Towards protein crystallization as a process step in downstream processing of therapeutic antibodies: screening and optimization at microbatch scale

Abstract

Crystallization conditions of an intact monoclonal IgG4 (immunoglobulin G, subclass 4) antibody were established in vapor diffusion mode by sparse matrix screening and subsequent optimization. The procedure was transferred to microbatch conditions and a phase diagram was built showing surprisingly low solubility of the antibody at equilibrium. With up-scaling to process scale in mind, purification efficiency of the crystallization step was investigated. Added model protein contaminants were excluded from the crystals to more than 95%. No measurable loss of Fc-binding activity was observed in the crystallized and redissolved antibody. Conditions could be adapted to crystallize the antibody directly from concentrated and diafiltrated cell culture supernatant, showing purification efficiency similar to that of Protein A chromatography. We conclude that crystallization has the potential to be included in downstream processing as a low-cost purification or formulation step.

Figure 1. Micrograph of crystals of mAb04c under polarized light.

3 µl microbatch. Conditions: 1.5 µl 20 mg/ml mAb04c in 20 mM Tris, 50 mM Histidine, pH 7 plus 1.5 µl 12% (w/v) PEG 8000, 0.4 M calcium acetate in 0.2 M Imidazol, pH 7, RT. The broken crystal (intentionally) indicates that the dimension of crystal is about 100∼150 µm×10∼20 µm×10∼20 µm.

Figure 2. Phase diagram of mAb04c with PEG 8000 as precipitant.

Buffer: 0.1 M imidazol, 0.2 M calcium acetate, pH 7.0, RT.

Figure 3. SDS-PAGE of HCP spiking experiment.

Silver-staining. Lanes: (1) mAb04c, standard; (2) mother liquor with mAb04c and lysozyme; (3) washed mAb04c crystals from mother liquor with lysozyme; (4) the supernatant from the crystallization with lysozyme; (5) mother liquor with mAb04c and BSA; (6) washed mAb04c crystals from mother liquor with BSA; (7) the supernatant from the crystallization with BSA.

Figure 4. Micrograph of crystalline mAb04c crystallized from clarified culture supernatant.

Crystallization condition: sitting drop. Reservoir: 0.1 M imidazol, 0.2 M calcium acetate, 9% w/v PEG 8000. 20 µL clarified culture supernatant (8.3 mg/ml mAb04c) plus 20 µl reservoir, RT. Scale bar 100 µm.

Figure 5. Coomassie blue stained none-reducing SDS-PAGE of mAb04c before and after crystallization or protein A purification.

7 µg of IgG was loaded per lane. Lanes: (1) clarified mAb04c culture supernatant; (2) washed mAb04c crystals, redissolved in 100 mM sodium acetate pH 4.0; (3) mAb04c, purified via protein A chromatography.

Figure 6. SE-Chromatogram of the sample before and after crystallization.

Elution buffer: 0.05 M Tris/0.15 M NaCl, pH 7; flow rate: 1 ml/min; wavelength: 225 nm. (A) Clarified mAb04c culture supernatant. Peak 3: mAb04c, 8.42 min; Peak 4: Contaminating Protein, 9.98 min; Peak 5: Histidine in buffer, 12.19 min. (B) washed mAb04c crystals, redissolved in 100 mM sodium acetate pH 4.0. Peak 2: mAb04c, 8.43 min.

Figure 7. Time course of microbatch crystallization.

Protein: 8 mg/ml mAb04c, crystallization buffer: 8% (w/v) PEG 8000, 0.2 M calcium acetate 0.1 M imidazol, pH 7.0, RT. Time after mixing: (A) 0 hrs, (B) 2 hrs, (C) 10 hrs, (D) 70 hrs. Scale bar 100 µm.