Changzhou Smart-Lifesciences Biotechnology Co.,Ltd.

Recombinant proteins are typically expressed and amplified using E. coli, yeast, and mammalian cells. In recent years, the use of insect and plant cells for recombinant protein production has become increasingly common.
After appropriate pretreatment methods such as clarification, lysis, ultrafiltration, extraction, and centrifugation, recombinant protein fermentation broth yields samples ready for purification. In the subsequent purification process, ion exchange and/or affinity chromatography are often used for crude and intermediate purification. For fine purification, hydrophobic interaction chromatography, size-exclusion chromatography, and multimodal chromatography are commonly employed. Small molecules like insulin and GLP-1 heavily rely on reversed-phase and size-exclusion chromatography resins. To ensure high activity, chiral separation purification is sometimes necessary.
For small molecule recombinant proteins, a fusion protein strategy can be used. This involves appropriate affinity chromatography and enzymatic cleavage to quickly obtain high-purity intermediates, typically meeting purity standards after 1-2 additional chromatography steps. If using the coupling ligand method, re-purification is required after coupling modification to remove mis-modified molecules and unbound monomers.

The vaccine preparation process is similar to that of general recombinant protein preparation. However, since the expressed proteins are antigens, they typically form virus-like particles (VLPs) (e.g., the HPV vaccine) or some type of polymer (e.g., the RSV vaccine). In the final purification stage, it is crucial to remove monomers and poorly formed VLPs. Some VLPs have larger particle sizes and can easily encapsulate impurities during formation, potentially causing side effects in the human body. Therefore, it is necessary to inhibit VLP formation in the early stages of purification and promote VLP formation by changing the liquid conditions when the purity is high, or remove VLP particles formed in the early stages. It is important to note that ion-exchange chromatography may disrupt the VLP structure during adsorption, leading to VLP disintegration. Thus, when designing antigen structures, surface charge interactions should be considered.

Antibodies are important molecules for achieving immune functions, mainly including IgG1-4, IgA, IgD, IgE, IgM, and IgY (avian). They can specifically bind to antigens to fulfill immune functions.
Due to their excellent specificity and bioactivity, antibodies also play irreplaceable roles in detection, biomolecular extraction, vaccine development, and targeted therapeutic drug development. To achieve higher activity/specificity or to realize specific functions, antibody modification is very common and has now become a highly developed field.
In the process of antibody purification, Protein A/G/L affinity chromatography is a critical step. Affinity chromatography requires high load capacity, long lifespan, low ligand leakage and impurity residue, and tolerance to high-concentration NaOH cleaning.
Fine purification needs to be flexibly designed according to quality requirements, often using "anion + cation" or "composite mode chromatography," with some cases employing single-step hydrophobic interaction chromatography.
At present, the purification process for bispecific antibodies is almost similar to that of monoclonal antibodies but more challenging. This increased difficulty is due to the higher likelihood of mismatching during expression and the potential for aggregate content to reach up to 50%.