Development of a Crystallization Step for Monoclonal Antibody Purification: Screening, Optimization and Aggregation Control

An intact monoclonal antibody was purified through crystallization. The purity after one step purification was 95%, which was comparable to protein A chromatography. But this process still needs to be improved because of a low recovery rate of antibody

WenTong HuoXue Cream Can Inhibit the Reduction of the Pain-Related Molecule PLC- β

WenTong HuoXue Cream (WTHX-Cream) has been shown to effectively alleviate clinical symptoms of diabetic peripheral neuropathy (DPN). This study investigated the gene and protein expression of the pain-related molecule PLC-β3 in the dorsal root ganglion (DRG) of DPN rats. 88 specific pathogen-free male Wistar rats were randomly divided into placebo (10 rats) and DPN model (78 rats) groups, and the 78 model rats were used to establish the DPN model by intraperitoneal injection of streptozotocin and were then fed a high-fat diet for 8 weeks. These rats were randomly divided into the model group, the high-, medium-, and low-dose WTHX-Cream + metformin groups, the metformin group, the capsaicin cream group, and the capsaicin cream + metformin group. After 4 weeks of continuous drug administration, the blood glucose, body weight, behavioral indexes, and sciatic nerve conduction velocity were measured. The pathological structure of the DRG and the sciatic nerve were observed. PLC-β3 mRNA and protein levels in the DRG of rats were measured. Compared with the model group, the high-dose WTHX-Cream group showed increased sciatic nerve conduction velocity, improved sciatic nerve morphological changes, and increased expression of PLC-β3 mRNA and protein in the DRG. This study showed that WTHX-Cream improves hyperalgesia symptoms of DPN by inhibiting the reduction of PLC-β3 mRNA and protein expression in the diabetic DRG of DPN rats

Towards Protein Crystallization as a Process Step in Downstream Processing of Therapeutic Antibodies: Screening and Optimization at Microbatch Scale

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

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

<p>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.</p

SE-Chromatogram of the sample before and after crystallization.

<p>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.</p

Micrograph of crystalline mAb04c crystallized from clarified culture supernatant.

<p>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.</p

Phase diagram of mAb04c with PEG 8000 as precipitant.

<p>Buffer: 0.1 M imidazol, 0.2 M calcium acetate, pH 7.0, RT.</p

Purity and Recovery of crystallized mAb04c.

<p>*Protein redissolved in 100 mM sodium acetate pH 4.0.</p

Micrograph of crystals of mAb04c under polarized light.

<p>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.</p