.30 min, 4ºC).
If cheese whey is the chosen material of choice rather than milk then we find that other proteins in whey such as α-lactalbumin and β-lactoglobulin are negatively charged whilst lactoferrin and lactoperoxidase (Lp) are positively charged. This charge difference allows for ready separation because it means the majority of the contaminating protein can be removed.
Lu et al., (2007) used a pretreatment step of ultrafiltration based on two steps prior to using cation exchange chromatography. A two-step ultrafiltration process was performed with membranes of nominal molecular weight cut-offs of 100 kDa for ultrafiltration (UF) in step 1 and then 10 kDa in the UF step 2. Ultrafiltration appears to be a commercially more effective method than even ion-exchange which we reference below.
3. Lactoferrin Extraction
This is the core step and can involve several alternative methods dependent on commercial effectiveness. As we will discuss, chromatography is the traditional method for separating proteins in whey but it has consistent disadvantages of fouling, long cycle times and a relatively high cost. Elution in many cases involves high salt which adds to the effluent load especially when it contributes to increasing salinity. Increasingly ultrafiltration finds greater uses and as a pressure-driven membrane filtration has become more cost effective. The issue is low selectivity.
a. Ion-Exchange Chromatography
-
Principle: Lactoferrin’s high isoelectric point allows selective binding to cation exchange resins at acidic pH to neutral pH. LF is positively charged and so will bind to negatively charged resins. The most common eluting material is 1M sodium chloride in an aqueous buffer.
-
Steps:
-
Apply clarified whey to the resin which is usually a cation-exchange resin. Good examples are SP-Sepharose and CM-Sepharose
-
Wash away unbound proteins.
-
Elute lactoferrin using a salt gradient or pH change.
-
-
Benefits: High purity and activity retention.
One advanced method used microfiltration followed by ultrafiltration then diafiltration to concentrate the lactoferrin fraction before application to a cation exchange expanded bed system (Maciel et al., 2020). The final concentration of LF was 17.4 mg/ml with a purity of 93% and recovery of 87%.
There are cases where DEAE- anion exchange has been used and the buffer conditions are above the pI of 8.0 to 8.7. In this case LF has a negative charge above this and will bind then to positively charged resin. The binding is usually very weak but it means less aggressive elution conditions are employed. It is conducted when casein is still present along with albumin which then behave very differently at high pH. It is also adopted if the cation exchange method is poor in resolution or as a final polishing step having conducted a caton exchange step. .
b. Affinity Chromatography (less common industrially)
-
Uses ligands that bind specifically to lactoferrin. One in particular is the triazinic dye, Red HE-3B dye.
Affinity chromatography is best when the concentration of the protein to be bound is in low concentration.
Relatively cheap dyes can be used such as Coomassie Blue bound to Sepharose (Babina et al., 2004). These dye ligands severely reduce the cost of affinity based separation
Wolman et al., 2007 boldly stated they could do a one-step purification from bovine whey and colostrum using affinity membrane chromatography. It is a good example of combining a mass separation method based on physical protein size with specific binding properties within the protein. In this example, hollow fibre membranes were prepared by grafting a glycidyl methacrylate/dimethyl acrylamide copolymer to polysulphone membranes and attaching Red HE-3B dye to them. These had high lactoferrin adsorption capacity. The maximum capacity of this membrane was 111.0 mg/ml compared to a similar ligan density on agarose beads (9.3 mg/ml). A similar dye, Yellow HE-4R, immobilized to Sepharose is also an effective ligand producing a yield of 71% and a purification factor of 61 (Baieli et al., 2014a & b).
Desorption was achieved using 2M NaCl in 25% ethylene glycol – about 99% with the hollow fibres and 80% with agarose beads.
An example of magnetic affinity separation has been employed. Heparin is a very effective ligand for LF. A micron-sized monodisperse superparamagnetic polyglycidyl methacrylate (PGMA) particle column was coupled with heparin (PGMA-heparin) (Chen et al., 2007). The magnetic properties of the column matrix was used to aid separation. The maximum LF binding capacity was 164mg/g.
Other alternatives use antibodies as ligands (Kawakami et al., 1987)but these are expensive to develop and are easily destroyed by proteases as well as being denatured by chemicals and extreme physical conditions.
For scale-up purposes, macroporous monolith columns are being used. A monolith is a single piece of highly porous material characterized by a highly interconnected network of channels with a diameter in the range of 10–4000 nm. Mass transport of materials is through convection.
c. Hydrophobic Interaction/Hydroxyapatite
- LF has been separated from whey in a one-column chromatographic system (Ng & Yoshitake, 2010). The column was based on ceramic hydroxyapatite (CHT™).
d. Salting Out and Precipitation
- The addition of salt to milk to produce a high-concentration salt solution can compete with LF for water molecules. Salting out means the protein solubility is reduced because the salt (NaCl or ammonium sulphate) disrupts the water shell around the protein helping to maintain solubility. It can destroy the water films on the surface of protein colloidal particles and thus cause protein precipitation (Wong, 2018). Certain proteins like lactoferrin are preferentially precipitated out.
