Novel Food Information – 2'-fucosyllactose (2'-FL) from Escherichia coli BL21 (DE3) Strain #1540

Health Canada has notified Jennewein Biotechnologie, GmbH, that it has no objection to the use of 2'-fucosyllactose (2'-FL) derived from a genetically modified strain of E. coli BL21 (DE3), "the production strain" or "the strain", as an ingredient for infant formula. The Department conducted a comprehensive assessment of this oligosaccharide according to its Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based upon internationally accepted principles for establishing the safety of foods with novel traits.

Background:

The following provides a summary of the notification from Jennewein Biotechnologie GmbH, and the evaluation by Health Canada. This document contains no confidential business information.

1. Introduction

Jennewein Biotechnologie, GmbH, has developed 2'-fucosyllactose from a genetically modified E. coli BL21 (DE3) strain, using recombinant-DNA techniques. The bacterially synthesized 2'-FL has a purity of ≥ 90% and is expected to be used in infant formula for term infants to a maximum use level of 1.2 g 2'-FL per litre of formula. Jennewein specified that the use level would be 1.0 g +/- 0.2 g 2'-FL per litre of infant formula, which would accommodate manufacturing tolerances and ensure compliance. 2'-FL is a naturally occurring oligosaccharide in human milk composed of L-fucose and D-lactose (a disaccharide of D- galactose and D- glucose). The bacterially synthesized 2'-FL produced by Jennewein was confirmed to be chemically equivalent to 2'-FL isolated from human milk. 2'-FL may support infant health as a prebiotic substrate for commensal bacteria as well as functioning as a modulator of the immune system. This evaluation did not include an assessment of any potential claims that could be made about 2'-FL. Genetic modifications were made to E. coli BL21 (DE3) for the purpose of inserting the genes involved in the entire 2'-FL biosynthetic pathway. The strain was engineered to produce 2'-FL from lactose as a substrate, and to secrete 2'-FL into the growth medium.

The safety assessment performed by Food Directorate scientific evaluators was conducted according to Health Canada's Guidelines for the Safety Assessment of Novel Foods. These guidelines are based on harmonization efforts with other regulatory authorities and reflect international approach in this area (e.g., Codex Alimentarius). The safety assessment considered: how the 2'-FL was developed; how the composition and nutritional quality of the 2'-FL compared to human milk oligosaccharides (HMOs); and, what the potential is for the 2'-FL to be toxic or cause allergic reactions. Jennewein has provided data which demonstrates that the 2'-FL is safe to use in infant formula for term infants.

The Food Directorate has a legislated responsibility for the pre-market assessment of novel foods and novel food ingredients, as detailed in Division 28 of Part B of the Food and Drug Regulations (Novel Foods). As this 2'-FL is produced by a genetically modified bacterium, it is considered to be a novel food under the following part of the definition of novel foods:

"c) a food that is derived from a plant, animal, or microorganism that has been genetically modified such that

iii. One or more characteristics of the plant, animal, or microorganism no longer fall within the anticipated range for that plant, animal, or microorganism."

2. Development of the Modified Microorganism

The production strain was derived from the well-characterized E. coli BL21 (DE3) parent strain which was genetically modified to introduce several gene constructs. The introduction of these constructs result in the production strain containing the complete biosynthetic pathway for 2'-FL. As a result, this strain is capable of converting lactose into 2'-FL, and exporting the 2'-FL into the medium. The strain was also subjected to chemical mutagenesis to improve 2'-FL yield. The production strain is not present in the final 2'-FL product.

The insertion of the 2'-FL biosynthetic pathway into the production strain involved several integration steps.

Synthesis of 2'-FL requires two co-substrates: lactose and β-GDP-L-fucose (GDP-Fucose). Lactose is imported from the growth medium into the cell by a permease encoded by lacY. GDP-Fucose is derived from the endogenous fucose biosynthesis pathway. The substrates are then linked by the enzyme 2-fucosyltransferase encoded by wbgL, resulting in 2'-FL.

