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Monoclonal antibodies (mAbs) are emerging biopharmaceuticals that have drawn great public attention due to their potential to treat many thorny diseases such as cancers, infections, autoimmune disorders and transplant rejection. However, because their production typically relies on biological means and their biological nature is giant and complex proteins, they are prone to be contaminated by impurities introduced during the production process or generated due to the instability of the products themselves. Both process-related and product-related contaminants need to be strictly monitored throughout the entire process from production to consumption. CESDS is a canonical method widely used to determine the purity of biopharmaceuticals like mAbs. Compared with early techniques such as SDS-PAGE, it can separate components of different sizes in the analytes in a quick and precise manner. In this review, we demonstrate the principle underlying the function of CE-SDS and summarize the applications of CE-SDS in assessing major types of impurities. We will also discuss the limitations and advantages of CE-SDS compared with other techniques to point out potential directions for future research. [ABSTRACT FROM AUTHOR]

Abstract Bispecific antibodies (bsAbs) are therapeutically promising due to their ability to bind to two different antigens. However, the bsAb byproducts and impurities, including mispaired homodimers, half-antibodies, light chain mispairings, antibody fragments and high levels of high molecular weight (HMW) species, all pose unique challenges to their downstream processing. Here, using two knob-into-hole (KiH) constructs of bsAbs as model molecules, we demonstrate the excellent removal of bsAb byproducts and impurities in a single Protein A chromatography under optimized conditions, including hole–hole homodimer mispaired products which are physicochemically very similar to the target bsAbs and still present even with the use of the KiH format, though at reduced levels. The removal occurs through the incorporation of an intermediate low-pH wash step and optimal elution conditions, achieving ~ 60% monomeric purity increase in a single Protein A step, without the introduction of sequence-specific bsAb modifications to specifically induce differential Protein A binding. Our results also suggest that the higher aggregation propensity of bsAbs may cause aggregation during the column process, hence an optimization of the appropriate loading amount, which may be lower than that of monoclonal antibodies (mAbs), is required. With the use of loading at 50% of 10% breakthrough (QB10) at 6-min residence time, we show that an overall high monomer purity of 92.1–93.2% can be achieved with good recovery of 78.4–90.6% within one capture step, which is a significant improvement from a monomer purity of ~ 30% in the cell culture supernatant (CCS). The results presented here would be an insightful guidance to all researchers working on the purification process development to produce bispecific antibodies, especially for knob-into-hole bispecific antibodies. Graphical Abstract

The preferred test methods for control of product-related impurities in medicinal products are high-performance liquid chromatography (HPLC) with a fine sorbent, and ultra-performance liquid chromatography (UPLC), which allow for better chromatographic separation of active substances and related impurities, reduction of time costs, and saving of material resources. The aim of the study was to develop HPLC and UPLC test procedures and assess the chromatographic separation capacity and efficiency in order to improve determination of the main vancomycin component and related impurities. Materials and methods: vancomycin hydrochloride lyophilisate for oral solution and solution for injection, and vancomycin hydrochloride reference standard (USP RS) were used as test objects. Agilent 1290 Infinity liquid chromatography system, and Chromolith® Performance RP-18e, Kinetex C18, Nucleodur C18 Isis, Zorbax RRHD Eclipse Plus C18, and LiChrospher® RP-18 columns were used for the testing. Results: HPLC analysis using a Chromolith® column (100×4.6 mm) reduces the testing time by 10 minutes compared to the USP test procedure, and by 15 minutes compared to the British Pharmacopoeia procedure. The proposed test procedure requires less eluent and increases chromatographic separation efficiency. UPLC analysis using a Kinetex C18 column (50×4.6 mm, 2.6 μm) made it possible to reduce the testing time by two thirds compared to the British Pharmacopoeia procedure. The use of isocratic elution greatly simplified the testing. The testing time under the proposed chromatographic conditions was 10 minutes. Conclusions: the selected HPLC and UPLC test conditions made it possible to significantly reduce the time of testing, minimise the use of expensive reagents, and increase efficiency of chromatographic separation in the determination of vancomycin impurities and the main component Vancomycin B.

