2. Induce Cu , Zn to inhibit Trx activity during cell harvest
Protein capture is a recurring topic, and researchers have designed different tags on proteins to achieve effective separation [ 18 ]. While the capture of mAbs has been well-established, the capture methods for complex fusion proteins, tag-free recombinant proteins, and vaccines can vary significantly, depending on the unique properties of each protein.
Affinity chromatography (AC) is widely used in the capture of mAbs, which relies on the specific binding between the target antibody with AC resin. As the complexity of molecular design increases, various antibody fragment-based fusion proteins have been developed, such as Fc-fusion proteins, ScFv-fusion proteins and VHH-fusion proteins.
As shown in Figure 2 , antibodies are composed of different structural domains, and different domains can be designed and produced as recombinant fusion proteins with therapeutic activity. In antibody process development, Protein A, Protein G and Protein L AC have been developed for the capture of antibodies at different sites [ 7 ]. Fc-fusion proteins are the most common design structure and can typically be captured using Protein A and Protein G chromatography. Protein A capture is made possible through the interaction between Protein A and the Fc region of the target molecule as well as the heavy chain variable domain (VH) region of the HC for targets belonging to the VH3 gene family [ 19 ]. Protein G recognizes the Fab and Fc antibody regions in a manner similar to Protein A but with different binding specificities. The protein designed based on ScFv is another form of composition, and these forms are achieved through different combinations of chains and linkers. Protein L and Protein A can specifically recognize the VL and VH regions of ScFv for selective capture.
Schematic representation of IgG and fusion proteins classified based on their affinity binding domain.
Selection of the optimal affinity resin is the primary task of protein capture. Theoretically, Protein A, Protein G and Protein L can all be used for capture of Fab fragment, they showed vastly different binding capability in applications. Different ligand structures, ligand densities and resin matrix led to significant difference in protein capture capability among same ligand types. While the principle can offer guidance for the selection of capture resins during process development, it is important to note that various resins may demonstrate distinct specificities for different molecules. Our own practice showed that Protein L resin demonstrated a superior capture ability for a Fab fragment, and Protein L resin from different sources displayed distinct capture capabilities. ( Supplementary Fig. S1 ).
Besides the selection of resin, the choice of buffer system is also crucial for protein capture, especially for unstable fusion proteins. AC typically employs acidic elution to separate the target protein from the resin, under which condition the protein may become unstable. In addition, for acidic fusion proteins, the adjustment of pH across its isoelectric point (pI) may lead to protein precipitation or aggregation. Thus, for fusion proteins that are sensitive to acidic conditions, it is recommended to select a resin with lower affinity and reduce the buffering capacity of the elution buffer to increase the elution pH. Our own practice showed that the effect of affinity resin, elution buffer concentration and pH on the capture of an acidic fusion protein ( Fig. 3 ). This protein precipitates when exposed to acidic conditions. Affinity resin#2 has been specifically designed to enable the elution of protein at high pH and avoid protein precipitation under acidic conditions. Additionally, the affinity resin#2 exhibits lower affinity to protein, allowing for high recovery during high pH elution. Reducing the buffering capacity of the elution buffer can further increase the pH of the protein eluate. After optimization, the pH of protein eluate remains above its pI throughout the entire capture process, thus avoiding post elution pH adjustment and product loss through aggregation.
Effect of affinity resin and elution conditions on the eluate pH and recovery of acidic fusion protein (pI = 5.4, Mw = 111 kDa).