3. Characterization of the Modified Microorganism

Molecular characterization of the strain was performed using whole genome sequencing (WGS) and Sanger sequencing. The number of copies of each construct which was integrated into the genome was determined as well as the locus of each insertion. Each construct was stably integrated into the genome as was demonstrated by sequencing the whole genome of the production strain intermediates as well as the final strain. Insertional scar sequences were also determined and a closed consensus sequence was achieved for the production strain. No genomic rearrangements were observed and all constructs remained in their expected insertion sites.

An open reading frame analysis and single nucleotide polymorphism (SNP) analysis was performed for all insertions in the strain. Any observed amino acid changes or frameshift mutations were reviewed and the petitioner concluded that any unintended modifications did not significantly affect the expression of 2'-FL. The petitioner indicated that the potential for unintended protein expression would not be a safety concern since the final 2'-FL product does not contain any proteins. This lack of protein in the final product was demonstrated for five batches of the 2'-FL.

The petitioner provided plasmid maps for the vectors which were used in the production of the strain. The vector backbone sequences were added to the reference genome during whole genome sequence analysis in order to verify that no vector backbone DNA was transferred to the production strain. Mapping the Illumina reads showed no sequence coverage for the plasmid-specific fragments which confirmed the absence of vector backbone sequences in the production strain.

The list of donor organisms used in the genetic engineering of the production strain was reviewed and deemed to be acceptable. All introduced genes do not pose a safety concern regarding the unintended transfer of unknown foreign DNA to the production strain.

4. Product Information

The 2'-FL is produced via a fed-batch axenic fermentation under sterile conditions at a facility compliant with current Good Manufacturing Practices (cGMP). Batch fermentation is performed in a chemically-defined, salt-based minimal medium which does not contain inhibitors or antibiotics. Once a certain level of 2'-FL is secreted into the medium, the culture supernatant containing 2'-FL is collected and the microbial biomass is removed by filtration. The filtrate containing 2'-FL is purified by a series of ion exchangers to remove proteins, DNA, organic acids and inorganic salts. The filtrate is then concentrated, decoloured, and purified. After filtration steps (including a 3 kDa filter to remove endotoxins), the final solution is packaged as a liquid concentrate containing 45 % (w/v) 2'-FL in water or it is spray-dried to produce a solid white powder.

5. Dietary Exposure

Human Milk Oligosaccharide (HMO) concentrations range from 20 g/L in the colostrum to 12 g/L in breast milk (Thurl et al. 2010). Breastfed infants would therefore be exposed to up to 12 g of HMO per day. As bovine milk contains less oligosaccharides than human milk, and specifically less 2'-FL, infants on unsupplemented bovine milk formulas are currently exposed to less oligosacharides and 2'-FL than human milk fed infants.

The dietary exposure for infants consuming 2'-FL in infant formula would be lower or consistent with current exposure for breastfed infants, as the proposed rate of addition of 2'-FL in formulas is lower than that in breast milk. Breast milk contains on average 2.38 g of 2'-FL/L (Erney et al. 2000), which is about two fold higher than the petitioner's proposed maximum specification of 1.2 g of 2'-FL/L (includes overage).

6. Nutrition

In infants, HMOs are transported into the lower parts of the intestine and they are either metabolized or excreted in the feces. Marcobal and Sonnenburg (2012) indicate that while infants are able to metabolize lactose, they lack the glycoside hydrolases in the small intestine as well as the necessary intestinal membrane transporters to metabolize HMOs. Two major groups of commensal bacteria in the infant gut, specifically Bifidobacterium and Bacteroides, are able to consume the oligosaccharides. The data reviewed suggests that 2'-FL acts similarly to other fermentable oligosaccharides in that it is metabolized to short-chain fatty acids or excreted. It is not expected that 2'-FL will have a negative effect on the absorption of nutrients.