4. Concentration and Purification
-
Ultrafiltration/Diafiltration: Concentrates lactoferrin and removes smaller molecules (e.g., salts, lactose).
-
Optional steps: Activated carbon treatment to remove off-colours or residual contaminants.
Membrane separation is an effective alternative to ion-exchange because it does not involve adsorption or elution steps. There are no costs for chromatographic materials, buffers or effluent disposal. It is not effective at separating similar sized proteins including LF and BSA. Nyström et al. studied the fractionation of several proteins with molecular weight between 15 kD and 80 kD. The best pH value for fractionation was such that one protein had its isoelectric point at this pH, and passed through the membrane. The other proteins were retained because of charge repulsion with the membrane. At a low pressure, BSA was retained and LF passed through whilst at higher pressures the selectivity was lost.
Ultrafiltration could be an effective method even with its relatively low selectivity compared to chromatography. Selectivity can be improved by combining it with electrodialysis. An electric field force has better selectivity than just a pressure-driven method. When the pressure driven force is removed, the compressive forces that once existed are removed. It means that the non-selective fouling cake does not form and block the pore membranes. Bazinet have patented a process for ultrafiltration and electrodialysis of peptides.
Lactoferrin has the highest isoelectric point of any of the whey proteins of any consequence in water or a low salt solution. When the ionic strength exceeds 30 mM at pH 6.5, then negative values of zeta potential are noted. The thinking is that there is a formation of net-negatively charged LF aggregates or micelles, which are observed to form at NaCl concentrations above 10 mM. At higher salt concentrations close to 100 mM salt concentrations was larger aggregates of 110nm in size. Lactoferrin will also polymerize in 10 mM calcium chloride with the predominant species being a tetramer of molecular weight of 300 kDa. Given whey or milk has an ionic strength of 30 mM or more because of the presence of calcium and sodium, then the net charge is not positive but negative. Hence, it is then difficult to separate lactoferrin from the other negatively-charged proteins such as BSA.
5. Drying
-
Spray Drying or Freeze Drying: Converts the lactoferrin solution into a stable powder.
-
Control points: Maintain temperature below thresholds to preserve protein activity.
6. Quality Control & Packaging
-
Testing includes:
-
Purity (via HPLC or SDS-PAGE)
-
Microbial load
-
Iron saturation level
-
Colour and solubility
-
-
Packaging: Done in controlled environments to prevent contamination and degradation.
The Key Factors
-
Purity target: Usually >90% for pharmaceutical or nutritional use.
-
Iron saturation: Can be adjusted post-extraction, depending on the application (apo- or holo-lactoferrin).
-
Stability: Must avoid high heat or proteolytic degradation.
One method that might reduce the number of steps is to exploit isoelectric separation using charged ultrafiltration membranes (Valino et al., 2014). In their study, they assessed three operational variables; (i) BSA/LF initial concentration ratio, (ii) protein isoelectric point (Ip), and (iii) membrane charge using three composite regenerated cellulose (CRC) membranes: negatively charged, positively charged, and unmodified. From this study, they identified the necessary conditions for optimum protein separation. LF was retained in the feed mixture when an uncharged membrane was used and BSA was able to pass through because its isoelectric point was a pI of 4.9. When a negatively charged membrane was used, BSA was retained and LF passed through because its pI was 9.0
The Production Of Lactoferrin by Precision Fermentation
Producing lactoferrin via precision fermentation is an innovative biotechnology approach that allows for scalable, animal-free production of bioidentical proteins — including human or bovine lactoferrin. Here’s how this process works:-
Lactoferrin Production by Precision Fermentation
1. Gene Editing / DNA Insertion
-
Goal: Introduce the lactoferrin gene (either human or bovine) into a microbial host.
-
Common Hosts:
-
Yeast (e.g., Komagataella phaffii (now Pichia pastoris) (Cavallo et al., 2025) or Saccharomyces cerevisiae).
-
Fungi (e.g., Aspergillus niger)
-
Bacteria (e.g., E. coli, though less common due to protein folding issues)
-
-
Method: Use recombinant DNA technology to insert the gene encoding lactoferrin into the host’s genome.
Lactoferrin is also a glycoprotein so it needs careful management in the cell to produce the glycosylation pattern. That requires a form of post-translational modification. Generally, there is N-linked glycosylation to potentially one of five asparagine residues. Without glycosylation, the affinity for iron chelation markedly drops off.
It is also antimicrobial which means it can impact the host cell and other cells around it once it has been released.
