Affinity chromatography for the purification of therapeutic proteins from transgenic maize using immobilized histamine

Abstract

Plant molecular pharming is a technology that uses plants as bioreactors to produce recombinant molecules of medical and veterinary importance. In the present study, we evaluated the ability of histamine (HIM), tryptamine (TRM), phenylamine (PHEM) and tyramine (TYRM) coupled to Sepharose CL-4B via a 1,4-butanediol diglycidyl ether spacer to bind and purify human monoclonal anti-HIV antibody 2F5 (mAb 2F5) from spiked maize seed and tobacco leaf extracts. Detailed studies were carried out to determine the factors that affect the chromatographic behaviour of mAb 2F5 and also maize seed and tobacco leaf proteins. All affinity adsorbents showed a reduced capacity to bind and a reduced ability to purify proteins from tobacco extract compared to maize extract. Under optimal conditions, HIM exhibited high selectivity for mAb 2F5 and allowed a high degree of purification (>95% purity) and recovery (>90%) in a single step with salt elution (0.4 M KCl) from spiked maize seed extract. Analysis of the purified antibody fraction by ELISA and Western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was assessed further using a second therapeutic antibody (human monoclonal anti-HIV antibody mAb 2G12) and a therapeutic enzyme (alpha-chymotrypsin). HIM may find application in the purification of a wide range of biopharmaceuticals from transgenic plants.

Antibody purification via affinity membrane chromatography method utilizing nucleotide binding site targeting with a small molecule

“…34,69-72 Moreover, increased number of injection cycles is not a concern because our results proved that the m-NBST column retained its function without any loss in its performance even after 200 injections. This is a significantly high number compared to any protein based column, especially considering that most of the protein based affinity columns start showing signs of degradation after just 10 cycles of injections, and require cleaning cycles regularly.…”

“…Therefore, small molecules and peptides are being examined, by us and others, to be used as an alternative to protein A (or G) for antibody purification with varying success. 27,28,30-32 Amino acid derivatives 33,34 , dyes 9 , and thiophilic compounds 35 are some of the small molecules that are mostly proposed for capturing antibodies. Additionally, computational studies were performed to determine small molecules or peptides, binds on different regions of antibodies, to be used for purification purposes.…”

Agroinfiltration as an Effective and Scalable Strategy of Gene Delivery for Production of Pharmaceutical Proteins

“…In addition, a variety of expression vectors are already available for these species, providing another advantage. However, most tobacco and related Nicotiana plants contain high levels of phenolics and toxic alkaloids, which complicate the downstream purification of pharmaceutical protein production [35,36]. Our laboratory explored the feasibility of using lettuce (Lactuca sativa) as an alternative plant host to produce pharmaceutical proteins, by agroinfiltration with deconstructed viral vectors.…”

Design, synthesis and application of benzyl-sulfonate biomimetic affinity adsorbents for monoclonal antibody purification from transgenic corn

“…However, it suffers from problems, such as ligand instability, leaching and high cost because Protein A and Protein G resins are very expensive and readily amenable to chemical and biological degradation (Jiang et al ., ; Ren et al ., ). On the other hand, alternative conventional chromatographic methods, such as ion exchange and biomimetic‐ligand affinity chromatography (Li et al ., ; Branco et al ., ) are suitable for large‐scale chromatography, because they are inexpensive, resistant to chemical or biological degradation, and display high protein‐binding capacity (Platis et al ., ; Platis and Labrou, ; Platis et al ., ; Liu et al ., ; Menegatti et al ., ).…”

“…One of the major challenges in the molecular pharming industry is the development of efficient purification protocols for the production of active recombinant mAbs from transgenic plants (Platis and Labrou, ; Platis et al ., ; Platis et al ., ; Barros et al ., ; McLean et al ., ). In order to satisfy this growing demand, to increase the production yields, and lower the production costs, alternative purification systems based on affinity chromatography are being actively developed (Horak et al ., ; Arnold et al ., ; Dinon et al ., ; Naik et al ., ; Lund et al ., ).…”