Bispecific antibodies (bsAbs) are therapeutically promising due to their ability to bind to two different antigens. However, the bsAb byproducts and impurities, including mispaired homodimers, half-antibodies, light chain mispairings, antibody fragments and high levels of high molecular weight (HMW) species, all pose unique challenges to their downstream processing. Here, using two knob-into-hole (KiH) constructs of bsAbs as model molecules, we demonstrate the excellent removal of bsAb byproducts and impurities in a single Protein A chromatography under optimized conditions, including hole–hole homodimer mispaired products which are physicochemically very similar to the target bsAbs and still present even with the use of the KiH format, though at reduced levels. The removal occurs through the incorporation of an intermediate low-pH wash step and optimal elution conditions, achieving ~ 60% monomeric purity increase in a single Protein A step, without the introduction of sequence-specific bsAb modifications to specifically induce differential Protein A binding. Our results also suggest that the higher aggregation propensity of bsAbs may cause aggregation during the column process, hence an optimization of the appropriate loading amount, which may be lower than that of monoclonal antibodies (mAbs), is required. With the use of loading at 50% of 10% breakthrough (QB10) at 6-min residence time, we show that an overall high monomer purity of 92.1–93.2% can be achieved with good recovery of 78.4–90.6% within one capture step, which is a significant improvement from a monomer purity of ~ 30% in the cell culture supernatant (CCS). The results presented here would be an insightful guidance to all researchers working on the purification process development to produce bispecific antibodies, especially for knob-into-hole bispecific antibodies. [ABSTRACT FROM AUTHOR]

Bispecific antibodies, a class of therapeutic antibodies, can simultaneously bind to two distinct targets. Compared with monospecific antibodies, bispecific antibodies offer advantages, including superior efficacy and reduced side effects. However, because of their structural complexity, the purification of bispecific antibodies is highly challenging. The purification process must strike a delicate balance between purity and productivity, eliminating a broad spectrum of contaminants, including product-related and process-related impurities, while also maximizing the yield wherever feasible. This review systematically describes the strategies for bispecific antibody capture, the elimination of product-related impurities, and the mitigation of the formation of process-related impurities, thereby, providing guidance for the development of downstream purification processes for bispecific antibodies. [Display omitted] • This manuscript introduces the latest research developments in bispecific antibody capture methods • This article systematically introduces strategies for removing products-related impurities of bispecific antibody. • This article systematically introduces strategies for removing process-related impurities of bispecific antibody. • This review looks ahead to new technologies for the downstream purification of bispecific antibodies [ABSTRACT FROM AUTHOR]

Bispecific protein scaffolds can be more complex than traditional monoclonal antibodies (MAbs) because two different sites/domains for epitope binding are needed. Because of this increased molecular complexity, bispecific molecules are difficult to express and can be more prone to physical and chemical degradation compared to MAbs, leading to higher levels of protein aggregates, clipped species, or modified residues in cell culture. In this study, we investigated cell culture performance for the production of three types of bispecific molecules developed at Amgen. In particular, we cultured a total of six CHO cell lines in both an approximately 12‐day fed‐batch process and an approximately 40‐day high‐density perfusion process. Harvested cell culture fluid from each process was purified and analyzed for product quality attributes including aggregate levels, clipped species, charge variants, individual amino acid modifications and host cell protein (HCP) content. Our studies showed that in average, the intensified perfusion process increased 15‐fold the integrated viable cell density and the total harvested product (and fivefold the daily volumetric productivity) compared to fed‐batch. Furthermore, bispecific product quality improved in perfusion culture (as analyzed in affinity‐capture pools) with reduction in levels of aggregates (up to 72% decrease), clipped species (up to 75% decrease), acidic variants (up to 76% decrease), deamidated/isomerized species in complementarity‐determining regions, and HCP (up to 84% decrease). In summary, the intensified perfusion process exhibited better productivity and product quality, highlighting the potential to use it as part of a continuous manufacturing process for bispecific scaffolds. [ABSTRACT FROM AUTHOR]

• Development of novel multimodal CEX resin with moderate hydrophobic properties. • Three antibodies of varying hydrophobicity successfully purified with novel resin. • Robust process design through high throughput partition coefficient screening. • High selectivity, generic approach indicates broad applicability of novel resin. • Cost-saving potential through replacement of CEX and HIC steps with a single column. Immunoglobulins of complex formats possess great potential for increased biopharmaceutical efficacy. However, challenges arise during their purification as the removal of numerous product-related impurities typically requires several expensive chromatographic steps. Additionally, many complex antibody formats have a high hydrophobicity which impairs the use of conventional mixed mode chromatography. In the present study, both of these challenges were addressed through the development of an innovative mixed mode resin with 2-amino-4methylpentanoic acid ligands that combines weak cation exchange with moderate hydrophobic interactions. Supported by high throughput partition coefficient screens for identification of preferable pH and salt concentration ranges in bind and elute mode, this mixed mode resin successfully demonstrated efficient impurity separation from an extremely hydrophobic bispecific antibody with a single unit operation. High purity (>97%) was obtained as a result of significant reduction of product-related impurities as well as process-related host cell proteins (>3 log scale), while maintaining satisfactory recovery (70%). This also supports that highly hydrophobic antibody formats can be efficiently purified using a resin with moderate hydrophobic characteristics. Studies involving additional antibodies possessing different formats and a wide range of hydrophobicity confirmed the broad applicability of the new resin. In view of its high selectivity and robust operating ranges, as well as the elimination of the need for an additional column step, the novel resin enables simplified downstream processing and economic manufacturing of complex antibody formats. [ABSTRACT FROM AUTHOR]

