Current Protein and Peptide Science - Volume 20, Issue 1, 2019

Process chromatography forms the core of purification of biotherapeutics. The unparalleled selectivity that it offers over other alternatives combined with the considerable robustness and scalability make it the unit operation of choice in downstream processing. It is typical to have three to five chromatography steps in a purification process for a biotherapeutic. Generally, these steps offer different modes of separation such as ion-exchange, reversed phase, size exclusion, and hydrophobic interaction. In the past decade, multimodal chromatography has emerged as an alternative to the traditional modes. It involves use of more than one mode of separation and typically combines ion-exchange and hydrophobic interactions to achieve selectivity and sensitivity. Over the last decade, numerous authors have demonstrated the significant potential that multimode chromatography offers as a protein purification tool. This review aims to present key recent developments that have occurred on this topic together with a perspective on future applications of multimodal chromatography.

Mixed mode chromatography offers a diversity of ligands, each providing a new selectivity. This allows the design of novel purification processes with reduced column steps. Structure of ligands is based on both hydrophobic and ionic groups. Thanks to its salt tolerance, crude extracts or post-IEX samples can be loaded directly without conditioning. The selectivity could be enhanced by modulating elution parameters or by using additives. More importantly, mixed mode chromatography could be as effective as affinity chromatography for mAb purification processes. Mixed mode chromatography opens the way to short and economical processes.

Mixed mode chromatography resins with salt tolerance, large design space and orthogonal selectivity requires a slightly more complex development than traditional resins. It is important to screen several ligands and several binding and elution conditions. This allows taking full advantage of these resins. High-Throughput Screening (HTS) for Process Development should be done with the help of Design of Experiment (DoE). It could be performed in filter plates or Robocolumns, and assisted by liquid handling automated workstation. Modeling of the results allows the choice of optimal parameters that can then be validated and scaled up. All this leads to a better knowledge and robustness of the purification step.

MEP (mercapto-ethyl-pyridine) HyperCel is one of the hydrophobic charge induction chromatography (HCIC) resins. Under normal operation, proteins are bound to the MEP resin at neutral pH, at which MEP is not charged, mostly via hydrophobic interaction. MEP has a pyridine group, whose pK is 4.8, and hence is positively charged at acidic pH range. Based on the binding mechanism (i.e., hydrophobic interaction) and the induced positive charge at acidic pH, there may be two ways to elute the bound proteins. One way is to bring the pH down to protonate both MEP resin and the bound protein, leading to charge repulsion and thereby elution. Another way is to use hydrophobic interaction modifiers, which are often used in hydrophobic interaction chromatography, to reduce hydrophobic interaction. Here, we summarize such two possible elution approaches.

Previously, we have reviewed in this journal (Arakawa, T., Kita, Y., Curr. Protein Pept. Sci., 15, 608-620, 2014) the interaction of arginine with proteins and various applications of this solvent additive in the area of protein formulations and downstream processes. In this special issue, we expand the concept of protein-solvent interaction into the analysis of the effects of solvent additives on various column chromatography, including mixed-mode chromatography. Earlier in our research, we have studied the interactions of such a variety of solvent additives as sugars, salts, amino acids, polymers and organic solvents with a variety of proteins, which resulted in mechanistic understanding on their protein stabilization and precipitation effects, the latter known as Hofmeister series. While such a study was then a pure academic research, rapid development of genetic engineering technologies and resultant biotechnologies made it a valuable knowledge in fully utilizing solvent additives in manipulation of protein solution, including column chromatography.

Multimodal or mixed-mode chromatography can be used to separate various proteins, including antibodies. The separation quality and efficiency have been improved by the addition of solutes, especially arginine. This review summarizes the mechanism underlying the effects of arginine on protein elution in multimodal chromatography with neutral, anionic or cationic resin ligands; the mechanism has been investigated using experiments and molecular dynamics simulations. Arginine is effective in facilitating protein elution compared to salts and protein denaturants such as guanidine and urea. The unique elution effect of arginine can be explained by the interplay among arginine, proteins and the resin ligands. Arginine exhibits multiple binding modes for the ligands and further affinity for protein aromatic residues through its guanidinium group. These properties make arginine versatile for protein elution in multimodal chromatography. Taking into account that arginine is an aggregation suppressor for proteins but not a protein denaturant, arginine is a promising protein-eluting reagent for multimodal chromatography.

The needs for purified nucleic acids for preparative and analytical applications have increased constantly, demanding for the development of new and more efficient methods for their recovery and isolation. DNA molecules harbour some intrinsic chemical properties that render them suitable for chromatographic separations. These include a negatively charged phosphate backbone as well as a hydrophobic character originating mainly from the major groove of DNA which exposes the base pairs on the surface of the molecule. In addition, single stranded DNA often allows for a free exposure of the hydrophobic aromatic bases. In this review, multimodal chromatography (MMC) has been evaluated as an alternative tool for complex separations of nucleic acids. MMC embraces more than one kind of interaction between the chromatographic ligand and the target molecules. These resins have often proved superior to conventional single-mode chromatographic materials for DNA isolation, including, e.g., the purification of plasmid DNA from crude cell lysates and for the preparation of DNA fragments before or after a polymerase chain reaction (PCR).

