Relying on years of research and development and purification application experience, NanoMicro Technologies has launched a general high-efficiency purification solution based on the specific selectivity of chromatography resin for monoclonal antibodies (mAb), bispecific antibodies (bsAb), and Fc fusion proteins (Fc fusp), etc (Fig. 1).

Fig. 1. General process for antibody purification.

mAb purification platform and resin recommendation

Antibody purification involves selective enrichment or specific separation of antibodies from serum, ascites, or cell culture supernatants of hybridoma cell lines. The impurities, including host proteins, DNA, viral particles, endotoxin, antibody fragments, aggregates and charge heterogeneity, which can be purified by filtration, chromatography. As a leading global supplier of monodisperse chromatography resin, we offer a platform solution for antibody purification with a wide selection of high-performance protein A affinity, ion exchange, hydrophobic interaction chromatography resins in capture, intermediate and final polishing steps (Fig. 2).

Fig. 2. Chromatography resin recommendation for antibody purification.

Protein A affinity chromatography resins

NanoMicro Technologies offers two series high-performance protein A affinity chromatography resins with agarose and polymethyl methacrylate matrices. The alkaline-stable recombinant protein A ligands are immobilized onto our proprietary base matrices to provide cost-effective solutions for fast-processing of monoclonal and polyclonal antibodies, bispecific antibodies, and Fc fusion constructs of high quality.

• Rigid particles with outstanding pressure-flow properties; easy linear scale-up

• Efficient mass transfer; high dynamic binding capacity; small elution volumes

•  Minimal non-specific binding; excellent antibody recovery

• Animal component-free production; full technical and regulatory support

Table 1. Characteristics of protein A affinity resins.

a.          10% Breakthrough capacity with 4 min residence time, mAbs at various concentrations.

b.          Contact time varied from 15 min to 24 hours, with or without NaCl.

c.           Depending on NaOH concentration and contact time; leached Protein A can be quantified by ELISA kits from commercial vendors such as Cygnus and Enzo etc.

High dynamic binding capacity of NanoMicro protein A affinity Resins

NMab Pro is an agarose matrix based protein A affinity resin with high dynamic binding capacity of different antibodies and has camparable performance to industry-leading products.

Fig. 3. Comparison of 10% Breakthrough dynamic binding capacity (DBC) of different resins (5 min residence time).

Application cases

Case 1: Mab purification with Protein A affinity + anion ion exchange (FT) + cation ion exchange

Fig. 4. Purification of a Mab by three step, capture: UniMab 50HC (a), flow through intermediate purification: NanoGel-50Q, final polishing: NanoGel-50SP (c)

Results

Purification a certain mAb with a three step method (Figure 4), UniMab50HC affinity chromatography was used to capture and improve the purity to 97.4%. After that, impurities such as HCP and DNA were removed from the mixture through the flow through mode of NanoGel-50Q anion exchange chromatography. Finally, trace impurities were polished by NanoGel-50SP cation exchange chromatography. The final product has a purity of 99.3% and an overall yield of 80.5% (Table 2).

Table 2. Results of purification of a certain mAb

Case 2: Cation exchange chromatography resin adjust the content of antibody acid-base charge heterogeneity

Fig. 5. Linear gradient elution of salt concentration with NanoGel-50SP cation exchange chromatography (a), collection section results (analysis column BioCore WCX, i.d. 4.6 mm, column height 250 mm) (b).

Results

Charge heterogeneity originates from post translational modifications of proteins, such as terminal changes, glycosylation, deamidation, isomerization, oxidation, splitting, polymerization, etc., Acidic heterogeneity leads to a decrease in the retention time of monoclonal antibodies and an increase of blood clearance rate; Alkaline heterogeneity often has a positive impact on drug efficacy, such as showing stronger affinity and stronger antibody dependent cell mediated cytotoxicity affects in many cases. The mixture of monoclonal antibody charge heterogeneity captured by affinity chromatography can be purified with NanoGel-50SP cation exchange chromatography, and the acid-base peak and main peak can be separated through linear gradient elution of salt concentration (Fig. 5). The content of the main peak in the collection section increased from 47.50% to 66.07%, with a yield of over 72%, meeting the requirements for drug purity and efficacy (Table 3).

Table 3. Comparison of charge heterogeneity content before and after purification.

Resin Recommendation