Eternity ™

Chemically Stable Merged Organic/Inorganic Silica Material for Purification of Peptides and Proteins in Reversed Phase Preparative ChromatographyIntroductionSilica-based chromatography materials are usually stable in the range of pH 2 to pH 8, thus limiting the possibilities to use high pH to achieve high selectivity, and subsequently high purity and yield. An extension of the pH window upwards can in many cases improve the selectivity significantly, and thereby allowing for substantial improvements in loading capacity, and productivity. Here we present a material developed for preparative scale chromatography, with increased chemical stability of the silica. An organosilane was merged into the silica surface to create an interfacial gradient, and the resulting organic/inorganic silica material combines improved chemical stability with maintaining superior mechanical stability and pore structure from the original silica matrix.Accelerated Chemical StabilityThe chemical stability of the material was tested for ~100 h and compared with classic silica by monitoring the change in k’ for Phenanthrene at elevated temperature (60°C) using 10 mMcarbonate buffer at pH 10.5.

To further show the stability of the material, chromatograms from the analytical injections for EternityXT 10-C18 are presented below with 12 hour hydrolysis with carbonate buffer between each analysis.

Cleaning in PlaceHydrophobic impurities can be eluted with 100% CH2Cl2 or by washing with 0.2 M acetic acid with some organic modifier. If these treatments fail, alkaline wash can be performed, but it is very aggressive to the phase, and will reduce the column life time. The new organosilane reinforced silica phases were subjected to sodium hydroxide concentrations between 0.1 and 1.0 M and tested for efficiency and anti-depressants. Anti-depressants are very sensitive to changes in the stationary phase and will shift in retention times as shown for standard C18 material in grey trace.

Fredrik Limé, Per Jageland (per.jageland@akzonobel.com)AkzoNobel, Separation Products, SE-445 80 Bohus, Sweden

PUBPxtcl

Cleaning in Place

Peptide Purification at High pHA ~3.5 kDa peptide was difficult to purify under conditions normally used with classic silica material, but by using organosilane reinforced silica, EternityXT 10-C18, the purification could be done at higher pH.

EternityXT 300 Å for Protein ChromatographyThe applicability of the EternityXT concept for proteins was tested by synthesizing an EternityXT C4 on a 10 μm 300 Å matrix, and running a variety of protein samples.

ConclusionsEternityXT organosilane reinforced silica functionalized with C4, C8 and C18 shows high chemical resistance under harsh alkaline conditions while maintaining the high performance. The stability at high pH significantly expands the working range of the material.

ConditionsBasic hydrolysis: acetonitrile / 10 mM ammonium carbonate pH 10.5 [10/90], 60°C, 0.2 mL/minAnalysis: acetonitrile / water [70/30], 25°C, 1.0 mL/minSubstance: phenanthrene

ConditionsBasic hydrolysis: acetonitrile / 10 mM ammonium carbonate pH 10.5 [10/90], 60°C, 0.2 mL/minAnalysis: acetonitrile / water [70/30], 25°C, 1.0 mL/minSubstance: sodium nitrate, dimethyl phtalate, toluene, biphenyl, phenanthrene

Bottom trace represents new column, then after 0.1, 0.5 and 1.0 M NaOHa) Substances: 1: dimethyl phtalate, 2: toluene, 3: biphenyl, 4: phenanthreneMobile phase: acetonitrile/water [70/30] Flow rate: 0.7 mL/min, Detection: UV @ 254 nmb) Substances : 1: sodium nitrate, 2: nortriptyline, 3: toluene, 4: amitriptylineMobile phase : methanol/20 mM KH2PO4 pH6 [80/20], Flow rate: 1.0 mL/min ,Detection: 215 nm

Reconstructed elution profile

Reconstructed elution profile

Pooled fractions at pH 8.0

Pooled fractions at pH 9.5

TRIS buffer pH 8.0

NH4HCO3 buffer pH 9.5

Purity: 96.1%

Recovery: 79.4%

Purity: 98.4%

Recovery: 98.0%

Competitor material was not possible to run using 1.0 M sodium hydroxide

Protein mix Crude EPO

ConditionsColumn: Kromasil EternityXT 300-10-C4, 4.6 x 250 mmFlow rate: 1 mL/min, Temperature: 25 °C, Detection: UV @ 220 nmMobile phase : acetonitrile / water + 0.1% TFA, Protein mix substances: 1: ribonuclease A, 2: cytochrome C, 3: lysozyme, 4: BSA, 5: myoglobin Protein mix gradient: 0 min: 25%, 30 min: 50% acetonitrileCrude EPO gradient: 0 min: 5%, 30 min: 15% acetonitrile