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Compositional guideline: Lacto-N-tetraose (LNT)

20 January 2022

Name of the ingredient

Lacto-N-tetraose (AAN #140330)

Definition of the ingredient

Lacto-N-tetraose is a purified, white to off-white amorphous powder produced by a microbial process that involves the genetically modified strain, Escherichia coli K-12 DH1.

Molecular formula: C26H45NO21

CAS Number: 14116-68-8

Structure of Lacto-N-tetraose

Table 1. Ingredient specific requirements
Test Method reference Acceptance criteria
Description
Appearance ISO 6658 Powder or agglomerates, powder with agglomerates
Colour ISO 6658 White, white to off-white, off-white
Characteristics
pH in 5% solution (20°C) Ph. Eur. 2.2.3 4.0 to 6.0
Water Karl-Fischer ≤ 6.0 % w/w
Identification
NMR Strecker et al. 1989[1] Recorded NMR spectra must be in full agreement with the structure of LNT
MS

Chai et al., 2001[2]

Pfenninger et al., 2002a[3]

Pfenninger et al., 2002a[4]

Base molecular ion peak matches molecular weight(s)
HPLC HPLC[5] The retention time of the main component corresponds to the retention time of the LNT standard ±3%
HPAEC HPAEC[6] The retention time of the main component corresponds to the retention time of the LNT standard ±3%
Assay
Lacto-N-tetraose
(water-free)
HPLC[5] and HPAEC[6] ≥70.0% w/w
D-Lactose HPAEC[6] ≤12.0% w/w
Lacto-N-triose II HPAEC[6] ≤10.0% w/w
para-Lacto-N-hexaose-2 HPAEC[6] ≤3.5% w/w
Lacto-N-tetraose fructose isomer HPLC[7] ≤1.0% w/w
Sum of other carbohydrates HPAEC[6] ≤5.0% w/w
Human-identical milk saccharides (water-free)a Calculated theoretically as the summation of percentages of lacto-N-tetraose, lactose, and lacto-NN-triose II. ≥90.0% w/w
Notes
aHuman-identical milk saccharides is defined here as the sum of lacto-N-tetraose, lactose, and lacto-N-triose II
Table 2. Incidental constituents
Test Method reference Acceptance criteria
Incidental metals and non-metals
Lead EN 13805:2002; EPA 6020A:2007 ≤ 0.1 mg/kg
Other organic or inorganic impurities or toxins
Ash, sulphated Ph Eur 2.4.14 ≤ 0.5% w/w
Residual Proteins by Bradford Assay Bradford Assay ≤ 0.01% w/w
Residual Endotoxins Ph Eur 2.6.14 ≤ 10 E.U./mg
Microbiology
aWhile substance manufacturers are encouraged to include limits for objectionable microorganisms, it is the product into which those substances are formulated that is subject to a legally binding set of criteria. The Therapeutic Goods Order No. 100 'Microbiological Standards for Medicines' mandates that any finished product that contains the ingredient, alone or in combination with other ingredients, must comply with the microbial acceptance criteria set by Clause 11 of the Order

Footnotes

Key to abbreviations

EU = Endotoxin units

EN = European norms

EPA = United States Environmental Protection Agency

HPLC = High-pressure liquid chromatography

HPAEC = High performance anion exchange chromatography

ISO = International organisation for standardisation

MS = Mass spectrometry

NMR = Nuclear magnetic resonance

Ph Eur = European Pharmacopoeia


Version history
Version Description of change Effective date
1.0 Initial 17/11/2021