Precision Capturing Publications

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Overcoming challenges in Ferritin extraction from blood plasma: precise affinity chromatography solutions

Ferritin is a critical protein complex essential for iron storage and release in living organisms. Ensuring high purity and effective isolation of Ferritin is vital for its various biomedical and industrial applications. However, due to its low presence in human plasma, the purification of Ferritin can be very challenging.

The small amount of Ferritin (13-400 ng/mL) present in plasma makes purification a significant challenge for purification techniques. However, we have successfully overcome this challenge. Our next-gen Affinity Chromatography (AC) methods enable the isolation and purification of Ferritin with exceptional precision, ensuring high purity and yield even from very small initial amounts. This achievement underscores our commitment to leveraging cutting-edge technology to solve complex biochemical problems.

The development of AC for Ferritin purification is a significant bioprocessing advancement. This technique improves the efficiency of Ferritin isolation and ensures the production of high-quality Ferritin for applications in medical diagnostics and therapy. It can serve as a blueprint for many other antibodies, non-antibodies and proteins with clinical potential facing purification obstacles.

The custom affinity resin was developed using the Precision Capturing® technology. The platform brings the benefits of Protein A affinity chromatography to the space of non-antibody proteins. Precision Capturing® enables a simpler process architecture resulting in optimized purity and yield. It is a unique and innovative affinity technology for DSP development to achieve a robust, predictable, scalable, and GMP-compliant purification procedure.

© 2024 Published by Navigo Proteins GmbH and PreviPharma Consulting GmbH

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Evaluation of mild pH elution protein A resins for antibodies and Fc-fusion proteins

Protein A affinity chromatography is widely used as a capture step for monoclonal antibodies (mAb) and molecules that possess an Fc-domain, such as fusion proteins and bispecific antibodies. However, the use of low pH (3.0–4.0) to elute the molecule and achieve acceptable yield (>85 %) can lead to product degradation (e.g. fragmentation, aggregation) for molecules sensitive to low pH. In this paper, we describe a comprehensive evaluation of two protein A resins with ligands designed to elute at a milder pH as a result of modified sequences in their Fc and VH3 binding regions. One of the evaluated resins has been made commercially available by Purolite and named Praesto Jetted A50 HipH. Results demonstrated that Jetted A50 HipH could elute the Fcfusion protein and most mAbs evaluated with an elution pH at or above 4.6. Elution and wash optimization determined run conditions for high recovery (>90 % monomer yield), reduction of high molecular weight (HMW) species (>50 %), and significant host cell protein (HCP) clearance at the mildest elution pH possible. For a pH-stable mAb and a pH-sensitive fusion protein, cell culture material was purified with optimized conditions and demonstrated the mild elution pH resins’ ability to purify product with acceptable yield, comparable or better impurity clearance, and significantly milder native eluate pH compared to traditional resins. The benefits of the mild elution pH resins were clearly exemplified for the pH-sensitive protein, where a milder elution buffer and native eluate pH resulted in only 2 % HMW in the eluate that remained stable over 48 h. In contrast, a traditional protein A resin requiring low pH elution led to eluate HMW levels of 8 %, which increased to 16 % over the same hold time. Additionally, these resins have high dynamic binding capacity and allow the use of traditional HCP washes. Therefore, Jetted A50 HipH is an ideal candidate for a platform protein A resin and provides flexibility for pH-sensitive proteins and stable mAbs, while preserving product quality, recovery, and seamless integration into a downstream process.

© 2024 The Authors. Published by Elsevier Ltd.

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Affinity purification of SARS-CoV-2 spike protein receptor binding domain produced in a C1 fungal expression system

The Receptor Binding Domain (RBD) of the spike protein of SARS-CoV-2 has shown promise for diagnosis, treatment, and development of vaccines for COVID-19. However, two problems persist with large scale production of RBD: 1) lack of high productivity upstream cell culture, 2) absence of a commercial, highly selective affinity resin. In an effort to overcome these limitations, we evaluated two novel technologies for the production and purification of RBD. Brieflccy, RBD was expressed using C1, an engineered fungal strain of Thermothelomyces heterothallica (Dyadic International1 ). The C1 platform expresses glycosylated antigens with high productivity, stability, and purity. RBD was purified using a novel affinity resin2 known to produce yields of 90% to 95% purity in one chromatography step. Affinity purification did not affect protein quality, as demonstrated by ACE-2 binding of RBD. The novel affinity resin showed excellent base stability, consistent product quality, and similar ACE-2 binding activity over 40 cycles. RBD produced in C1, in conjunction with affinity purification using a novel affinity resin, provides a breakthrough in the large-scale production of affordable COVID-19 protein-based vaccines.

© 2021 Dutta et. al. 

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Affinity chromatography for vaccines manufacturing: Finally ready for prime time?

Affinity chromatography is among the most powerful separation techniques, achieving the finest separation with high yields even in the most challenging feed streams. Incorporating affinity chromatography in vaccine purification has long been attempted by researchers to improve unit yield and purity with the secondary goal of reducing the number of downstream process operations. Despite the success in laboratory-scale proof of concept, implementation of this technique in pilot or cGMP manufacturing has rarely been realised due to technical and economic challenges in design and manufacturing of ideal ligands as well as availability of high-productivity chromatography media. This paper reviews evolving technologies in engineered ligands and chromatography media that are encouraging companies to revisit the possible use of affinity chromatography in larger scale vaccine purification. It is postulated that commercial-scale implementation of high throughput single-use affinity chromatography can significantly simplify process architecture, improve productivity and flexibility, and reduce cost of goods.

© 2018 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license.

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Affinity Membrane Chromatography For Robust And Cost-Efficient Industrial Vaccine Purification

Due to the outdated manufacturing technologies used for majority of legacy products, many current vaccines processes suffer from a lack of control and robustness; hence, the highest levels of quality are maintained only at the expense of extensive QC release testing and high lot rejection rates. While manufacturers are engaged in life cycle management projects, the use of process chromatography is gaining its popularity but the implementation is still limited mostly due to the low throughput and vastly capital expense associated with resin columns. This work presents an innovative process architecture strategy taking advantage of the purification power of affinity chromatography and the high throughput of hydrogel membranes, while leveraging all the proven benefits of disposable technologies. Proof of concept studies combining Scil Affilin ligands and Natrix hydrogel membranes technologies are presented. The potential for a robust, high yield and generic vaccine affinity-based purification platform is discussed. Relying on a full process economic model independently developed by a customer, the impact of this approach on cost of goods of the drug substance for a current vaccine candidate is demonstrated.

© 2015 Zhao et al