application note
poRoS ®
a20 analytical Hplc columns for the Quantitation of Monoclonal antibodies
System Setup
A pre-column filter should be utilized to protect against column fouling. A filter pore size in the range of
0.5 μm to 2 μm is recommended—for example, Upchurch Part# A-315, A-316, A-318, A-355, or A-356.UV monitoring can be performed at 280 nm, or for incread nsitivity , 214 nm. Samples can be assayed at room
temperature. If numerous samples are being loaded in a quence, autosampler temperature control should be t at 2–8ºC. Sample Preparation/Injection
Standards and samples should be 0.2 μm filtered prior to loading in sample vials,
as centrifugation of samples may not be sufficient to ensure clarity. Bubbles residing at the bottom of a sample vial should be shaken free.
Recommended injection volume for
standards and samples is between 20 μL and 100 μL. Injection volume should be
equivalent for all standards and samples, and should not exceed the column bed volume. Sample injection volumes can be incread if samples are too dilute. Highly concentrated samples can be diluted with equilibration buffer prior to injection.
Since the A20 assay is bad on recombinant Protein-A affinity , most sample solution
语文老师作文components will not interfere with binding or
Introduction
poRoS ®
a20 columns, containing
20 µm poRoS ® beads functionalized with recombinant protein-a, enable the rapid and preci quantitation of monoclonal antibodies and other Fc-containing biomolecules from sample solutions such as harvested cell culture fluid and downstream product pools. the practicality, high throughput, and robustness of assays developed using the columns has led to their widespread adoption in biopharmaceutical analytical procedures.
this application note provides
recommended operating conditions that will help maximize assay performance and column lifetime. However , since antibodies and sample solutions differ widely between various applications, operating conditions should be optimized to accommodate the specific needs of each unique assay.
Figure 1. Typical Chromatogram for Injection of Harvested Cell Culture Fluid Containing Monoclonal Antibody (mAb). 20 μL injection, 1 mg mAb/mL.
elution. Common excipients that should not interfere include: ≤1% Tween 20, ≤1% Tween 80, <1% Triton X-100, <20% polysaccharides, and <20% glycols. If desired, the addition
of 10 mM EDTA can help with the chelation of ferric compounds that often exist in cell culture samples and can result in nonspecific binding of impurities to the A20 column.
Figure 1 shows a typical chromatogram for a 20 μL injection of harvested cell culture fluid containing monoclonal antibody at
1 mg mAb/mL.
Standard Curve
Standard curves should be generated from purified material, and unique standards should be ud for each molecule being assayed. Standard curve data are typically
fit by linear regression. Linear correlation coefficients and assay precision are typically excellent (R2 >0.98, coefficient of variation
<10%). Dynamic range will be dependent in part on instrumentation (e.g., UV detector linearity at high antibody concentration).
Figure 2 shows standard curves generated with purified monoclonal IgG. Table 1 outlines method conditions ud.
Buffer Preparation
A simple two-buffer system is typically ud
for column operation. A typical equilibration/ wash buffer (Buffer A) is 25–100 mM phosphate, pH 6.6–7.5, with or without sodium chloride up to 150 mM. A typical elution buffer (Buffer B) is the same buffer at
pH 2.0–3.5. Other elution buffer components that may be ud include hydrochloric acid, glycine, citrate, acetate, or other components that buffer well at low pH.
Preparing the elution buffer with a concentration equal to or greater than that of the equilibration buffer will help ensure a good pH transition during elution. Preparing the elution buffer with a salt concentration equal to the equilibration/wash buffer will minimize shifts in the UV baline during elution that are particularly evident when UV monitoring is performed at 214 nm and whena股
analyzing dilute samples. The characteristics of the balines achieved during equilibration and elution can be assd by running blank samples. Figure 3 shows the influence of elution buffer composition on peak height, peak width, and retention time. Table 1 outlines the method conditions ud.NOTE:It is important that complete elution
is attained. If only partial elution is attained,
then antibody will remain bound to the column
between injections, column fouling will
begin, and carryover may affect results. The
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火印读后感completeness of elution is assd by recovery
of the standards.
