scholarly journals Current trends and challenges in the downstream purification of bispecific antibodies

2021 ◽  
Author(s):  
Serene W Chen ◽  
Wei Zhang
Keyword(s):  
Affinity Chromatography ◽  
High Purity ◽  
Process Development ◽  
Downstream Processing ◽  
Bispecific Antibodies ◽  
Ph Gradients ◽  
Current Trends ◽  
Purification Methods ◽  
Culture Supernatants ◽  
Differential Affinity

Abstract 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 multi-step elutions in various modes of purification. Finally, a perspective towards future process development is offered. Statement of significance: This review aims to present the key structural properties of bsAbs and their associated byproducts, outlining the current major purification methods of bsAbs and highlighting the corresponding solutions that have been proposed to circumvent the challenges, as well as to offer a perspective towards future process development.

scholarly journals A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

2020 ◽  
Author(s):  
Cecy Xi ◽  
Arianna Arianna Di Fazio ◽  
Naveed Nadvi ◽  
Karishma Patel ◽  
Michelle Xiang ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
High Purity ◽  
Specific Activity ◽  
Specific Binding ◽  
Exchange Chromatography ◽  
Cell Surface Receptor ◽  
Spike Protein ◽  
High Yield ◽  
Purification Procedure ◽  
Dipeptidyl Peptidase 4

Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulfate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor binding domain (RBD) were measured using surface plasmon resonance. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.

scholarly journals Sustainable strategies based on glycine–betaine analogue ionic liquids for the recovery of monoclonal antibodies from cell culture supernatants

2019 ◽  
Vol 21 (20) ◽  
pp. 5671-5682 ◽  
Author(s):  
Emanuel V. Capela ◽  
Alexandre E. Santiago ◽  
Ana F. C. S. Rufino ◽  
Ana P. M. Tavares ◽  
Matheus M. Pereira ◽  
...  
Keyword(s):  
Cell Culture ◽  
Monoclonal Antibodies ◽  
Downstream Processing ◽  
Aqueous Biphasic Systems ◽  
Phase Partitioning ◽  
Hybrid Processes ◽  
Three Phase ◽  
Aqueous Biphasic ◽  
Biphasic Systems ◽  
Culture Supernatants

Ionic-liquid-based aqueous biphasic systems, three phase partitioning methods and hybrid processes combined with ultrafiltration are sustainable strategies for the downstream processing of monoclonal antibodies.

scholarly journals A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

Molecules ◽  
2020 ◽  
Vol 25 (22) ◽  
pp. 5392
Author(s):  
Cecy R Xi ◽  
Arianna Di Fazio ◽  
Naveed Ahmed Nadvi ◽  
Karishma Patel ◽  
Michelle Sui Wen Xiang ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Affinity Chromatography ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
High Purity ◽  
Specific Activity ◽  
Cell Surface Receptor ◽  
Spike Protein ◽  
High Yield ◽  
Purification Procedure

Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of the MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29–766) produced in insect cells. Purification used differential ammonium sulphate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion-exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor-binding domain (RBD) were measured using surface plasmon resonance and ELISA. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected by surface plasmon resonance or ELISA. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.

scholarly journals Optimization and in Silico Analysis of a Cold-Adapted Lipase from an Antarctic Pseudomonas sp. Strain AMS8 Reaction in Triton X-100 Reverse Micelles

Catalysts ◽  
2018 ◽  
Vol 8 (7) ◽  
pp. 289 ◽  
Author(s):  
Fatin Abd. Jalil ◽  
Raja Raja Abd. Rahman ◽  
Abu Salleh ◽  
Mohd Mohamad Ali
Keyword(s):  
In Silico ◽  
Reverse Micelles ◽  
Catalytic Domain ◽  
In Silico Analysis ◽  
Downstream Processing ◽  
Cold Adapted ◽  
Sodium Phosphate Buffer ◽  
Purification Technique ◽  
Purification Methods ◽  
Triton X

A moderate yield of a purified enzyme can be achieved by using the simple technique of reverse micellar extraction (RME). RME is a liquid–liquid extraction method that uses a surfactant and an organic solvent to extract biomolecules. Instead of traditional chromatographic purification methods, which are tedious and expensive, RME using the nonionic surfactant Triton X-100 and toluene is used as an alternative purification technique to purify a recombinant cold-adapted lipase, AMS8. Various process parameters were optimized to maximize the activity recovery of the AMS8 lipase. The optimal conditions were found to be 50 mM sodium phosphate buffer, pH 7, 0.125 M NaCl, and 0.07 M Triton X-100 in toluene at 10 °C. Approximately 56% of the lipase activity was successfully recovered. Structural analysis of the lipase in a reverse micelle (RM) was performed using an in silico approach. The predicted model of AMS8 lipase was simulated in the Triton X-100/toluene reverse micelles from 5 to 40 °C. The lid 2 was slightly opened at 10 °C. However, the secondary structure of AMS8 was most affected in the non-catalytic domain compared to the catalytic domain, with an increased coil conformation. These results suggest that an AMS8 lipase can be extracted using Triton X-100/water/toluene micelles at low temperature. This RME approach will be an important tool for the downstream processing of recombinant cold-adapted lipases.

