GENERAL RECOGNITION

Ren et al. 2019.Gut and immune effects of bioactive milk factors in preterm pigs exposed to prenatal inflammation. Am J PhysiolGastrointestLiver Physiol. 15 [Epubahead of print] 

(https://www.ncbi.nlm.nih.gov/pubmed/31091150)

Donovan 2019.Human milk proteins: Composition and physiological significance. Nes le Nutr Inst Workshop Ser. 90: 93-101 (https://www.ncbi.nlm.nih.gov/pubmed/30865978)

Jiang & Lönnerdal 2019.Osteopontinin human milk and infant formula affects infant plasma osteopontinconcentrationsPediatrRes. 85(4): 502-505

(https://www.ncbi.nlm.nih.gov/pubmed/30636771)

Chen et al. 2018.Osteopontin-enriched formula feeding improves the T-cell-dependent humoral immune response in infant rats. Int J Food Sci Nutr. 69(8): 969-975

(https://www.ncbi.nlm.nih.gov/pubmed/30001650)

Bruun et al. 2018.Osteopontinlevels in human milk vary across countries and within lacta ion period: Data from a multicenter study JPGN. 67(2): 250-256

(https://www.ncbi.nlm.nih.gov/pubmed/29668569)

Demmelmair et al. 2017.Benefits of lactoferrin, osteopon inand milk fat globule membranes for infants. Nutrients. 9(8) ( https://www.ncbi.nlm nih.gov/pubmed/28788066)

Lönnerdal 2017.Bioactive protein in human milk – potential benefits for preterm infants. Clin Perinatol. 44(1): 179-191 (https://www.ncbi.nlm.nih.gov/pubmed/28159205)

Jiang & Lönnerdal 2017. Biological roles of milk osteopon in.CurrOpinClinNutr MetabCare. 19(3): 214-219 (https://www.ncbi.nlm.nih.gov/pubmed/27504516)

Lönnerdal 2016. Human milk: Bioactive proteins/peptides and func ional properties. Nest é Nutr Inst Workshop Ser. 86: 97-107 (https://www.ncbi.nlm.nih.gov/pubmed/27337145)

Christensen & Sørensen 2016.Structure, function and nutri ional potential of milk osteopontin.Int DairyJ. 57: 1-6 (https://www.sciencedirect.com/science/article/pii/S0958694616300437)

Lönnerdal 2016. Bioactive proteins in human milk: Health, nutrition, and implications for infant formulas JPediatr. 173: S4-S9 (https://www.ncbi.nlm.nih.gov/pubmed/27234410)

Lönnerdal et al. 2016. Growth, nutrition, and cytokine response of breast-fed infants and infants fed formula with added bovine osteopontin.JPGN. 62(4): 650-657

(https://www.ncbi.nlm.nih.gov/pubmed/26465791)

Donovan et al. 2014.Bovineosteopontinmodifies the intestinal transcriptome of formula-fed infant rhesus monkeys to be more similar to those that were breas fed. JNutr. 144(12): 1910-1919

(https://www.ncbi.nlm.nih.gov/pubmed/25320184)

Kvistgaard et al. 2014.Pre-clinical in vitro and in vivo safety evaluation of bovine whey deriv d osteopontin,Lacprodan® OPN-10. Food Chem Toxicol. 73: 59-70

(https://www.ncbi.nlm.nih.gov/pubmed/25072164)

Lönnerdal 2014. Infant formula and infant nutrition: bioactoveproteins of human milk and implications for composition of infant formulas Am J ClinNutr. 99(3): 12S-17S

(https://www.ncbi.nlm.nih.gov/pubmed/24452231)

Chatterton et al. 2013.Anti-inflammatory mechanisms of bioac ive milk proteins in the intestine of the newborns. In J BiochemCell Biol. 45(8): 1730-1747

(https://www.ncbi.nlm nih.gov/pubmed/23660296)

Lönnerdal 2011. Biological effects of novel bovine milk fractio s Nes léNutr Workshop Ser PediatrProgram. 67: 41-54 (https://www.ncbi.nlm.nih.gov/pubmed/21335989)

Schacket al. 2009.Considerable varia ion in the concentration of osteopon inin human milk, bovine milk, and infant formulas. J Dairy Sci. 92(11) 5378 -5385

(https://www.ncbi.nlm.nih.gov/pubmed/19841198)

4

Publicationof OPN in earlylife nutrition
GENERAL RECOGNITION

• Q3 2019 we will submit a Novel Food application in EU to enhance general recognition

• We willrequest138 mg/L as novel food approval dose

• We havediscussedin scientific forums and alsowith individual pediatricMD immunologistson some 

of the concerns raised by FDA in scientific memo

• We can arrangearound table discussionof bOPNsafety that includesOPN structural experts, pediatric

immunologistsand nutrition expertstogether with scientists fromFDAfor discussion

• The outcomesand minutesof this meeting could be added tofuture bOPNsubmission for no questions on 

GRASto FDA

5 14 June 2019

Next step

TOXICOLOGY AND SAFETY

• Pre-clinical in vitro and animal safety study (Kvistgaard et al 2014) showed no adverse events on non - immune parameters