Method Timetable and Flow Rate
In order to ensure effective pH transition,
elution should consist of a step to 100%
Buffer B. Elution should not consist of a
gradient or a blend of Buffer A and Buffer B
that results in less than 100% Buffer B being
ud for elution.
Figure 2. Standard Curves for Monoclonal IgG Eluted With Various Elution Buffers. Data points are mean values of replicate samples (maximum CV = 3.5%). Method conditions are listed in Table 1.
Table 1. Method Conditions Ud for Figures 2 and 3.
System:Agilent 1200 HPLC
UV Detection:280 nm
Equilibration/Wash Buffer
A:
50 mM phosphate, 150 mM NaCl, pH 7.0
Elution Buffer B, one of:100 mM citrate, pH 2.5
100 mM acetate, pH 2.5
100 mM phosphate, pH 2.5
100 mM glycine, pH 2.5
12 mM HCl, pH 1.9
Method Timetable:Time (min)Gradient (%B)
0.000Equilibration/Wash, 0.5 min,
14 CV
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0.500
0.51100Elution, 1.0 min,
29 CV
1.5100
1.510Reequilibration, 1.5 min,
43 CV
3.000
The duration of wash, elution, and
equilibration stages should provide sufficient column volumes of flow to allow for complete pH transitions and establishment of UV balines.
Flow rate can range from 800 to 8,600 cm/hr . Columns are packed at 180 bar , and thus operating pressure limits are high. To ensure bed stability , columns should not be operated over 180 bar .
Column Reu and Cleaning
Columns are generally very robust; lifetimes of 3,000 injections per column have been reported. Extended reu should be accompanied by monitoring of column
backpressure and an assay control sample.
If backpressure increas or control sample recovery changes, the column should be cleaned to remove residual material from the column frits and from the POROS ® A20 media.
Typical cleaning solutions include 2–6 M
guanidine hydrochloride, 1 M acetic acid, 20% ethanol, 1 M acetic acid plus 20% ethanol, 20% isopropanol, elution buffer titrated to pH 1.5–2.0, and elution buffer plus 1–2 M sodium chloride.
A cleaning cycle should involve 2 or 3
injections of cleaning solution at a volume equal to the column bed volume, followed by 2 or 3 injections of equilibration buffer—for example, 2 x 100 μL cleaning solution,
2 x 100 μL equilibration buffer . Alternatively , multiple column volumes of a desired solution can be run over the column. If desired, the cleaning can be run in rever flow to help clean the top frit, with flow direction returned to normal after cleaning. If the system does not allow for flow reversal, the column can be plumbed in rever, cleaned, and returned to normal after
cleaning. The cleaning quence should be monitored for removed protein peaks.Conclusions
The flexibility and robustness of POROS ® A20 columns makes the lection of suitable operating conditions a straightforward process. Fine-tuning of assay parameters within the broad guidelines outlined above is encouraged to facilitate optimized results and to enable maximum resource efficiency for each unique analytical application. When properly ud, POROS ® A20 columns enable a long lifetime of rapid, accurate quantitation of monoclonal antibodies.
Scientific Contributors
David K. Peng, PhD, POROS ® Senior Applications Specialist
Christine Gebski, MS, POROS ® Director of Applications and R&D
Paul Lynch, POROS ® Senior Product Manager POROS ® Chromatography Media Group, Applied Biosystems, Bedford, MA
Figure 3. Chromatograms for Monoclonal IgG Standard Eluted With Various Elution Buffers. 20 µL injection, 1.8 mg mAb/mL. Method conditions are listed in Table 1.
800700
60050040030020010001
0.5
1.52
12 mM HCI, pH 1.9
100 mM glycine, pH 2.5100 mM phosphate, pH 2.5
100 mM acetate, pH 2.5100 mM citrate, pH 2.5
2.5
3.0
Time (min)
A b s o r b a n c e (m A u a t 280 n m )研究生助学贷款
For Rearch U Only. Not intended for any human or animal therapeutic or diagnostic u, unless otherwi stated.
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