1/20/2024 0 Comments Cytiva capto adhere![]() ![]() With increased selectivity and a broader pH working range, MMC is preferable to use over IEC and hydrophobic-interaction chromatography (HIC) methods. Multimodal or mixed-mode chromatography (MMC) is another widely used technique for removal of oligomers from protein product streams. Also, additional steps of buffer exchange can be averted with introduction of salt-tolerant membranes. Technologies such as anion-exchange membrane absorbers are becoming attractive because of their high throughput and binding capacities as well as cost-effectiveness. Oligomers generally carry charges that are more positive or negative than that of the monomer at such pH levels. A working pH that is closer to a protein’s pI gives the molecule near-neutral charge. Ion-exchange chromatography (IEC) can be useful for oligomer-content reduction at production scale both in binding and nonbinding modes, depending on the type of ion-exchange resin used. Size-exclusion chromatography (SEC) is an efficient unit operation for removing oligomers, but it is not used widely because of low resolution, sample-volume limitations, long process times, and high costs. Protein molecules experience ranges of pH, ionic strength, diafilterability, and concentration factors in downstream processing, all of which can influence the degree of oligomer formation. Most pharmacopoeias specify oligomer content in a range from 0.5% to 3.0% depending on the nature and origin of a therapeutic protein as well as duration of therapy.įor biotherapeutics, oligomer presence not only is undesirable, but also introduces the risk of immunogenic reactions upon injection. Proteins in such drug products can develop aggregation as high concentrations promote associations among them, ultimately decreasing drug stability and compromising shelf life. Sometimes, highly concentrated liquid formulations are required to achieve a desired therapeutic dose. Chemical aggregation pathways include disulfide-bond formation or exchange due to oxidation, deamidation, and formation of free thiol groups. Protein aggregation can be initiated by a number of physical factors, including temperature (e.g., freezing and thawing), ionic strength, and interacting microenvironment exposure in a bioprocess (e.g., mixing during a change in pH or temperature). Depending on the physicochemical characteristics of a protein, oligomers can compromise product quality - specifically in safety, efficacy, delivery, dosing, and/or marketability. Protein aggregation can be reversible or irreversible because the oligomers formed may be soluble or insoluble, covalent or noncovalent, and native or nonnative. Thus, techniques are needed that can reduce oligomers to well below acceptance limits and ensure optimal safety levels. Much research is ongoing in this area, with limited successes in purifying proteins from oligomers. ![]() A downstream process should be robust and reproducible at scale-up with minimum impact on recovery. ![]() To minimize the yield loss during extensive reduction of oligomers, we considered the use of novel techniques with process optimization. With those pI similarities, removal of oligomers to a considerable extent by ion exchangers can compromise protein recovery, thus causing unacceptable product loss during processing. Proteins such as hormones have pI ranges similar to their oligomers and thus can be difficult to separate out using a conventional polishing chromatographic step such as ion exchange. Sometimes it is difficult to purify monomeric proteins from oligomers because of similarities in their isoelectric points (pIs). Aggregation is a common cause of protein instability, which renders a biologic product unfit for therapeutic use. ![]()
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