Classical ion-exchange chromatography using a linear salt gradient to elute the adsorbed protein at fixed pH is the most common method to separate product-related impurities during downstream processing of biopharmaceuticals. Linear pH gradient elution provides a useful alternative by separating proteins in a linear pH gradient at fixed salt concentration. Although linear pH gradient elution provides excellent selectivity, it is rarely encountered in industrial purification processes. Here, a stoichiometric displacement model is used to characterize pH gradient elution based on simple linear gradient elution experiments. Protein retention behavior is described with respect to the pH dependencies of the characteristic binding charge and the equilibrium constant of the ion exchange reaction. Furthermore, the influence of solvent composition using PEG as a mobile phase modifier is investigated. Validity and applicability of the model are demonstrated for the purification of a conventional monoclonal antibody from soluble aggregates and for a novel bispecific antibody format containing a unique product-related impurity profile. pH step elution protocols are derived from model calculations without further optimization experiments necessary. [ABSTRACT FROM AUTHOR]

Background: Significant challenges exist in downstream purification of bispecific antibodies (BsAbs) due to the complexity of BsAb architecture. A unique panel of mispaired species can result in a higher level of product-related impurities. In addition to process-related impurities such as host cell proteins (HCPs) and residual DNA (resDNA), these product-related impurities must be separated from the targeted BsAb product to achieve high purity. Therefore, development of an efficient and robust chromatography purification process is essential to ensure the safety, quality, purity and efficacy of BsAb products that consequently meet regulatory requirements for clinical trials and commercialization., Methods: We have developed a robust downstream BsAb process consisting of a mixed-mode ceramic hydroxyapatite (CHT) chromatography step, which offers unique separation capabilities tailored to BsAbs, and assessed impurity clearance., Results: We demonstrate that the CHT chromatography column provides additional clearance of low molecular weight (LMW) and high molecular weight (HMW) species that cannot be separated by other chromatography columns such as ion exchange for a particular BsAb, resulting in ≥98% CE-SDS (non-reduced) purity. Moreover, through Polysorbate-80 (PS-80) spiking and LC-MS HCP assessments, we reveal complete clearance of potential PS-80-degrading HCP populations in the CHT eluate product pool., Conclusions: In summary, these results demonstrate that CHT mixed-mode chromatography plays an important role in separation of product- and process-related impurities in the BsAb downstream process., (© The Author(s) 2022. Published by Oxford University Press on behalf of Antibody Therapeutics. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Abstract: Product-related impurities constitute a major burden in the production of recombinant viral vectors for gene therapy and vaccination; it impairs not only the biological efficacy of the preparation but the process yield/productivity. Recombinant baculovirus was used as an enveloped virus model to address this issue. Given that ion-exchange chromatography is a process of choice for purification of viral vectors, the analysis of the electrostatic behavior can be instrumental for the improvement of impurity removal. The main species, product (infective virus particle) and product-derived impurities (dsDNA-, glycoprotein-, and envelope-deprived baculovirus particles), were isolated and correspondent ζ potentials were analyzed through dynamic light scattering. A model of the virus based on the viral components critical for biological function is proposed. The contribution of these viral components to the overall particle electrostatic interaction energy profile (calculated between the particle and a putative ion-exchange surface) was assessed as a function of ionic strength and pH. This resulted in a deterministic tool capable of distinguishing the electrostatic properties of the infective virus particle from the major virus-related impurities. Within an ion-exchange bind-elute process, this knowledge helps narrow the optimization space in early stage process development for viral vectors by predicting the best selectivity conditions. [Copyright &y& Elsevier]

Bispecific antibodies (bsAbs) represent a highly promising class of biotherapeutic modality. The downstream processing of this class of antibodies is therefore of crucial importance in ensuring that these products can be obtained with high purity and yield. Due to the various fundamental structural similarities between bsAbs and monoclonal antibodies (mAbs), many of the current bsAb downstream purification methodologies are based on the established purification processes of mAbs, where affinity, charge, size, hydrophobicity and mixed-mode-based purification are frequently employed. Nevertheless, the downstream processing of bsAbs presents a unique set of challenges due to the presence of bsAb-specific byproducts, such as mispaired products, undesired fragments and higher levels of aggregates, that are otherwise absent or present in lower levels in mAb cell culture supernatants, thus often requiring the design of additional purification strategies in order to obtain products of high purity. Here, we outline the current major purification methods of bsAbs, highlighting the corresponding solutions that have been proposed to circumvent the unique challenges presented by this class of antibodies, including differential affinity chromatography, sequential affinity chromatography and the use of salt additives and pH gradients or multistep elutions in various modes of purification. Finally, a perspective towards future process development is offered., (© The Author(s) 2021. Published by Oxford University Press on behalf of Antibody Therapeutics.)