• Clinical safety study ( Lönnerdalet al 2016) that looked into immune markers, infection rates and 

vaccination response showed reduction in pyrexia, no changes in vaccination response and showed 

immune marker levels typically observed infant formulas

• Lymphocyte subsets looked in a subset of population from the clinical study showed (West et al 2017

controlled T-cell activation that is not different from breast fed infants showing bOPN’srole is to bridge 

the gap in formula fed infants

• New information to be discussed (Jiang and Lönnerdal2019) would show that even in formula fed 

infants there is high circulating levels of hOPNthat may contribute to immunological well being of all 

infants

7 14 June 2019

-What we know today including new information
PLASMA OPN CONCENTRATIONS 

8 14 June 2019 Schack et al (2009), J. Dairy Sc . 92, 5378-5385.
MEASURINGOPN INPLASMA- STUDYDESIGN

• Samples:

• Plasma was isolated from blood samples obtained from 1 -, 4-, and 6-month-old infants in a clinical trial in 

China (Lönnerdalet al 2016)

• Infants were either exclusively breast-fed (BF group) or fed one of the following formulas: unsupplemented

formula (F0 group), formula supplemented with 65 mg/L bOPN(F65 group), or formula supplemented with 

130 mg/L bOPN(F130 group) up to 6 months of age

• OPN assays:

• Human OPN (hOPN) concentrations in breast milk and in all infant plasma samples were measured by an ELIS

kit (Human OsteopontinDuoSetELISA, R&D Systems, Minneapolis, MN) following the manufacturer’s 

instructions

• Bovine OPN was also measured in plasma samples by an ELISA kit (Bovine OsteopontinELISA,LifeSpan

BioSciences, Seattle, WA) following the manufacturer’s instructions

• There was no cross-reactivity between the two kits; i.e., theOPNELISA did not recognize bOPNnor did the 

bOPNELISA recognize hOPN

9 14 June 2019 Jiang and Lonnerdal,(2019),Pediatr. Res. 85(4), 502-505
CIRCULATING HOPINN BREAST AND FORMULA FED INFANTS

10 14 June 2019 Jiang and Lonnerdal,(2019),Pediatr. Res. 85(4), 502-505

MEASURINGHOPNANDBOPNIN US INFANTS TO ADDRESS 

LONG TERM CONCERNS

• Need guidance from FDA on what is long term to assess safety of bOPN

Study Possibilities:

• Recruit infants and collect random blood sampling from 3 months to 12 months to measure endogeno

hOPNandbOPNlevels (breast fed at least for first 4 months and exclusively formula fed infants)

• Have a long term follow-up to the just completed Building Block Nutritionals Study (now the infants are - 2 year old)

• In a subset of subjects, Collect health history, one blood sample to measure hOPN,bOPNand possible 

vaccination response

• Question:

• Does demonstration of high levels of endogenous hOPNin formula fed infants alleviate FDA concern that bOPNis

not the cause for long term immune programming effects?

• To date, our pre-clinical safety study on bOPNwith conventional non-immune end points did not show any toxicity

• All interventions of exogenous bOPNin animal models of inflammation and cancer have yielded only beneficial outcome

• Given the endogenous levels of OPN in circulation in early life, it will be difficult to demonstrate specific bOPNrelated

effects in animal models and human setting

12 14 June 2019
TOXICOLOGICAL AND SAFETY

• High levels of OPN both in breast milk and in circulation indicate that OPN may have a key physiolog

role in infants

• High circulating hOPNlevels are noted in formula fed infants at 1, 4 and 6 months. bOPNlevels

measured in plasma at the same time points are at least 20 fold less compared to hOPN

• Data indicates that any immune programming/ inflammatory / safety concern for infants will predominantly 

result from high hOPNlevels compared to bOPNlevels in formula fed infants

• Immune effects outlined in scientific memo mainly discussed hOPNas the cause for pathological and 

autoimmune diseases

• Supplementation of bOPNin infant formula may help infants in two possible ways

• Local effects in gastrointestinal tract

• DietarybOPNpromotes endogenous circulating levels of hOPN. This phenomena was also noted in a mouse study

• It will be difficult to dissect the endogenous versus exogenous effect of OPN in any pre-clinical model

13 14 June 2019

Conclusion
ADDITIONAL TOXICOLOGY STUDIES

• Two different toxicology models have been explored

• A mini pig model with dosing and immune assessment

• A rat model with a virus challenge

• Both models have pros and cons

• Mini Pigs:

• Post birth dosing, physiologically close to human

• Questionable if data can be translated to human response

• Rat Model

• Virus challenge model to measure DIT

• Rat physiology is not similar to human

• Immunological response and pathway may differ

14 14 June 2019

HOPNIN HUMAN MILK

• OPN is like any other molecule in human milk that varies from early lactation to late lactation on 

levels and may be on post-translational modifications (Froehlich et al 2011)

• Post-translational modifications are dependent on the enzyme activity in mammary gland and 

may vary in the same individual mother day to day

• Given the structural homology and conserved phosphorylation sites, the phosphorylation sites 

are similar between human and bovine OPN (Christensen et al 2005)

