Biozen™ Biologics LC Columns
Biocompatible Flow Path with BioTi™ Hardware
4 Advanced Particle Platforms
All four of the Biozen particle platforms were individually designed and built by Phenomenex to take advantage of integral levels of performance, ruggedness, and reproducibility for protein characterization applications. Individually, each platform differs in the proprietary processing techniques used to control particle size and morphology.
8 Particle Chemistries
Inside Biozen BioTi Hardware
Inside Biozen Particle Platforms
Thermally Modified Fully Porous
Through a proprietary thermal processing series of steps, we eliminate micropores and further improve consistency, column efficiency, inertness, ruggedness, and reproducibility.
Core-Shell Technology
Using sol-gel processing techniques that incorporate nano structuring technology, a durable, homogeneous porous shell is grown on a solid silica core. This highly optimized process combined with industry-leading column packing technology, produces highly reproducible columns that generate extremely high efficiencies and sensitivity.
Monosized Polymeric Non-Porous
Meticulously controlled monosized particle technology secures incredible particle consistency that leads to improved and reliable efficiency. This innovative non-porous particle serves as the perfect backbone for complex ion-exchange chemistries.
Pore Controlled Technology
The Biozen dSEC columns are packed with low pore volume silica coupled with a proprietary hydrophilic diol-type bonded surface chemistry that prevents the silica surface from interacting with protein samples.
Peptide Mapping
Digested mAbs or ADCs typically include a large body of compounds that are crucial to understanding post translation modifications. So we designed two Biozen Peptide columns to offer highly useful and unique retention profiles. Each allows for fast and effective elution windows by utilizing either high-efficiency core-shell or thermally modified fully porous particles to gain sharper peaks, better peak capacities, and overall higher sensitivity.
Trastuzumab Biosimilar Peptide Map
Infliximab Biosimilar Peptide Map
Conditions for all columns:
Biozen 1.6 µm Peptide PS-C18
Biozen 2.6 µm Peptide XB-C18
B: 0.1 % Formic Acid in Acetonitrile
Aggregate Analysis
With mAb aggregate often at very low levels (<0.1% by peak area compared to a monomer) and fragment separation a requirement, adequate resolution and peak shape have become even more crucial method outcomes. To address this need, the robust set of bioZen SEC columns was developed with a combination of UHPLC efficiency and higher sensitivity to drive resolution and identification of even lower-level targets.
| Column: | Biozen 1.8 µm dSEC-2, 200 Å |
| Dimension: | 300 x 4.6 mm |
| Part No. : | 00H-4787-E0 |
| Mobile Phase : | 200 Potassium Phosphate + 250 mM KCl, pH 6.2 |
| Part No. : | 00H-4787-E0 |
| Flow Rate : | 0.35 mL/min |
| Injection Volume : | 10 µL |
| Temperature : | 25 °C |
| Detection : | UV @ 280 nm |
| Sample: | Various,10 mg/mL |
Charge Variant Analysis
Biozen WCX was crafted to consistently decipher between native protein variants that arise from PTMs within a therapeutics creation and development. The linear polycarboxylate chains grafted to monosized non-porous polymeric particles envelop and separate proteins from acidic and basic protein variants. With such a highly tuned and controlled manufacturing process, Biozen WCX media affords scientists a way to reproducibly characterize heterogeneity while taking advantage of excellent recovery through high particle inertness and bioinert titanium BioTi™ column hardware.
