Purification of Modified Therapeutic Proteins Available on the Market: An Analysis of Chromatography-Based Strategies

Abstract

Proteins, which have inherent biorecognition properties, have long been used as therapeutic agents for the treatment of a wide variety of clinical indications. Protein modification through covalent attachment to different moieties improves the therapeutic’s pharmacokinetic properties, affinity, stability, confers protection against proteolytic degradation, and increases circulation half-life. Nowadays, several modified therapeutic proteins, including PEGylated, Fc-fused, lipidated, albumin-fused, and glycosylated proteins have obtained regulatory approval for commercialization. During its manufacturing, the purification steps of the therapeutic agent are decisive to ensure the quality, effectiveness, potency, and safety of the final product. Due to the robustness, selectivity, and high resolution of chromatographic methods, these are recognized as the gold standard in the downstream processing of therapeutic proteins. Moreover, depending on the modification strategy, the protein will suffer different physicochemical changes, which must be considered to define a purification approach. This review aims to deeply analyze the purification methods employed for modified therapeutic proteins that are currently available on the market, to understand why the selected strategies were successful. Emphasis is placed on chromatographic methods since they govern the purification processes within the pharmaceutical industry. Furthermore, to discuss how the modification type strongly influences the purification strategy, the purification processes of three different modified versions of coagulation factor IX are contrasted.

Chemical Synthesis and Semisynthesis of Lipidated Proteins

“…The use of columns with longer alkyl chains such as C 18 complicate purification because of the higher affinity of the lipid modification to these stationary phases. [99] Thec hoice of organic eluent and modifier for RP-HPLC is also critical, as is the temperature.N otably,o ptimized purification conditions usually need to be identified for each protein individually (or at least for ac lass of proteins) and one must ensure that the use of low pH and/or high temperatures during purification does not lead to decomposition of the proteins or cleavage of lipid chains. [100] In many cases,t he addition of organic solvents miscible with water, such as acetonitrile or fluorinated alcohols,help to solubilize lipidated peptides and proteins before and during purification.…”

“…Here, the use of short‐chain stationary phases with sufficiently large pore size is recommended, for example, a C 4 stationary phase with a pore size of 30 nm. The use of columns with longer alkyl chains such as C 18 complicate purification because of the higher affinity of the lipid modification to these stationary phases [99] . The choice of organic eluent and modifier for RP‐HPLC is also critical, as is the temperature.…”

Chemische Synthese und Semisynthese von lipidierten Proteinen

“…B. eine C 4 ‐Säule mit einer Porengröße von 30 nm. Die Nutzung von Säulen mit längeren Alkylketten, wie beispielsweise C 18 , verkompliziert die Aufreinigung durch eine erhöhte Affinität der Lipidmodifikation zu diesen stationären Phasen [99] . Die Wahl des organischen Eluenten und Additivs für RP‐HPLC sowie die Temperatur sind wichtige weitere Parameter.…”

“…Die Nutzung von Säulen mit längeren Alkylketten, wie beispielsweise C 18 ,v erkompliziert die Aufreinigung durch eine erhçhte Affinitätder Lipidmodifikation zu diesen stationären Phasen. [99] Die Wahl des organischen Eluenten und Additivs fürR P-HPLC sowie die Temperatur sind wichtige weitere Parameter.E si st wichtig anzumerken, dass normalerweise fürj edes Protein (oder zumindest für verschiedene Proteinklassen) individuelle optimierte Aufreinigungsbedingungen identifiziert werden müssen. Außerdem muss sichergestellt werden, dass die Nutzung eines niedrigen pH und/oder hoher Temperaturen während der Aufreinigung nicht zu einer Zersetzung des Proteins oder zur Abspaltung der Lipidketten führt.…”

Highly Expressed Soluble Recombinant Anti-GFP VHHs in Escherichia coli via Optimized Signal Peptides, Strains, and Inducers

“…Recombinant protein production via prokaryotic system has brought hundreds of therapeutic proteins into clinical applications or clinical trials over the past few decades ( Dimitrov, 2012 ; Sánchez-Trasviña et al, 2021 ). Recombinant proteins are frequently soluble.…”