11


Ultrapure water was injected with and without the addition of buffer substances to determine the purity of this water. The buffer substances were added to the ultrapure water as follows:


• TE buffer = 1% 1 M Tris-(hydroxymethyl)-aminomethane (TRIS), 0.2% 0.5 M Tetrasodium ethylenediaminetetraacetate dihydrate (EDTA), 0.01% Sodium dodecyl sulphate (SDS), pH 8, conductivity 1.4 mS/cm


• Citrate = 20 mM citric acid monohydrate, pH 3.6, conductivity 1.7 mS/cm


• Sodium phosphate/sodium sulphate = 0.1 M sodium phosphate/sodium sulphate, pH 6.6, conductivity 23 mS/cm, as well as


• arium® ultrapure water without any buffer additives, for use as a control


Test Procedure and Results for monoclonal antibody aggregation states Determination


The column and the precolumn are washed with the mobile phase at 1 mL/min until a stable baseline is achieved. A UV detector measures the absorbance of the samples at wavelengths of 220, 260 and 280 nm in milli absorbance units (mAU). The column performance is checked using proteins of a known molecular size (see Figure 4 and Table 3).


Table 3. Standard Proteins Used Peak Protein


1 2 3


4 5


Thyroglobulin (monomer peak)


Gamma globulin (monomer peak)


OV albumin (monomer peak)


Ribonuclease Adenine


Manufacturer Order Number


Sigma Sigma Sigma


Sigma Sigma


MW [kDa] Ret. [min] T 1126 670 G 5009 150 A 5503 45


R 5000 14 A 8626 0.14


6.73 8.44 9.89


10.65 15.35


A standard curve can be generated based on the logarithmic molecular sizes of standard proteins and on the retention times determined. This curve can then be used to calculate unknown samples on the basis of their retention times.


The column effi ciency is regularly checked using a standard protein mixture. This performance is affected by the purity of the additives to the eluant, the interaction of the eluant with the column matrix and by the composition of the sample analysed. The buffers used may not interact at all with the stationary phase.


Figure 3. HPLC-SEC analysis of various buffers and of arium® pro VF ultrapure water. Mobile


phase: 0.1 M sodium phosphate/sodium sulphate, pH 6.6, conductivity 23 mS/cm, in ultrapure water (conditions described in Table 2).


The chromatograms of the SEC run were recorded (Figure 3) and show that the ultrapure water used did not interact at all with the stationary phase. The addition of 0.1 M sodium phosphate/sodium sulphate for the mobile phase likewise showed a straight baseline without peaks. By contrast, the addition of citrate and TE indicated that contaminants or substances are contained that interact with the column matrix, which is expressed by peaks and can distort the actual chromatogram as a result.


After it had been clarifi ed in the trial runs that arium® VF ultrapure water can be used as


a solvent for the buffer substances employed to prepare the eluant, actual analysis of the samples was conducted.


To obtain reproducible results in SEC analysis, freshly prepared eluants must always be used. The pH and the ionic strength need to be optimised for the samples available for analysis [1]. Before storing the column, it should be rinsed with eluant fi rst, then fl ushed with ultrapure water containing 0.05% sodium azide and stored in this condition [1].


The mobile phase (0.1 M sodium phosphate/0.1 M sodium sulphate, pH 6.6, conductivity 23 mS/cm) used for the column was prepared with ultrapure water, which originally had a conductivity of 0.055 µS/cm or 18.2 MΩ (megohms) compensated to 25°C. SEC- HPLC was run using the settings/parameters listed in Table 2. A 0.05% azide solution in ultrapure water was employed for washing and storing the column after use in order to prevent bacterial growth. To prepare both solutions for the HPLC-SEC run, they were each degassed by fi ltration through a Sartolab BT 500 Bottle Top 0.2 µm vacuum fi ltration unit. The samples to be injected were prepared by prefi ltering them through Sartorius Minisart® RC4 (17821, 0.2µm) and fi lled in vials (WICOM WIC 42000).


SEC analysis is employed in quality control and is also used in the optimisation and development of purifi cation processes.


The following shows SEC chromatograms recorded during downstream process steps. The monoclonal antibodies are purifi ed in a series of consecutive steps. Cell harvesting, along with clarifi cation and concentration steps, is performed fi rst, followed by a capture step (protein A affi nity chromatography). After that, an intermediate step is carried out to remove contaminants (e.g., cation exchangers), and a polishing step is then completed (anion exchangers). Subsequently, virus fi ltration and fi nal ultrafi ltration [see also 7] are performed.


To determine the purity of the antibody, samples are run in SEC columns during downstream processing. The chromatograms of the runs performed after initial clarifi cation of the cell harvest (Figure 5) and protein A affi nity chromatography (Figure 6) are shown below.


Figure 5. HPLC-SEC analysis of a monoclonal antibody after cell harvesting and initial clarifi cation during downstream processing (conditions are described in Table 2).


The HPLC analytical run after initial clarifi cation of the cell harvest (Figure 5) shows a number of contaminants, e.g., host cell DNA, host cell proteins, endotoxins, components of the medium and impurities caused by the antibody product itself, as well as aggregates, which need to be removed by the subsequent downstream processing steps.


In the following step, protein A chromatography, the specifi c affi nity of protein A to immunoglobulin G (IgG) is used. A Sartorius Sartobind®


protein A (93PRAP06HB-12-


Figure 4. HPLC-SEC analysis of a protein mixture (standard proteins) (conditions are described in Table 2).


-A) adsorber is employed in this step. Following initial clarifi cation (see Figure 5 for analysis), the sample is fi ltered through the protein A unit. IgG binds to the protein A adsorber, while contaminants pass through the adsorber. The elution of protein A is examined by HPLC-SEC analysis (see Figure 6).


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