Trastuzumab (MES Salt Gradient)
| Column: | Biozen 6 μm WCX | |
| Dimension: | 250 x 4.6 mm | |
| Part No. : | 00G-4777-E0 | |
| Mobile Phase : | A: 20 mM MES (pH 5.6) B: 20 mM MES + 300 mM NaCl (pH 5.6) |
|
| Gradient : | Time(min) | %B |
| 0 | 15 | |
| 1 | 15 | |
| 31 | 45 | |
| 31.1 | 100 | |
| 34 | 100 | |
| 35 | 15 | |
| Flow Rate : | 1 mL/min | |
| Temperature : | 30 °C | |
| Detection : | UV @ 280 nm | |
| Sample: | Trastuzumab |
Trastuzumab (pH Gradient Buffer)
| Column: | Biozen 6 μm WCX | |
| Dimension: | 250 x 4.6 mm | |
| Part No. : | 00G-4777-E0 | |
| Mobile Phase : |
A: CX -1 (pH 5.6) pH Gradient Buffer* B: CX -1 (pH 10.2) pH Gradient Buffer* |
|
| Gradient : | Time(min) | %B |
| 0 | 0 | |
| 1 | 0 | |
| 21 | 100 | |
| 23 | 100 | |
| 24 | 0 | |
| Flow Rate : | 1 mL/min | |
| Temperature : | 30 °C | |
| Detection : | UV @ 280 nm | |
| Sample: | Trastuzumab |
Glycan Analysis
The unique selectivity of the Biozen Glycan was designed to provide higher-order separations of released and labeled glycans. With a 2.6 μm core-shell particle size, customers using either HPLC or UHPLC systems can draw upon a high-efficiency Biozen Glycan particle run at higher linear velocities to easily provide sharper peak shapes and faster elution windows without high UHPLC pressures. Under HILIC-FLR or HILIC-MS conditions, the bioZen Glycan excels with increased polar retention and selectivity.
| Column: | Biozen 2.6 µm Glycan | |
| Dimension: | 150 x 2.1 mm | |
| Part No. : | 00F-4773-AN | |
| Mobile Phase : | A: 100 mM Ammonium Formate, pH 4.5 B: Acetonitrile |
|
| Gradient : | Time(min) | %B |
| 0 | 78 | |
| 10 | 74.5 | |
| 24 | 72 | |
| 38.5 | 55.9 | |
| 38.6 | 40 | |
| 40.6 | 40 | |
| 40.7 | 78 | |
| 48 | 78 | |
| Flow Rate : | 0.5 mL/min | |
| Temperature : | 50 °C | |
| Detection : | FLD ex/em 285/345 nm | |
| Sample: | As noted |
Peptide Quantitation
When quantitating signature peptides from biological matrices, you need a sharp peak shape and sufficient retention of hydrophilic peptides to prevent any signal loss from matrix suppression regions. Both Biozen Peptide columns were developed to deliver excellent selectivity for even closely related peptides. Additionally, they build on this body of valuable characteristics with unique ways of delivering sharper peak shape for basic peptides; Biozen Peptide XB-C18 blocks secondary surface interactions via isobutyl side chains, while the Biozen Peptide PS-C18 contains a positively charged weak base that repels other basic species.
Conditions that are the same for both samples:
Column:
Biozen 3 µm Peptide PS-C18
Flow Rate:
0.5 mL/min
Dimensions
50 x 2.1 mm
Temperature:
22 °C
Part No.:
LC System:
ExionLC™ AD HPLC
Mobile Phase:
A: 0.1 % Formic Acid in Water
B: 0.1 % Formic Acid in Acetonitrile
Detection:
MS/MS
Detector:
SCIEX QTRAP® 5500
Flow Rate:
1.85 mL/min
Sample:
As noted
Intact and Fragment Analysis
Impurity profiling and characterization of intact biologic fragments is a challenging undertaking because of the need to identify very small differences between variants. Both bioZen Intact columns. contain skillfully manufactured large pore core-shell particles that provide narrower, taller peaks in conjunction with higher resolution between the target HC/LC, Fc/Fab, or isoforms.