K. phaffii is also notable for its ability to achieve high cell densities and, consequently, increased protein yield, in addition to not secreting many proteins inherent to it, which simplifies the downstream process. Exploiting the AOX1 promoter in K. phaffii meant the achievement of a significant production of LF with a yield of up to 3.5 g/L of recombinant bovine lactoferrin.
Attention is now being paid to human LF. The fungus Aspergillus awamori can produce up to 2 g/L (Ward et al., 1995). The main issue in producing human LF is reproducing the N-glycosylation pattern without it becoming random. Glycosylation of human lactoferrin occurs predominantly at three sites: Asn-156, Asn-497, and Asn-642 (Zlatina et al., 2021). The yeast’s own glycosylation pathways are removed followed by introduction of five active eukaryotic proteins, including mannosidases I and II, N-acetylglucosaminyl transferases I and II, and uridine 5′-diphosphate (UDP)-N-acetylglucosamine transporter. This method helps increase the life of LF in vivo and removes any potential allergenic issues (Hamilton et al., 2003).
2. Fermentation (Bioreactor Cultivation)
-
Bioreactors are used to grow the engineered microbes in controlled environments.
-
Inputs: Sugars (glucose), nitrogen sources, minerals, and oxygen.
-
Process:
-
Microbes grow and express lactoferrin as they metabolize the feedstock.
-
Conditions such as pH, temperature, and oxygen are tightly regulated to maximize yield.
-
3. Protein Secretion or Cell Lysis
-
Depending on the host, lactoferrin is:
-
Secreted directly into the fermentation broth which is preferred for easier downstream processing, or
-
Retained intracellularly, requiring cell lysis to release it.
-
4. Purification
-
Similar to dairy-derived lactoferrin:
-
Filtration (e.g., ultrafiltration)
-
Ion-exchange chromatography to isolate and purify lactoferrin based on charge.
-
Polishing steps like size-exclusion or hydrophobic interaction chromatography (if needed).
-
5. Drying & Formulation
-
The purified protein is dried (spray-drying or freeze-drying) and then formulated into powders, capsules, or liquids.
-
Final product is bioidentical to natural lactoferrin but produced without animals.
Advantages of Precision Fermentation
-
Animal-free, sustainable production.
-
Customizable (e.g., human lactoferrin for infant formula).
-
Scalable and consistent.
-
Avoids risks of pathogens or allergens from dairy.
Companies
1. TurtleTree (Singapore/USA)
TurtleTree has developed LF+, a precision fermentation-derived lactoferrin, and achieved the world’s first self-GRAS (Generally Recognized as Safe) status from the U.S. FDA for this product. LF+ is being incorporated into various applications, including infant formulas, plant-based dairy alternatives, and sports nutrition products. The company has partnered with brands like Cadence Performance Coffee and Strive to bring lactoferrin-enhanced products to market .
2. FrieslandCampina Ingredients & Triplebar (Netherlands/USA)
Dutch dairy giant FrieslandCampina Ingredients has collaborated with U.S.-based biotech company Triplebar to produce lactoferrin via precision fermentation. This partnership aims to scale up production and reduce costs, making lactoferrin more accessible for various applications beyond infant nutrition, such as adult supplements and functional foods .
3. All G Foods (Australia)
Sydney-based startup All G Foods plans to launch bovine lactoferrin produced through precision fermentation in 2025, followed by human lactoferrin in 2026. The company is focusing on high-value dairy proteins to meet the growing demand for sustainable and ethical alternatives in the food industry.
These companies are at the forefront of utilizing precision fermentation to produce lactoferrin, contributing to a more sustainable and ethical food system.
The Antibacterial Peptides Of Lactoferrin
Lactoferrin could be a valuable source of antibacterial peptides called lactoferricins. It has been shown to be active as an antimicrobial against Escherichia coli (Dionysius & Milne, 1997). These peptides are generated from a pepsin digest. All these peptides are cationic and come from the N-terminus of lactoferrin. The three antimicrobial peptides are small molecular weight.
References
Babina, S. E., Kanyshkova, T. G., Buneva, V. N., & Nevinsky, G. A. (2004). Lactoferrin is the major deoxyribonuclease of human milk. Biochemistry (Moscow), 69(9), pp. 1006-1015.
Baggiolini, M., D. de Duve, P. L. Masson, and J. F. Heremans. (1970). Association of lactoferrin with specific granules in rabbit heterophil leukocytes. J. Exp. Med. 13 pp. 559.
Baieli, M.F., Urtasun, N., Miranda, M.V., Cascone, O., Wolman, F.J., 2014a. Bovine lactoferrin purification from whey using Yellow HE-4R as the chromatographic affinity ligand. J. Sep. Sci. 37, pp. 484–487.