• The minor in vitrobinding differences noted in Christensen and Sorensen 2014 is an 

undigested bOPN and the difference is not seen upon thrombin cleavage of bOPN(a likely 

scenario in humans)

14 June 2019 Froehlich et al (2011), Anal Biochem 408, 136-146

Christensen et al (2005) Biochem J 390, 285-292

Christensen and Sorensen (2014)J Dairy Sci 97, 136-146

16

Structural homology between bovine and human OPN
HOPNIN HUMANM ILK

• When consulted with experts, it was argued that there are more functional and, to a certain 

degree, structural differences between hOPNin breast milk compared to hOPNin extracellular 

secretions (urine) and in organs

• We also looked for FDA guidance targeted for approval of generic biological molecules. FDA 

acknowledges minor structural differences in post translational modifications exist and the 

molecules will be considered similar, if these structural variations do not result in adverse 

functional differences (“Guidance to Industry: Scientific Considerations in Demonstrating Biosimilarity to a Reference product” was used for guidance)

• bOPNhas not been shown to have any different biological activity in direct comparison to hOPN

17 14 June 2019

Structural homology between bovine and human OPN
HOPNIN HUMANMILK

• hOPNandbOPNare substantially homologous at amino acid levels, functional binding sequences 

areconserved, and relatively similar post translational modifications

• The minor differences noted in sequence do not result in adverse physiological consequences ( Rittlinget

al2014)

• Based on recent observations (Jiang and Lönnerdal2019), in infants at any given time point there is 20

fold or higherhOPNin circulation compared tobOPN. Any physiological consequences during infancy will 

beattributed to the predominanthOPNin circulation

• Given the variability in human milk post translational modifications (similar to other bioactive proteins 

likelactoferrin, Bile Salt Stimulated Lipase), it is possible to detect minor variabilities based on modern 

daytechnology

18 14 June 2019

CONCLUSIONS ON HOMOLOGY


HISTORY OF CONSUMPTION

• Besides the launch in China from 2009 -2012, and in Korea in 2008-current, where no safety concerns 

have been raised, OPN have been launched in several countries since our GRAS were submitted to F

• Launched in stage 1 and stage 2 in EU in Fall 2018

• Launched in stage 1 and stage 2 China in spring 2018

• Potentially exposed to 1.000.000 babies through commercially available products, no safety concerns

raised

• Used in a clinical trial in the United States, providing an experimentan infant formula enriched in OPN

to 128 babies, no safety concerns observed

21 14 June 2019

CONCLUSION

• We would like to thank FDA for the diligence in looking into our OPN self -affirmed GRAS dossier and 

comments on the scientific aspects and bioactive potential

• It certainly challenged us to look the molecule differently and discuss with pediatric, immunological and OPN

structural experts to further understand the functional and structural similarities/ differences between hOPN

andbOPN

• We at Arla Foods Ingredients, and many scientific experts we spoke to believe that OPN at physiologic

level would benefit all infants

• We appreciate FDA’s continued support and guidance on this topic

23 14 June 2019
DISCUSSIONPOINTSAND NEXT STEPS

• ArlaFoods Ingredients would like to have guidance on

• General Recognition:

• What additional measures would ensure general recognition of safety of bOPNingredient?

• Canwe assemble around tablepanelwith pediatric, immunology andOPNstructural experts to discuss short and 

long-term safety that would also include FDA scientists?

• Toxicology and Safety:

• Given information presented on high levels of endogenous hOPNearly in infancy, is there a reason to consider 

additional animal safety studies ?

• Does measurement ofhOPNin USAinfantsadd value toexistingknowledge and evaluation ofour application?

• Homology between hOPNand bOPNand hOPNlevels in milk:

• Experts acknowledged that differences in post translational modifications are noted within lactation period and 

between breast milk and other secretions, the minor differences noted may not be functionally consequential. 

Human milk average for other bioactive ingredients have been used by industry to fortify infant formula

• Given this complexity, is there anything we could do that would help FDA’s concerns?

24 14 June 2019
BACK-UP SLIDES

25 14 June 2019
DEVELOPMENTAL COMPARISO–NH UMAN VS PIG

26 14 June 2019
(b) (4)
(b) (4)
(b) (4)
(b) (4)
(b) (4)
ARLA’SPREFERENCE

• Arlawould prefer the mini pig model as it is more physiological, measures all key parameters at various

time points, would give us a direction on long term consequences of bOPNon top of endogenous 

pOPNfeeding.

• It may not accurately reflect human conditions

• Very expensive to conduct (current estimates 1.5 to 2 million).

• Still would not dissect the effects between endogenous pOPNversus exogenous bOPN

• Would like to seek guidance from FDA based on March 1 st 2018 and June 29th 2018 memos.

• Do we need additional pre-clinical safety studies?

• Consensus and general recognition of bOPNsafety by pediatric experts to precede this activity?

• Does it have to be a GLP safety or well controlled animal study that answers the question for eventual FDA 

approval?

32 14 June 2019