A clinical growth and tolerance study of chemically synthesized 2'-FL in infants from soon after birth to 4 months of age carried out by Marriage et al. (2015) was reviewed. It had two test groups. One group consumed 0.2 g/L of 2'-FL and a second group received 1.0 g/L 2'-FL, along with GOS such that the total content of dietary fibre was 2.4.g/L. There was no addition of dietary fibres to the control group. A human milk fed group was included as a reference group. There were no significant differences among any groups for weight, length, or head circumference growth. All of the formulas were well tolerated and comparable for average stool consistency, number of stools per day, and percent of feedings associated with spitting up or vomit. This study provides evidence of the safety and tolerance of 2'-FL in infants. The Bureau of Chemical Safety (BCS) concluded that the chemically synthesized form was chemically equivalent to the fermented one by Jennewein using GM E.coli. Therefore, Jennewein's 2'-FL would demonstrate growth and tolerance similar to human milk fed infants.

There are no nutritional safety concerns with the use of Jennewein 2'-FL in term infant formula at a maximum level up to 1.2 g per litre of formula as fed (inclusive of overage). Further assessment of the proposed new infant formula with 2'-FL will be conducted when the premarket notification is submitted, as per Division 25 of the Food and Drug Regulations.

7. Microbiology

The host strain E. coli BL21 (DE3) is a well-characterized, non-pathogenic strain which has been used extensively in the biopharmaceutical industry as the host for expression of recombinant proteins and has been used in the manufacture of at least one food ingredient (USFDA GRN 485, β-galactosidase). E. coli BL21 (DE3) does not possess the fertility plasmid indicating that it is incapable of transferring its DNA to other organisms. It is also incapable of colonizing the human gastrointestinal system.

E. coli BL21 (DE3) does not express long-chain lipopolysaccharides and the endotoxins it produces are not toxic as was confirmed by acute oral toxicity tests in mice (reviewed by PTAS, section H). The 2'-FL manufacturing process includes a filtration step using a 3 kDa membrane which would remove any potential endotoxins. The endotoxin specification (≤ 300 EU/g) and validated test method used for testing each lot of 2'-FL powder prior to release were deemed to be acceptable. There are no safety concerns regarding endotoxins in the final 2'-FL product.

The production strain possesses antimicrobial resistance genes integrated into its genome as a result of the strain development process. However, these antimicrobial resistance genes do not pose a safety concern since the final 2'-FL product is devoid of transgenic DNA as was demonstrated by real-time qPCR analysis of five batches of 2'-FL product. The qPCR results confirmed the absence of the respective resistance genes in the final 2'-FL product. In addition, there are no concerns regarding the expression products of the antimicrobial resistance genes since the final 2'-FL product is devoid of proteins as was demonstrated by testing five batches of 2'-FL product.

The microbiological specifications and validated testing methods were provided for the final 2'-FL powder as well as for the liquid concentrate. Certificates of analysis were provided for the 2'-FL product during the product development stage as well as for the final product to be released for commercial use. The specifications, methods and test results were all deemed to be acceptable. The specifications included all of the microorganisms and microbial toxins relevant to infant formula. The petitioner successfully demonstrated the absence of the production strain in the final product. There are no concerns regarding the specifications or microbial safety of the 2'-FL product.

8. Chemistry

The petitioner provided specifications for their 2'-fucosyllactose (2'-FL; CAS RN 41263-94-9) product, including limits on trace elements and residual sugars, that they have established in the absence of specifications for this substance set out in the Food Chemicals Codex or in the Combined Compendium of Food Additives Specifications prepared by the Joint FAO/WHO Expert Committee on Food Additives (JECFA).

2'-FL produced by fermentation of the production strain contains small amounts of other carbohydrates that were not removed during the various purification steps in the manufacturing process. These carbohydrates are sugars that occur naturally in human milk, or in the case of fucosylgalactose, as a breakdown product in the human body.

Results of analyses of the toxic trace elements arsenic, cadmium, lead, and mercury were provided by the petitioner, demonstrating that 2'-FL consistently meets the limits for these trace elements set out in the petitioner's specifications, which are deemed to be suitably low. The addition of 2'-FL to the foods specified by the petitioner at the proposed level of use would contribute negligible amounts of lead, arsenic, mercury, and cadmium to infant formula in which it is used.

