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bioZen LC Protein and Peptide UHPLC/HPLC Columns: Phenomenex
bioZen UHPLC/ HPLC columns offer maximized recovery and sensitivity for biomolecules and offer optimized performance for biologic applications such as aggregate analysis, peptide quantification, peptide mapping, charge variant analysis. bioZen UHPLC/ HPLC selectivities include reversed phase ( C18, C8 and C4), ion-exchange (WCX), and GFC/SEC.
bioZen HPLC/UHPLC Columns
Explore a Bio State of Zen

With a new titanium BioTi™ biocompatible hardware to minimize priming, three particle platforms for optimal versatility and nine particle chemistries to maximize selectivity and sensitivity, bioZen UHPLC/HPLC columns are seamlessly designed to bring peace of mind to your analysis of biologics through:

  • Charge Variant Analysis
  • Aggregate Analysis
  • Peptide Mapping
  • Intact Mass
  • Intact and Fragment Analysis
  • Glycan Analysis
  • Peptide Quantitation
  • Drug Antibody Ratio (DAR)
  • Oligonucleotide Characterization

Phenomenex bioZen LC Columns for Biologics Analysis

bioZen HPLC short and long column
Biocompatible Flow Path with BioTi™ Hardware

Keep your mind at ease knowing that we’ve minimized the need for priming with a new titanium infused biocompatible hardware and frit that doesn’t interfere with protein or peptide integrity!


BioTi cutaway
3 Advanced Particle Platforms
Thermally Modified Fully Porous
Core-Shell Technology
Monosized Polymeric Non-Porous
All three 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.
9 Particle Chemistries
Intact
bioZen Intact XB-C8
3.6 µm
bioZen WidePore C4
2.6 µm

Large pore core-shell particle for fast intact and subunit biologic entry. C8 provides highly useful moderate hydrophobic selectivity.


Core-shell particle with butyl stationary phase and optimal wide pore size distribution for better resolution of large biologics, including monoclonal antibodies and subunit analysis.



Size Exclusion (SEC)
bioZen SEC-2
1.8 µm
bioZen SEC-3
1.8 µm

Large pore core-shell particle for fast intact and subunit biologic entry. C8 provides highly useful moderate hydrophobic selectivity.


Extremely inert, high density fully porous particle with high efficiency and high molecular weight (HMW) separation range of 10 k – 700 kDa.


Peptide
bioZen Peptide XB-C18
1.7 µm and 2.6 µm
bioZen Peptide PS-C18
1.6 µm and 3 µm

Overall retention of both acidic and basic peptides through C18 stationary phase with di-isobutyl side chains.


Excellent retention by combined positively charged surface ligand and C18 ligand.


Ion-Exchange Glycan
bioZen WCX
6 µm
bioZen Glycan
2.6 µm

Monosized particles grafted with linear polycarboxylate chains to envelop and separate proteins from acidic/basic variants

Provides optimal combination of high efficiency and selectivity for released glycans.


Oligonucleotides
bioZen Oligo
1.7 µm and 2.6 µm

Organo-silica core-shell particle bonded with a C18 stationary phase offers high selectivity for even minute oligo differences alongside high and low pH robustness.

Inside bioZen BioTi Hardware

Overlaid Successive Injections - Protein Priming Comparison

Conditions for all columns:

Columns:
Dimension:
150 x 4.6 mm
Mobile Phase:
50 mM Dipotassium Phosphate + 100 mM Sodium Sulfate, pH 5.0
Flow Rate:
0.3 mL/min
Temperature:
Ambient
Detection:
UV @ 280 nm
Sample:
1. γ-Globulin, 5 mg/mL
2. Ovalbumin, 1 mg/mL

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.

Uniform Particle Size Distribution

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.

Uniform Particle Size Distribution

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.


Monosized Particle
Peptide Mapping

Digested mAbs or ADCs typically include a large body of com pounds which 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
Trastuzumab Biosimilar Peptide Map

Infliximab Biosimilar Peptide Map
Infiximab Biosimilar Peptide Map


Conditions for all columns:
Column:
  bioZen 1.6 µm Peptide PS-C18
  bioZen 2.6 µm Peptide XB-C18
Dimensions
150 x 2.1 mm
Part No.:
Mobile Phase:
A: 0.1 % Formic Acid in Water
B: 0.1 % Formic Acid in Acetonitrile
Gradient:
Time (min)
0
0.5
50
55
56
 
% B
1
1
50
50
95
Flow Rate:
0.3 mL/min
Temperature:
40 °C
Detection:
QTOF (SCIEX®  X500B)
Aggregate Analysis
With mAb aggregate often at very low levels (<0.1% by peak area compared to 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 were developed with a combination of UHPLC efficiency and higher sensitivity, to drive resolution and identification of even lower level targets.