Intact Trastuzumab at 70, 80, and 90 °C
Conditions for all columns:
| Column: | Biozen 3.6 µm Intact XB-C8 | |
| Dimension: | 250 x 4.6 mm | |
| Part No. : | 00F-4766-AN | |
| Mobile Phase : |
A: 0.1 % TFA in Water B: 0.1 % TFA in Acetonitrile |
|
| Gradient : | Time(min) | %B |
| 0 | 20 | |
| 1 | 20 | |
| 3 | 25 | |
| Flow Rate : | 0.5 mL/min | |
| Temperature : |
70 °C 80 °C 90 °C |
|
| Detection : | UV @ 280 nm | |
| Sample: | 1. Trastuzumab |
Infliximab F(ab)2
| Column: | Biozen 3.6 µm Intact XB-C8 | |
| Dimension: | 150 x 2.1 mm | |
| Part No. : | 00F-4766-AN | |
| Mobile Phase : |
A: 0.1 % TFA in Water B: 0.1 % TFA in Acetonitrile |
|
| Gradient : | Time(min) | %B |
| 0 | 20 | |
| 1 | 20 | |
| 13 | 60 | |
| Flow Rate : | 0.5 mL/min | |
| Temperature : | 80 ° | |
| Detection : | UV @ 280 nm | |
| Sample: | 1. Infliximab F(ab)2 |
Cetuximab
| Column: | Biozen 3.6 µm Intact XB-C8 | |
| Dimension: | 150 x 2.1 mm | |
| Part No. : | 00F-4766-AN | |
| Mobile Phase : |
A: 0.1 % TFA in Water B: 0.1 % TFA in Acetonitrile |
|
| Gradient : | Time(min) | %B |
| 0 | 20 | |
| 1 | 20 | |
| 13 | 45 | |
| Flow Rate : | 0.5 mL/min | |
| Temperature : | 80 ° | |
| Detection : | UV @ 280 nm | |
| Sample: | 1. Cetuximab |
Intact Mass
Intact mass can indicate not only relative abundance of glycoforms but also stability as degraded mAbs will not give a good charge envelope by ESI-MS. Intact mass with a high-resolution MS to identify PTMs, especially the relative abundance of glycoforms , combines extremely well with the fast run times and tight peak shapes provided by the Biozen Intact XB-C8 and bioZen WidePore C4.
Intact Mass of Trastuzumab Biosimilar using a Biozen Intact XB-C8 and SCIEX® X500B
| Column: | Biozen 3.6 µm Intact XB-C8 | |
| Dimension: | 150 x 2.1 mm | |
| Part No. : | 00F-4766-AN | |
| Mobile Phase : |
A: 0.1 % Formic Acid in Water B: 0.1 % Formic Acid in Acetonitrile / Isopropyl alcohol (50:50) |
|
| Gradient : | Time(min) | %B |
| 2.5 | 0 | |
| 10 | 65 | |
| 110.1 | 95 | |
| Flow Rate : | 0.3 mL/min | |
| Temperature : | 90 ° | |
| Detection : | QTOF (SCIEX X500B) | |
| Sample: | Trastuzumab |
Drug Antibody Ratio (DAR)
With a direct effect on efficacy and safety, conjugation for each ADC must be well understood. The Biozen Intact XB-C8 provides an excellent vehicle for determining drug load distribution and DAR for ADCs. Its large pore size allows intact ADCs to interact with a moderately retentive stationary phase while the core-shell particle supplies increased efficiency to deliver the required resolution between ADC species with differing drug loads.
Herceptin—vcMMAE using Biozen 3.6 µm Intact XB-C8
Herceptin—mcMMAF using Biozen 3.6 µm Intact XB-C8
BioTi versus Traditional Stainless Steel Hardware
Oligos can chelate to trace heavy metals in stainless steel column hardware, leading to poor recovery, inconsistent chromatography and problematic carryover. The Biozen Oligo bio-inert hardware provides greater sensitivity as well as improved recovery, demonstrating this column’s optimal utility for oligonucleotide characterization and quantitation.
Intact mAbs and Subunit Analysis
Impurity profiling and characterization of intact biologic fragments is a challenging undertaking because of the need to identify very small differences between variants. Biozen WidePore C4 columns contain skillfully manufactured large pore core-shell particles that provide narrower, taller peaks in conjunction with higher resolution between the target HC/LC, Fc/Fab, or isoforms and are ideal for large biologics to optimize analysis.
Diverse mAb Comparison with Chromatographic Performance Suitable for Intact MS Analysis
| Sample | Retention Time | Width @ 50% |
|---|---|---|
| Rituximab | 3.580 | 0.0233 |
| Infliximab Biosimilar | 3.606 | 0.0272 |
| Cetuximab | 3.696 | 0.0270 |
| Column: | Biozen 2.6 µm WidePore C4 | |
| Dimension: | 100 x 2.1 mm | |
| Part No. : | 00D-4786-AN | |
| Mobile Phase : |
A: 0.1 % Formic Acid in Water B: 0.1 % Formic Acid in Acetonitrile |
|
| Gradient : | Time(min) | %B |
| 0 | 10 | |
| 4 | 90 | |
| Flow Rate : | 0.3 mL/min | |
| Temperature : | 80 ° | |
| Detection : | UV @ 280 nm | |
| Sample: | mAbs, Various (1 mg/mL) |