scholarly journals Current Trends in Protein Purification : A Review

2020 ◽  
pp. 279-310
Author(s):  
Angela Boxi ◽  
Isha Parikh ◽  
Radhika B S ◽  
Shryli K S
Keyword(s):  
High Pressure ◽  
Liquid Chromatography ◽  
Ion Exchange ◽  
Affinity Chromatography ◽  
Protein Purification ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Current Trends ◽  
Comparative Information

The present review is based on papers published between 1990 and 2020 and gives Comparative information about the most common protein purification techniques Gel-Filtration, Chromatography, Ion-Exchange Chromatography, Electrophoresis, Affinity Chromatography, and Dialysis, High-Pressure Liquid Chromatography. and their applications.

scholarly journals Current tendencies of migration process development: global features and implications for Ukraine

2020 ◽  
Keyword(s):  
Human Capital ◽  
Process Development ◽  
Donor Country ◽  
Recipient Country ◽  
Negative Consequences ◽  
Global Features ◽  
Current Trends ◽  
Main Effects ◽  
The Eu

The article considers and summarizes the main global features and consequences of migration processes, including Ukraine. The purpose of the article is to establishing current trends in the development of migration processes, namely the global features and consequences for Ukraine. The grouping and generalization methods are used in the article (to represent the main effects of migration processes for donor countries, intermediate countries and recipient countries). The graphic method is applied to reflect the dynamics of changes in the number of emigrants from Ukraine, who were granted the first residence permits in the EU from 2009 to 2018. Methods of concretization and synthesis were used in determining the main consequences of migration processes for Ukraine. As a result of the research, the classification of world countries depending on the directions of migration flows (donor countries, countries of intermediate location and recipient countries) was determined. The list of the largest donor countries, recipient countries in the world with the indication of the number of migrants in these countries was determined. The main consequences of migration processes for world countries were determined, concretized and grouped according to the degree of their influence. The list of countries that are the largest centers of emigration for Ukrainian citizens (Poland, USA, Germany, Canada, Czech Republic) was determined. The main reasons for the increase in the number of emigrants from Ukraine in the periods from 2009 to 2012 and from 2012 to 2018 have been identified. The main consequences of migration processes for Ukraine, as a country-donor of human capital, a country of intermediate location and a recipient country, have been identified and grouped. The predominance of negative consequences of migration processes for Ukraine, as a donor country of human capital, a country of intermediate location, have been determined.

scholarly journals Process Development and Scale-up Optimization of the SARS-CoV-2 Receptor Binding Domain-Based Vaccine Candidate, RBD219-N1C1

2020 ◽  
Author(s):  
Jungsoon Lee ◽  
Zhuyun Liu ◽  
Wen-Hsiang Chen ◽  
Junfei Wei ◽  
Rakhi Kundu ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Process Development ◽  
Low Cost ◽  
Scale Up ◽  
Neutralizing Antibody ◽  
Protein Antigen ◽  
Purification Process ◽  
Good Manufacturing Practices ◽  
Purification Methods ◽  
Selection Of

AbstractA SARS-CoV-2 RBD219-N1C1 (RBD219-N1C1) recombinant protein antigen formulated on Alhydrogel® has recently been shown to elicit a robust neutralizing antibody response against SARS-CoV-2 pseudovirus in mice. The antigen has been produced under current good manufacturing practices (cGMP) and is now in clinical testing. Here, we report on process development and scale-up optimization for upstream fermentation and downstream purification of the antigen. This includes production at the 1 and 5 L scale in the yeast, Pichia pastoris, and the comparison of three different chromatographic purification methods. This culminated in the selection of a process to produce RBD219-N1C1 with a yield of >400 mg per liter of fermentation with >92% purity and >39% target product recovery after purification. In addition, we show the results from analytical studies, including SEC-HPLC, DLS, and an ACE2 receptor binding assay that were performed to characterize the purified proteins to select the best purification process. Finally, we propose an optimized upstream fermentation and downstream purification process that generates quality RBD219-N1C1 protein antigen and is fully scalable at a low cost.

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