AMT - Advanced Materials Technology / HALO Protein

HALO Protein

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HALO protein columns from AMT (Advanced Material Technology) are made of core-shell material and offer 400 Å and 1000 Å pore sizes for unrestricted pore access of large proteins. With these pore sizes and core-shell technology, ultra-fast and high-resolution separations can be achieved. The particles are available with a diameter of 2.7µm and 3.4µm and with ES-C18, C4 and diphenyl modifications.

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Technical Data

Phase	Modification	Particle size	Pore size	Endcapping	C-content	USP	Surface area	pH range	Tmax
HALO Protein ES-C18	C18	2.7 µm 3.4 µm	1000 Å 400 Å	+	1.4 %C 1.0 %C	L1	15 m²/g	1.0-8.0	90°C^a 40°C^b
HALO Protein C4	C4	2.7 µm 3.4 µm	1000 Å 400 Å	+	0.6 %C 0.4 %C	L26	15 m²/g	2.0-9.0	90°C^a 40°C^b
HALO Protein Diphenyl	Diphenlmethyl	2.7 µm 3.4 µm	1000 Å 400 Å	+	N/A 1.0 %C	L11	15 m²/g	2.0-9.0	90°C^a 40°C^b

a Low pH; b High pH

Applications

Higher resolution with HALO 400 Å diphenyl compared to FPP 300 Å diphenyl

HALO 400 Å diphenyl outperforms an FPP 300 Å diphenyl column with higher resolution and 2.7 times lower back pressure. The 400 Å pores allow better access to the bound phase.

TEST CONDITIONS

Columns:
HALO 400 Å Diphenyl, 3.4 μm, 2.1 x 150 mm
FPP 300 Å Diphenyl, 1.8 μm, 2.1 x 150 mm

Flow rate: 0.2 mL/min
Temperature: 60 °C
Injection volume: 2 μL of 2 mg/mL denosumab
Instrument: Shimadzu Nexera
Detection: PDA at 220 nm
Mobile Phase A: 88/10/2 Water/ACN/n-Propanol + 0.1% DFA
Mobile Phase B: 70/20/10 n-Propanol/ACN/Water + 0.1% DFA
Gradient: 18-28% B in 20 min

High temperature comparison of the HALO 1000 Å phases

Three bound phases (C4, ES-C18 and diphenyl) are now available on surface porous HALO 1000 Å particles. Each phase offers unique selectivity for mAbs, as shown in this high temperature separation of trastuzumab.

TEST CONDITIONS

Columns:
HALO 1000 Å C4, 2.7 μm, 2.1 x 150 mm
HALO 1000 Å ES-C18, 2.7 μm, 2.1 x 150 mm
HALO 1000 Å Diphenyl, 2.7 μm, 2.1 x 150 mm

Flow rate: 0.4 mL/min
Temperature: 80 °C
Injection volume: 2 μL with 2 mg/mL
Sample: trastuzumab in water/0.1% TFA
Instrument: Shimadzu Nexera
Detection: PDA at 280 nm
Mobile Phase A: Water/0.1% TFA
Mobile Phase B: ACN/0.1% TFA
Gradient:
32-40% B in 16 min (C4 und ES-C18)
32-38% B in 12 min (Diphenyl)

Comparison of HALO 1000 Å Diphenyl with the competitor product PolyPhenyl at 40 °C

HALO 1000 Å diphenyl shows improved resolution, retention and peak area compared to a competitor column of 450 Å SPPs with polyphenyl phase. The increased retention clearly shows the advantage of unhindered large pore access to the bound phase. The HALO diphenyl phase can also be operated at 40 °C without any loss of peak area.