Due to the lack of affinity domains, tag-free recombinant proteins can only rely on non-specific capture, typically based on the protein’s physicochemical properties such as charge, hydrophobicity and molecular weight. Figure 4 recommends the purification strategy for tag-free fusion proteins. Ion exchange chromatography (IEX) typically provides high resolution under mild conditions with high binding capacity, and is, therefore, recommended for capturing recombinant proteins. Excessive adsorption of pigments on anion exchange chromatography (AEX) resin causes a decline in product purity and makes it challenging to regenerate the resin, consequently reducing its operational lifespan. Hence, cation exchange chromatography (CEX) is used as the preferred ion exchange capture method. Additionally, the sample pH should differ by at least 0.5 unit from the isoelectric point of the target molecule to ensure product stability. The pH and conductivity need to be fine-tuned to maximize the binding capacity and separation. Hydrophobic interaction chromatography (HIC) resins have been employed in protein purification steps and offer major advantages such as high adsorption capacity, high selectivity, mild elution conditions, and low cost [ 20 ]. By selecting a resin with suitable hydrophobic strength and optimizing the high-salt loading condition, better separation effects can be achieved. Furthermore, HIC elution is performed under low-salt conditions, making it relatively friendly for subsequent processes.
The purification process of tag-free recombinant protein.
Our own practice demonstrated that the capture strategy of an acidic recombinant protein with a pI of 5.9 ( Fig. 5 ). The protein solubility was influenced by pH and conductivity, with precipitation occurring at low pH and low conductivity. On the other hand, to enable capture by CEX, pH should be kept below the protein’s pI and conductivity should be low to maintain sufficient electrostatic interaction. Therefore, the loading condition needs to be carefully adjusted to enable protein capture while maintaining protein stability. From Figure 5(B) , pH had a greater impact on recovery than conductivity within the test range. Based on these results, the loading conditions were set at pH 5.5 and conductivity of 11 mS/cm to achieve optimal capture capability.
Counter plot of solution conductivity and pH versus recovery. (A) Recovery from protein precipitation at different pH and conductivity. (B) The CEX recovery at different loading pH and conductivity.
Ensuring protein stability throughout the process is the primary consideration of protein purification, as most proteins are susceptible to changes in temperature, pH, or salt concentration [ 11 ]. Due to the complexity of artificially designed non-natural proteins, they are prone to issues such as precipitation and reduced quality during conventional purification process. The following summarizes the factors causing protein instability and the strategies to address this problem during protein purification process.
Inappropriate pH condition is the most common factor leading to protein instability, which results in rapid protein aggregation and fragmentation [ 21 ]. The elution buffer for AC is typically acidic, and the prolonged exposure to this condition leads to a sustained decline in protein activity. Therefore, upon elution, the pH should be adjusted back to neutral as soon as possible to prevent long-term activity reduction. In addition, the buffer system and protein concentration are also crucial for stability. For loosely folded proteins, inappropriate solution conditions can lead to partial exposure of hydrophobic regions, thereby accelerating the formation of aggregates.
During the virus inactivation process, organic solvents have been employed as a solution to address the instability issue caused by low pH. However, the addition of inappropriate organic reagents can induce higher degree of aggregation formed through non-covalent binding. Addition of PS80 alone with proper concentration and treatment time can achieve the desired viral inactivation [ 22 ]. Surfactants not only have the ability to inactivate viruses but also helps to maintain protein stability. During the process development, significant precipitation occurred during the affinity step for proteins with strong hydrophobicity. Considering the relatively extended molecular structure and the presence of exposed hydrophobic regions in the molecule, a surfactant was introduced in the process buffer to stabilize the protein structure and enhance the product recovery significantly.
Heat and shear forces can cause irreversible protein aggregation, while freezing and dehydration may result in conformational changes in some intricate protein structure and affect their activities. Although excipients are typically added to stabilize the drug substance (DS) stability during freeze-thaw, adding extra excipients in the process intermediate is not desirable during the purification process. Therefore, shortening the purification process duration and minimizing the sample freeze–thaw is the most effective and convenient approach.
Table 2 summarizes the factors causing protein structural changes during the purification process and their corresponding resolution strategies.