Proteins often generate structure isoforms naturally or artificially due to, for example, different glycosylation, disulfide scrambling, partial structure rearrangement, oligomer formation or chemical modification. The isoform formations are normally accompanied by alterations in charged state or hydrophobicity. Thus, isoforms can be fractionated by reverse-phase, hydrophobic interaction or ion exchange chromatography. We have applied mixed-mode chromatography for fractionation of isoforms for several model proteins and observed that cation exchange Capto MMC and anion exchange Capto adhere columns are effective in separating conformational isoforms and self-associated oligomers.

TOYOPEARL particles are cross-linked hydroxylated methacrylic polymers available in different pore and particle sizes. They are conjugated with different ligands to generate ion-exchange, hydrophobic interaction and affinity resins. They have excellent physical and chemical properties. A mixed-mode resin, TOYOPEARL MX-Trp-650M, is made of this particle with tryptophan conjugated via N-terminal amino group and hence has both hydrophobic/aromatic side chain and carboxyl group. In this review, I will summarize the properties of the TOYOPEARL particles and MX-Trp-650M resin and application of this resin for purification of proteins and in some detail the separation of disulfide (SS)- scrambled oligomers of insulin-like growth factor-1 (IGF-1). For this particular application, the intact IGF-1 was used to examine binding and elution conditions of TOYOSCREEN MX-Trp-650M column. Strong binding was obtained at pH 4.0, at which arginine, but not NaCl, resulted in elution. Both NaCl and arginine resulted in elution at pH 6.5. In addition, a pH gradient from 4.0 to 8.5 was effective. When applied to SS-scrambled IGF-1 oligomers, both pH and arginine gradient exhibited an efficient separation of the oligomers.

Background: Mixed-mode chromatography is becoming an important tool for downstream process purification, as it provides the selectivity and robustness unmatched by conventional singlemode chromatographic methods. The joint action of multiple functionalities present on the ligands of mixed-mode chromatography matrices effectively enhances the separation of target molecules from impurities. Material and Methods: Using Nuvia cPrime as an example, we elucidate the separation principles of hydrophobic cation exchange mixed-mode chromatography and its difference from traditional strong cation exchangers. We have developed a Nuvia cPrime based polish purification step specifically for the removal of a major process contaminant, which has an isoelectric point similar to that of the target monoclonal IgM molecule. Additional purification was accomplished using a second mixed-mode chromatography column packed with Ceramic Hydroxyapatite. Conclusion: The monoclonal IgM prepared with this new process fully retained its biological activity and was free of high molecular weight aggregates, a product quality that was not achievable in previous attempts using traditional ion exchange or hydrophobic interaction chromatography.

Background: Retention mechanism of proteins in hydroxyapatite chromatography (HAC) was investigated by linear gradient elution experiments (LGE). Materials and Methods: Several mobile phase (buffer) solution strategies and solutes were evaluated in order to probe the relative contributions of two adsorption sites of hydroxyapatite (HA) particles, C-site due to Ca (metal affinity) and P-site due to PO4 (cation-exchange). When P-site was blocked, two basic proteins, lysozyme (Lys) and ribonuclease A(RNase), were not retained whereas cytochrome C(Cyt C) and lactoferrin (LF) were retained and also retention of acidic proteins became stronger as the repulsion due to P-site was eliminated. The number of the binding site B values determined from LGE also increased, which also showed reduction of repulsion forces. Conclusion: The selectivity (retention) of four basic proteins (RNase, Lys, Cyt C, LF) in HAC was different from that in ion-exchange chromatography. Moreover, it was possible to tune the selectivity by using NaCl gradient.

Programmed death ligand 1 (PD-L1) is a cell membrane protein that binds to programmed cell death protein 1 (PD-1) on the effector T cells and transduces immunosuppressive signals. It is now clear that the expression of the PD-L1 protein on the tumor cell surface is critical for tumor cells to escape immunosuppression. At present, more attention is focused on the transcriptional regulation of PDL1 mRNA. However, PD-L1 protein is the functional unit involved in immunotherapy response. It is essential to deeply understand how this membrane protein is regulated post-transcriptionally in tumors and immune cells. In this review, we summarize the recent progress on the translation, modification and transport of PD-L1 protein.

Diabetic retinopathy (DR) remains the leading cause of blindness in working-aged adults around the world. The proliferative diabetic retinopathy (PDR) and diabetic macular edema (DME) are the severe vision threatening stages of the disorder. Although, a huge body of research exists in elaborating the pathological mechanisms that lead to the development of DR, the certainty and the correlation amongst these pathways remain ambiguous. The complexity of DR lies in the multifactorial pathological perturbations that are instrumental in both the disease development and its progression. Therefore, a holistic perspective with an understanding of these pathways and their correlation may explain the pathogenesis of DR as a unifying mechanism. Hyperglycemia, oxidative stress and inflammatory pathways are the crucial components that are implicated in the pathogenesis of DR. Of these, hyperglycemia appears to be the initiating central component around which other pathological processes operate. Thus, this review discusses the role of hyperglycemia, oxidative stress and inflammation in the pathogenesis of DR, and highlights the cross-talk amongst these pathways in an attempt to understand the complex interplay of these mechanisms. Further, an effort has been made to identify the knowledge gap and the key players in each pathway that may serve as potential therapeutic drug targets.