Fig 1 Electropherograms of an ADC sample in formulation buffer A b - PDF document

Fig. 1: Electropherograms of an ADC sample in formulation buffer, A: before histidine removal; B: after removal of histidine using a HisSep column. Sample recovery: 91%. Fig. 2: Electropherograms of an mAb sample in formulation buffer, A: before histidine removal; B: after removal of histidine using a HisSep column. Sample recovery: 96%. pI Marker 5.85 pI Marker 9.46 Histidine Gap pI Marker 5.85 pI Marker 9.46 pI Marker 5.85 pI Marker 9.46 Histidine Dip pI Marker 5.85 pI Marker 9.46 A: Before B: After A: Before B: After SampleName: Meso_after2- Absorbance 0.00 0.02 0.04 0.06 0.08 pI 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60 SampleName: Meso_Before2- Absorbance -0.02 0.00 0.02 0.04 0.06 0.08 0.10 0.12 pI 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60 SampleName: CXCR-Before- Absorbance 0.00 0.10 0.20 0.30 0.40 0.50 0.60 pI 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60 SampleName: CXCR-Before- Absorbance -0.010 0.000 0.010 0.020 0.030 0.040 pI 7.40 7.60 7.80 8.00 8.20 8.40 8.60 SampleName: CXCR-after- Absorbance 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 pI 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60 SampleName: CXCR-after- Absorbance 0.000 0.010 0.020 0.030 0.040 7.40 7.60 7.80 8.00 8.20 8.40 8.60 Introduction: Biologics, especially monoclonal antibodies (mAbs) and antibody drug conjugates (ADCs), have had a profound impact medicine in recent years. development has become a major focus many major pharmaceutical and biotechnology companies.Approximately products are predicted reach the worldwide market . Histidine is commonly used Histidine Removal from Formulation Buffers Using HisSepColumns Introduction: Biologics, especially monoclonal antibodies (mAbs) and antibody drug conjugates (ADCs), have had a profound impact medicine in recent years. development has become a major focus many major pharmaceutical and biotechnology companies.

Approximately products are predicted reach the worldwide market . Histidine is commonly used biologics formulation buffers.However, histidine containing buffer interferes with certain bioanalytical methods, such Amino Acid Analysis and Imaged Capillary Isoelectric Focusing (icIEF). Amino Acid Analysis using HPLC, high concentration histidine may increase the amount histidine the final results, consume too much labeling reagents, which may generate inaccurate results. icIEF analysis, histidine produces a “histidinegap” a “histidinedip” a about 7.5 electropherograms, and thus interferes with real sample peaks having 7.0 7.6, depending the ratio the ampholytes. Therefore, removing histidine prior these analyses is necessary in order generate accurate results. Currently histidine removal can done using molecular weight cut-off membrane spin columns. This type spin columns can adsorb proteins membrane leading unpredicted and disproportional loss proteins. addition, the need for high g-force, multiple spins with these spin columns adds substantial stress samples which may introduce misleading information in stability studies and formulation development. have developed a spin column product, HisSep, optimized for the removal histidine from biologics formulation buffers. Compared with current membrane-based spin columns, HisSep has significant benefits: additional stress placed the proteins solution Rapid 4 minute process . minute process Greater than % recovery Proteins remain stable buffer medium Compatible with most downstream analysis Applications: Histidine removal from ADC and mAb samples in formulation buffer ADC and a therapeutic were formulated a formulation buffer containing histidine.The charge variants the ADC and were analyzed using icIEF iCEBefore removal histidine, a “histidinegap” the ADC sample and a “histidinedip” the sample are observed around 7.0 (Fig. 1A and 2. The histidine gap and the histidine dip interfere with the icIEF profiles the ADC and the . The histidine gap and dip were completely removed after histidine removal using HisSep columns (Fig. 1B and 2B). Histidine Removal from Formulation Buffers Using HisSepColumn