Page 19 - Pharmaceutical- Guide to Biopharmaceutical

Culture Purification Characterization

Automating Steps from Preparative Purification to Product Evaluation Prominence Inert LC System + LH-40 Liquid Handler

Seamless Analysis from Purification of IgG in benefits

Human Blood Plasma to SEC Evaluation Cell Line Optimization
• By selecting the target peak after fractionation, fractions can be automatically reinjected for analysis.
• A column switching valve allows automatic switching between columns for
Operating Principle and Features purification or analysis.
Table 1 Analytical Conditions of Affinity Purification
Sodium chloride and other halogen ions essential for biopharmaceutical Column: HiTrap rProtein A FF (1 mL) • Useful for optimizing cultivation parameters or other scenarios that involve
analysis are highly corrosive to metals. To eliminate this concern, the (made by Cytiva)
Prominence inert LC system uses PEEK or other polymer materials for Mobile phase A: 10 mmol/L (sodium) phosphate comparing large numbers of samples. Culture
all parts in contact with liquids, thereby ensuring worry-free operation. Mobile phase B: buffer pH 6.9
100 mmol/L (sodium) citrate
The liquid handler (LH-40) is an integrated autosampler and fraction buffer pH 4.0 Compatible with 96-well plates and a wide
collector. That means samples acquired during the first analysis can Time Program (B. Conc. ): 0% (0 – 10 min) variety of test tubes and other containers
be injected directly into the second analysis without having to transfer 100% (10.01 - 20 min)
them from a fraction collector. For example, with this system, the Flowrate: 0% (20.01 – 35 min)
1.0 mL/min
target protein is purified by an affinity column and fractionated at the Column Temp.: 15 °C
first step, and the fractionated protein is re-injected for SEC analysis at Injection Volume: 5 mL
the second step. These two steps can be done by just specifying the Detection: SPD-20A (280 nm)
method and fraction. Flow Cell: Inert flow cell
Measurement Method and Conditions Table 2 Analytical Conditions of SEC analysis Purification
Column: Shim-pack Bio Diol-300
5 mL of commercial human plasma was diluted 5-fold with mobile (300 mm × 4.6 mm I.D., 5 µm)
phase in a 15 mL tube and the tube was placed in the LH-40 rack. Guard Column: Shim-Pack Bio Diol-300 (G)
This sample was purified by affinity chromatography with an IgG (30 mm × 8.0 mm I.D., 5 µm)
purification column following the conditions in Table 1. The elution Mobile phase A: 10 mmol/L (sodium) phosphate
buffer pH 6.9
fractions were collected on the 96-deep-well plate set in the liquid Flowrate: 0.5 mL/min
handler. Then, 100 μL of the fraction involving the peak's top point Column Temp.: 15 °C
was analyzed by size exclusion chromatography (SEC) following the Injection Volume: 100 µL
conditions in Table 2. Detection: SPD-20A (280 nm)
Flow Cell: Inert flow cell Characterization
Specifications
Results
System controller CBM-20A
mAU
The IgG peak obtained from affinity purification in step 1 and the 250 Peak 2 UV 280nm Solvent delivery unit Two LC-20Ai units
peak obtained by SEC analysis in step 2 (Fig. 1) were evaluated by 225
performing SDS-PAGE. That resulted in detecting H and L-chain bands 200 Degassing unit DGU-20A5R
175
for the target IgG (Fig. 2). 150 Peak 1 Column oven CTO-20AC
125
Conclusion 100 Prominence inert LC system Mixer PEEK mixer 228-45093-92
75
50 UV-VIS detector SPD-20A Quality Control
By simply setting a sample to the liquid handler (LH-40) installed in 25
the LC system, the system can not only purify the sample, but also 0 Flow cell for inert LC system 228-33338091
seamlessly further analyze fractions from the sample. For routine 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min High-pressure flow channel switching FCV-12AHi
work with a prespecified target, it is possible to analyze only the Fig. 1 Chromatogram of SEC analysis valve
target fraction. By adding a column switching valve and increasing the VP option box 228-65512-58
number of columns, the system can also be used to screen purification Human After affinity SEC
parameters or purify samples in multiple steps. After fractions are plasma purification Peak1 Peak2 LH-40 liquid handler, main unit 228-65506-41
collected in a 96-well plate, they can be used directly for SDS-PAGE, kDa LH valve kit, preparative 228-75605-42
ELISA, or various other analytical methods. 220
170 Syringe kit, 20 mL 228-64173-44 Pharmacokinetics
Application Examples 116 Sample coil, 5 mL 228-39389-94
76
H-chain LH-40 liquid handler Analysis kit 228-75587-41
• Discovering and checking the quality of proteins in cell cultures 53
• Optimizing cultivation parameters Rinse pump 228-75586-41
• Evaluating proteins in blood L-chain Sample rack 228-75268-41
Rack kit, D16 228-75604-49
Rack kit, MTP 228-75604-40 Others

Examples of SEC analytical Shim-Pack Bio Diol-300 (300 mm × 4.6 mm I.D., 5 μm) 227-31010-04
column
Fig. 2 SDS-PAGE (Reducing) Results Shim-Pack Bio Diol-300 (G) (30 mm × 8.0 mm I.D., 5 µm) 227-31010-06

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