Page 14 - Shimadzu Journal vol.9 Issue2

Biopharmaceutical

aggregates and isomers, and other assessments as shown in Table 1. surfaces, and mobile phase. But in the SEC columns containing silica-
These assessments are extremely important at every step of pharma- based packing materials with chemically-bound common diol
ceutical development, from early stages to product shipment. groups, electrostatic interaction may arise between proteins and
residual silanol groups on the packing material surface causing pro-
mAbs form dimeric or multimeric aggregates depending on pro- teins to adsorb to the silica gel. This results in peak tailing, delayed
duction and storage conditions. Aggregates in antibody drugs not elution times, and other phenomena. Electrostatic interactions can
only cause a decrease in pharmacological action but also elicit an be suppressed by adding sodium chloride to the mobile phase to
immune response, thus affecting the efficacy and safety. For this negate these negatively charged silanol groups. The strength of
reason, ICH-Q6B requires the separation of impurities such as these interactions with column packing material varies with the type
monomers and aggregates in antibody drugs and determines their of protein; it is necessary to determine the appropriate analytical
content. This article introduces an analysis of mAb impurities and methods for each protein.
fragments by size-exclusion chromatography (SEC).
This article presents an optimization of an analytical method for
Table 1. Examples of Quality Assessment Tests for Antibody Drugs mAb aggregates and fragments that investigates the effects of mobile
phase salt concentration, flow rate, and pH on separation with a 150
Item Tested Purpose Analysis Technique
mm long Shim-pack™ Bio Diol-300 column with 2 µm diameter
Aggregates/ Determine levels of Size-exclusion chromatography,
Fragments aggregates and fragments Micro flow imaging etc.
packing material.
Ion-exchange chromatography,
Characterization and
Charge variants Imaged capillary isoelectric
monitoring of charge variants
focusing etc.
Hydrophilic chromatography,
Sugar chain Evaluate consistency of Reversed-phase
structures sugar chain structures chromatography,
Mass spectrometry etc.
Evaluate molecular structure Reversed-phase
Structure and specificity characteristics chromatography,
of bulk drug Mass spectrometry etc.
Antibody-drug Calculate coupling ratio of Hydrophobic chromatography,
conjugates antibody-drug conjugate Mass spectrometry etc. Intensity
Affinity chromatography,
Potency Quantify biological activity Enzyme-linked immunosorbent
assay etc.
Time
Size-Exclusion Chromatography Fig. 1. Principle of Separation in SEC Analysis

Size-exclusion chromatography (SEC) is a technique that separates
molecules based on their size (Fig. 1). The column packing mate- Analysis
rial contains numerous pores. Smaller molecules permeate deeper
into these pores and take longer to pass through the column, on 3-1. Analytical Conditions
the other hand, larger molecules are unable to permeate the pores. Table 2 shows the analytical conditions that are common to all anal-
Consequently, molecules are eluted from the column in order yses. The mobile phase compositions are described in each section.
from largest to smallest and effectively sorted according to size.
Conventional SEC analysis uses long columns 300 mm in length Table 2. Analytical Conditions
and low flow rates with long elution times from several tens of min- System Nexera XS inert
Shim-pack Bio Diol-300 * 1
utes up to an hour to ensure full separation between components. Column (150 mm×4.6 mm I.D., 2 µm)
Recent column development has reduced the sizes of packing ma- 0.2 mL/min (Fig. 2, Fig. 3, Fig. 9)
Flow rate 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4 mL/min (Fig. 5)
terials to achieve excellent separation with shorter elution times. 0.25 mL/min (Fig. 7)
Column Temp. 25 ˚C
Monoclonal Antibody Standard
Sample
However, further improvements in separation and sensitivity re- (Conc. 500 µg/mL)
2
Vial TORAST™-H Glass Vial * (Shimadzu GLC Ltd.)
quire not just smaller column packing materials but optimization
Injection vol. 5 µL
of the analytical conditions. In an ideal SEC separation, there is Detection 280 nm (SPD-M40 inert cell)
no chemical interaction between the molecules, packing material *1 P/N : 227­31010­01 *2 P/N : 370­04301­01

Shimadzu Journal vol.9 Issue2 47