The factors affecting protein stability during the purification process and the corresponding resolution strategies
Influence factors | Stage | Strategies |
---|---|---|
Molecular structure | All | Add stabilizers such as PS80 For proteins that are sensitive to metal ions, ensure the purity of the reagents and optimize the amount added |
pH | AC | Shorten the storage time in low pH condition Choose high pH elution affinity resin or optimize elution buffer system |
VIN | Use S/D inactivation or other inactivation reagents | |
Ionic conditions | HIC | Screen highly hydrophobic resin to reduce the conductivity required during sample loading Try flow-through mode to avoid high conductivity loading |
All | Maintain optimal salt concentration for salt solubilized proteins | |
Buffer system | All | Choose the optimal buffer system |
Freezing | Intermediates | Shorten the processing time to avoid freezing Add cryoprotectants without affecting the process |
DS | Add cryoprotectants | |
Temperature | All | Avoid high-temperature conditions, thaw at low temperature |
Shear force | UFDF | Use low shear equipment, reduce operation pressure and add protectants |
Protein aggregation is one of the most challenging aspects in protein purification. Part of the aggregates originate from the protein formation during cell culture processes due to covalent or non-covalent interaction. The late-stage aggregation is largely dependent on buffer conditions, including pH, ionic strength, buffer system and additives [ 23 , 24 ]. Different modes of chromatography are utilized to remove aggregates, depending on the variations in the physical and chemical properties between the target protein and the aggregates.
Both monomers and aggregates can be captured by affinity resins. The differences in affinity can yield distinct separation effects on aggregates, as Protein A can simultaneously bind to the Fc and Fab regions of the antibody, and the binding affinity depends on the design of the resin and antibody [ 25 ]. As shown in Figure 6 , the affinity between Protein A and antibody depends on the structure of both. Native-Protein A has weak interaction with the Fab region as well as strong binding with the Fc region. In contrast, recombinant Protein A lacks interaction with Fab. Studies found that each monomer can bind to only one Protein A ligand, while each dimer can bind to two ligands [ 26 ]. Therefore, aggregates of one mAb are more strongly retained compared with the mAb monomer. On the other hand, during aggregate formation, the Fc regions might be buried inside or partially lost [ 25 ]. For this type of aggregates, the interaction with recombinant Protein A becomes weaker than the interaction between mAb monomer and protein A. Frequently, incorporating suitable wash steps before elution can effectively eliminate these aggregates at an early stage.
Protein A-mAb interaction. The polymer can simultaneously bind multiple ligands of the resin, while the mAb can only bind a single ligand. The binding site and binding capacity of aggregate and affinity resin depend on protein A and aggregate types.
Additives are commonly used to separate aggregates and target proteins. The addition of calcium chloride/polyethylene glycol (PEG) or sodium chloride/PEG combinations to the wash and elution buffers can effectively enhance the separation of aggregates and target antibodies [ 27 ]. The Hofmeister series of salts can improve resin selectivity by adjusting the hydrophobic interactions between antibody species and Protein A ligands. The use of magnesium chloride and calcium chloride as elution additives has resulted in excellent separation between bispecific products and Fc-Fc homodimers [ 28 ].
Aggregation gives rise to protein surface coverage, leading to disparities in surface charge when compared to individual monomers. Although aggregates are usually more tightly bound to the CEX resin than monomeric proteins, the separation between the two is generally difficult, especially when the aggregates are dimers [ 29 ]. Besides screening different types of cationic resin, the separation of monomers and aggregates can be improved by adding excipients to the mobile phase [ 30 ]. The utilization of a pH-conductivity hybrid gradient elution method has demonstrated its efficacy in eliminating aggregates from samples, if a consistent, linear and reproducible pH profile is provided [ 31 ].
HIC presents notable benefits in aggregate removal, owing to the increased surface hydrophobicity exhibited by aggregates in comparison to monomers [ 32 ]. The efficacy of separation through HIC can be influenced by protein hydrophobicity, salt type and concentration, and hydrophobicity of the resin. By reducing the salt concentration or using resins with stronger hydrophobicity, the target protein can flow through while the aggregates and other impurities are retained, thus avoiding protein instability caused by high conductivity [ 33 ]. Additives with different polarities can alter the hydrophobic interactions among proteins, impurities and the resin, thereby improving resolution and achieving better separation.