Baieli, M.F., Urtasun, N., Miranda, M.V., Cascone, O., Wolman, F.J., 2014b. Isolation of lactoferrin from whey by dye-affinity, chromatography with Yellow HE-4R attached to chitosan mini-spheres. Int. Dairy J. pp. 39
Bishop, J. G., F. L. Schanbacher, L. C. Ferguson, and K. L. Smith. (1976). In vitro growth inhibition of mastitis-causing coliform bacteria by bovine apo-lactoferrin and reversal of inhibition by citrate and high concentrations of apo-lactoferrin. Infect. Immun. 14 pp. 911
Cavallo, J., Raynes, J., Mandacaru, S., Agarwal, D., Condict, L., & Kasapis, S. (2025). Physicochemical and functional comparison of food-grade and precision-fermented bovine lactoferrin. Food Hydrocolloids, 166, 111380.
Chandan, R.C., Kilara, A. Shah, N.P. Edt. (2015) Dairy Processing and Quality Assurance. 2nd Edt. Wiley Blackwell
Chen, L., Guo, C., Guan, Y., & Liu, H. (2007). Isolation of lactoferrin from acid whey by magnetic affinity separation. Separation and Purification Technology, 56(2), pp. 168-174.
Cui, S., Lv, X., Sun, G., Wu, W., Xu, H., Li, Y., … & Liu, L. (2022). Recent advances and prospects in purification and heterologous expression of lactoferrin. Food Bioengineering, 1(1), pp. 58-67.
Dionysius, D. A., & Milne, J. M. (1997). Antibacterial peptides of bovine lactoferrin: purification and characterization. Journal of Dairy Science, 80(4), pp. 667-674.
Dyrda-Terniuk, T., & Pomastowski, P. (2023). The multifaceted roles of bovine lactoferrin: Molecular structure, isolation methods, analytical characteristics, and biological properties. Journal of Agricultural and Food Chemistry, 71(51), pp. 20500-20531
, and . (2024). Impact of Ultrafiltration on the Physicochemical Properties of Bovine Lactoferrin: Insights Into Molecular Mass, Surface Morphology, and Elemental Composition. Journal of Dairy Science 107 (12): pp. 10280–10298.
, , , , and . (2024). Purification of Lactoferrin Through a Sequential Filtration and Magnetic Separation Process: A Processing Example for Acid Whey Valorization. Future Foods 9: 100342. .
Lu, R. R., Xu, S. Y., Wang, Z., & Yang, R. J. (2007). Isolation of lactoferrin from bovine colostrum by ultrafiltration coupled with strong cation exchange chromatography on a production scale. Journal of Membrane Science, 297(1-2), pp. 152-161.
Maciel, K. S., Santos, L. S., Bonomo, R. C. F., Verissimo, L. A. A., Minim, V. P. R., & Minim, L. A. (2020). Purification of lactoferrin from sweet whey using ultrafiltration followed by expanded bed chromatography. Separation and Purification Technology, 251, 117324. .
Ng, P. K., & Yoshitake, T. (2010). Purification of lactoferrin using hydroxyapatite. Journal of Chromatography B, 878(13-14), pp. 976-980
Valiño, V., San Román, M. F., Ibañez, R., & Ortiz, I. (2014). Improved separation of bovine serum albumin and lactoferrin mixtures using charged ultrafiltration membranes. Separation and Purification Technology, 125, pp. 163-169 (Article)
Wakabayashi, H., Yamauchi, K., & Takase, M. (2006). Lactoferrin research, technology and applications. International Dairy Journal, 16(11), pp. 1241-1251.
Wang, Q., Chen, G. Q., & Kentish, S. E. (2020). Isolation of lactoferrin and immunoglobulins from dairy whey by an electrodialysis with filtration membrane process. Separation and Purification Technology, 233, 115987.
Ward, P.P.; Piddington, C.S.; Cunningham, G.A.; Zhou, X.; Wyatt, R.D.; Conneely, O.M. A. (1995) System for Production of Commercial Quantities of Human Lactoferrin: A Broad Spectrum Natural Antibiotic. Biotechnology 13, pp. 498–503
Wolman, F. J., Maglio, D. G., Grasselli, M., & Cascone, O. (2007). One‐step lactoferrin purification from bovine whey and colostrum by affinity membrane chromatography. Journal of Membrane Science, 288, pp. 132–138
Wong, D. W.(2018). Proteins. In D. W. S. Wong (Ed.), Mechanism and theory in food chemistry . Springer International Publishing. (2nd Edition, pp. 55–122
Yoshida, S., Wei, Z., Shinmura, Y., & Fukunaga, N. (2000). Separation of lactoferrin-a and-b from bovine colostrum. Journal of Dairy Science, 83(10), pp. 2211-2215
Zlatina, K.; Galuska, S.P. (2021) The N-glycans of lactoferrin: More than just a sweet decoration. Biochem. Cell Biol. 99, pp. 117–127
Leave a Reply