A list of materials used in the production of 2'-FL by fermentation of the production strain was included in the submission. The acceptability of particular uses of substances for a technological purpose in manufacturing the 2'-FL is separate from the acceptability of the food itself. More generally, the petitioner is responsible for ensuring that the presence of contaminants in the product and the use of any substance in the manufacture of 2'-FL does not result in a violation of section 4 of the Food and Drugs Act. In this regard, the petitioner noted that all of the materials used in the production of 2'-FL are of food grade.

The petitioner provided data to sufficiently demonstrate that 2'-FL produced by fermentation of the production strain is chemically equivalent to 2'-FL isolated from human milk and the 2'-FL obtained from chemical synthesis that was used in clinical growth and tolerance studies which were evaluated as part of the submission. There are no chemical safety concerns with the proposed level of use of 2'-FL produced by the strain in infant formula for term infants.

9. Toxicology

The production organism is not expected to contain or produce a toxic substance since the parent strain E. coli BL21 (DE3) is used for the manufacture of pharmaceutical preparations that can be injected into patients and is not associated with any toxicological effect.

The presence of the E. coli endotoxin, lipopolysaccharide (LPS), was assessed in mice. Animals were administered an acute oral dose equal to 3.7 X 107 endotoxin units (EU)/kg bw of E. coli BL21 (DE3) LPS. No mortality, body weight differences or clinical signs of toxicity were observed over 14 days following treatment. No gross lesions or pathology was reported at necropsy. Under the conditions of this study, a dose of 3.7 X 107 EU /kg bw of E. coli BL21 (DE3) LPS did not produce toxicity in mice.

The specification shows that the 2'-FL preparation is tested for the absence of bacterial endotoxins (lipopolysaccharides; ≤ 300 EU/g of preparation; equal to ≤ 360 EU per L formula) and toxic by-products associated with molds (aflatoxins; < 0.025 µg/kg). The concentrations of these impurities are considered low enough not to pose a toxicological concern.

The 2'-FL is expected to be metabolized by gut bacteria into short-chain fatty acids or excreted intact in the feces. Less than 5% of the 2'-FL may be absorbed, intact, by the infant. The absorption, distribution, metabolism and excretion profile of the 2'-FL preparation is expected to be typical to that of other oligosaccharides, and is not expected to pose a toxicological concern.

A subchronic (90-day) oral toxicity study was performed with ten male and ten female Sprague-Dawley rats. The study was conducted in accordance with OECD Guidelines (Test No. 408). The rats were provided 2'-FL at a constant dietary concentration of 0 or 10% in the diet. Male and female rats consumed an equivalent dose of 7.66 and 8.72 g/kg bw per day, respectively, for 13 weeks. Animals in the control groups were provided the basal diet. All animals survived to necropsy. Some statistically significant changes in food consumption (increase of 7% in treated males), and clinical chemistry (alkaline phosphatase; increase of 24% in treated females) were observed in 2'-FL treated rats when compared to rats given basal diet control. No historical control data was provided. However, these effects were not accompanied by changes in body weight, changes in liver weight or histology and were not observed consistently between genders; thus, these effects were not considered to be toxicologically relevant. A no observed adverse effect level (NOAEL) of 7.66 g/kg b.w. per day was determined for 2'-FL by the study authors.

Results from an in vitro bacterial reverse mutation assay (Ames test) showed that 2'-FL is not mutagenic in S. typhimurium (strains TA98, TA100, TA102, TA1535 and TA1537; 5 mg/plate; OECD test 471). Further 2'-FL provided at doses of up to 2 g/kg bw was not genotoxic to mouse immature erythrocytes, in vivo (OECD Test 474). Under the conditions of these studies, 2'-FL was not considered genotoxic.