Cetuximab

Trastuzumab

Rituximab

Infliximab—abda

Conditions same for all samples:
Column:
Dimension:
300 x 4.6 mm
Part No.:
00H-4772-E0
Mobile Phase:
50 mM Potassium Phosphate +
250 mM Potassium Chloride (pH 6.8)

Flow Rate:
0.35 mL/min
Temperature:
30 °C
Detection:
UV @ 280 nm
Sample:
As noted

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
Dimensions
250 x 4.6 mm
Part No.:
Mobile Phase:
A: 20 mM MES (pH 5.6)
B: 20 mM MES + 300 mM NaCl (pH 5.6)
Gradient:
Time (min)
0
1
31
31.1
34
35
 
% B
15
15
45
100
100
15
Flow Rate:
1 mL/min
Temperature:
30 °C
Detection:
UV @ 280 nm
Sample:
Trastuzumab


Trastuzumab (pH Gradient Buffer)

Column:
bioZen 6 μm WCX
Dimensions
250 x 4.6 mm
Part No.:
Mobile Phase:
A: CX -1 (pH 5.6) pH Gradient Buffer*
B: CX -1 (pH 10.2) pH Gradient Buffer*
Gradient:
Time (min)
0
1
21
23
24
 
% B
0
0
100
100
0
Flow Rate:
1 mL/min
Temperature:
30 °C
Detection:
UV @ 280 nm
Sample:
Trastuzumab

* From Thermo Fisher Scientific® Inc.

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.

Conditions for all columns:

Column:
bioZen 2.6 µm Glycan
Dimensions
150 x 2.1 mm
Part No.:
Mobile Phase:
A: 100 mM Ammonium Formate,  pH 4.5
B: Acetonitrile
Gradient:
Time (min)
0
10
24
38.5
38.6
40.6
40.7
48
 
% B
78
74.5
72
55.9
40
40
78
78
Flow Rate:
0.5 mL/min
Temperature:
50 °C
Detection:
FLD ex/em 285/345 nm
Sample:
As noted

Sample Preparation Solutions

N-Gycan Clean-Up HILIC Solid Phase Extraction (SPE)

High recovery of labeled released N-glycans in a microelution format allowing for streamlined processing and clean-up of small sample volumes.

Peptide Quantitation

When quantitating signature peptides from biological matrices, you need 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 same for both samples:
Column:
bioZen 3 µm Peptide PS-C18
Dimensions
50 x 2.1 mm
Part No.:
Mobile Phase:
A: 0.1 % Formic Acid in Water
B: 0.1 % Formic Acid in Acetonitrile
Gradient:
Time (min)
0
1
4.5
 
%B
3
3
25
Flow Rate:
0.5 mL/min
Temperature:
22 °C
LC System:
ExionLC™ AD HPLC
Detection:
MS/MS
Detector:
SCIEX QTRAP® 5500
Flow Rate:
1.85 mL/min
Sample:
As noted

MagBeads Streptavidin Coated

Higher binding capacity magnetic particles result in faster and reliable purification, clean-up, and isolation of proteins and peptide molecules.

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

Intact Trastuzumab
Conditions for all columns:

Column:
bioZen 3.6 µm Intact XB-C8
Dimensions
150 x 2.1 mm
Part No.:
Mobile Phase:
A: 0.1 % TFA in Water
B: 0.1 % TFA in Acetonitrile
Gradient:
Time (min)
0
1
13
 
% B
20
20
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
Dimensions
150 x 2.1 mm
Part No.:
Mobile Phase:
A: 0.1 % TFA in Water
B: 0.1 % TFA in Acetonitrile
Gradient:
Time (min)
0
1
13
 
% B
20
20
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
Dimensions
150 x 2.1 mm
Part No.:
Mobile Phase:
A: 0.1 % TFA in Water
B: 0.1 % TFA in Acetonitrile
Gradient:
Time (min)
0
1
13
 
% B
20
20
45
Flow Rate:
0.5 mL/min
Temperature:
80 °
Detection:
UV @ 280 nm
Sample:
1. Cetuximab

Intact Mass
Intact Mass can give indications not only of relative abundance of glycoforms , but also stability as degraded mAbs will not give good charge envelope by ESI-MS. Intact Mass with a high resolution MS to identify PTMs, especially 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
Dimensions
150 x 2.1 mm
Part No.:
Mobile Phase:
A: 0.1 % Formic Acid in Water
B: 0.1 % Formic Acid in Acetonitrile / Isopropyl alcohol (50:50
Gradient:
Time (min)
2.5
10
110.1
 
% B2
0
65
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

Axia column and packing technology is patented by Phenomenex. U.S. Patent No. 7, 674, 383