The petitioner provided a tolerability study involving neonatal piglets provided 2'-FL fortified infant formula for a period of 3 weeks. The study was conducted according to several GLP standards. Four to eight animals per sex per group were treated with 0, 200, 500 or 2000 mg of Jennewein 2'-FL/ml formula on lactation day 2 (equal to a dose of 0.29 g/kg bw per day) until lactation day 23. All animals survived to necropsy. No clinical or physical changes were observed in the piglets that were attributed to the test substance. There were no differences in mean body weight, food consumption, or food efficiency between groups. Watery feces were observed in males and females of the high-, mid- and low-dose groups. Some animals in the high dose group showed a lack of appetite. However, the petitioner stated that the effect did not show a dose-response and the animals did not experience changes in growth or overall food intake. The authors concluded that these adverse effects were not toxicologically relevant. During necropsy, authors noted mild to moderate inflammation of the epithelium in the non-glandular portion of the stomach in two animals of the high-dose group and one female of the mid-dose group. The authors argued that the animals of the control group showed minimal and mild acute inflammation of the stomach as well (5 male/1 female and 1 female, respectively). The authors concluded that these microscopic findings were incidental, unrelated to test substance administration and are typical of swine of this age and strain. No toxicity was observed at the highest dose tested, 0.29 g/kg bw per day.

The petitioner provided a randomized, controlled, growth and tolerance study with healthy, full-term infants given chemically synthesized 2'-FL at concentrations of either 0.2 g/L or 1 g/L (N = 101-109 infants per group) for 119 days. Infants in the control groups were fed formula containing galactooligosaccharides (2.4 g of GOS/L; N = 189) or maternal breast milk (N = 106; ~ 2 g of 2'-FL/L milk). The human study provided infants with chemically synthesized 2'-FL at concentrations less than the intended level of 2'-FL inclusion (1.2 g/L) by the petitioner. Infants given up to 1 g/L of 2'-FL were observed to tolerate the infant formula and grew normally. The incidences of infection appeared to be statistically significantly higher in the high-dose group; however, a clear dose-response was absent. This adverse effect was considered incidental and not treatment-related. In general, adverse effects and gastrointestinal symptoms were similar between infants in the treatment and control groups. Results from this study suggest that a relatively pure 2'-FL preparation would be well tolerated by infants when consumed with infant formula at 1 g/L.

On a body weight basis, new born infants are the greatest consumers of the foods that could contain the 2'-FL. The 90th percentile of consumers in this subpopulation (newborn infants consuming up to 260 ml/kg bw per day of infant formula) could be exposed to 0.31 g of 2'-FL/kg bw per day.

The 2'-FL was calculated to have a margin of exposure (MOE) that is approximately 25-fold less than the NOAEL reported in the 90-day subchronic oral toxicity study (the highest dose tested in male rats). The margin of exposure is considered sufficient from a safety perspective, based on the absence of toxicologically relevant adverse effects in rats, piglets and human clinical trials. Based on the evidence provided, the 2'-FL produced by the production strain will not pose a safety concern, from a toxicological perspective, when provided at up to 1.2 g/L in infant formula.

10. Allergenicity

2'-FL is a human milk oligosaccharide that is not proteinaceous and is not expected to elicit an IgE mediated allergic response in consumers. The production strain was modified to express a number of transgenes; however, the petitioner stated that the preparation is devoid of proteins due to the use of i exchangers during the filtration process, as determined by a modified version of the Bradford assay (Nanoquant; ≤ 100 µg protein/g preparation). As the preparation is essentially devoid of proteins, including potential protein allergens, the 2'-FL preparation will not pose a safety concern to consumers, from an allergy perspective.

Conclusion:

Health Canada's review of the information presented in support of the use of 2'-FL derived from the production strain in infant formula does not raise concerns related to food safety.
Health Canada's opinion refers only to the use of 2'-FL as an ingredient in infant formula.

This Novel Food Information document has been prepared to summarize the opinion regarding the subject product provided by the Food Directorate, Health Products and Food Branch, Health Canada. This opinion is based upon the comprehensive review of information submitted by the petitioner according to the Guidelines for the Safety Assessment of Novel Foods.

(Également disponible en français)

For further information, please contact:

Health Canada
Novel Food Section
Food Directorate
Health Products and Food Branch
251 Sir Frederick Banting Driveway
PL2204E
Ottawa, Ontario, K1A 0K9