Some proteins and aggregates cannot be separated effectively by using chromatography with a single mechanism. Multimodal chromatography allows for separation based on charge, hydrophobicity, hydrogen bonding, and other factors in a single step, and therefore provides better selectivity. Ceramic hydroxyapatite (CHT™) is one type of multimodal resin with a dual exchange mechanism based on phosphate groups for cation exchange and calcium ion metal chelation. Experimental results have demonstrated the excellent efficacy of CHT™ resin in removing aggregates, DNA, endotoxins, and other impurities [ 34 ]. Therefore, in the development of BsAbs, fusion proteins, and recombinant proteins, CHT™ resin can be considered for the removal of challenging impurities.
The formation mechanism of aggregates is complex, which leads to heterogeneous structural characteristics. Therefore, the removal of aggregates requires thorough optimization efforts. Table 3 summarizes the methods and optimization strategies for removing protein aggregates during the purification process.
Strategies for aggregate removal using different types of chromatography
Type of chromatography | Mode | Optimization strategy |
---|---|---|
AC | B-E | 1. Affinity resin screening 2. Wash buffer selection 3. Elution buffer selection 4. Additive selection in the elution buffer, such as polyols, amino acids and high-valent metal salts. 5. Elution pH optimization |
IEX | F-T | Increasing pH to remove aggregates based on weak binding mode. |
B-E | 1. Investigation of sample pH and conductivity to modulate protein binding. 2. Elution methods selection, exploring the resolution differences between salt elution and pH elution. 3. Combination of elution pH and conductivity as optimization factors. 4. Additive selection in the elution buffer, such as polyols and amino acids. | |
HIC | B-E | 1. Selection of resin, where strong hydrophobic resins can be used for lower conductivity loading, and weak hydrophobic resins for higher conductivity loading. 2. Additive selection in the elution buffer, such as polyols and amino acids |
F-T | 1. Selection of resin depending on hydrophobicity. 2. Sample pH and conductivity optimization. |
Note: B-E, bind-and-elute; F-T, flow-through.
HCPs are heterogeneous mixtures of proteins secreted by living cells and intracellular proteins released upon cell death and lysis, with the majority of HCP impurities being acidic proteins. Over 6000 HCPs derived from CHO cells have been identified through proteomics and other methods [ 35 ]. In downstream processes, protein purification typically involves the use of orthogonal chromatographic separation methods to achieve HCP removal. Nevertheless, achieving higher titers of such proteins during cell cultivation proves difficult, which consequently leads to higher HCP contents for tag-free recombinant proteins (> 10 000 ppm) and BsAb or fusion proteins (a few thousand ppm) as compared with typical mAb (< 2000 ppm) after capture.
Depth filtration (DF) is used for solid–liquid separation and can also be employed for HCP removal utilizing electrostatic interaction between the charged binder and proteins. Therefore, two separation strategies are employed: first, controlling the conditions to promote HCP precipitation; and second, removing soluble HCPs through electrostatic interaction with the depth filter material. The pI of the majority of CHO cell-derived HCPs falls within the pH range of 4.5–7.5, and they become less soluble during the neutralization process, leading to aggregation and precipitation. In mAb development, controlling the pH after low pH inactivation allows HCPs to precipitate from solution, enabling their removal during intermediate depth filtration [ 36 ]. An optimal process with pH adjustment can be designed to achieve selective precipitation of HCPs while minimizing product loss.
AC is an ideal tool for protein capture, as the target protein can be strongly bound to the resin in a specific manner. However, some HCPs are co-eluted with the products due to non-specific interaction with the Protein A resin or non-specific binding with the target proteins [ 37 ]. A common approach is to use wash buffer with high salt concentration or low pH (5–5.5) to remove a part of the HCP prior to elution, but these conditions often do not completely disrupt the binding between HCPs and the target protein. Recent studies have found that using high pH and high conductivity wash buffer solutions can remove HCPs more effectively, as the electrostatic interactions between the mAb and most HCPs are repulsive under alkaline conditions (pH > 8) [ 36 ]. Since HCPs can bind to proteins through various mechanisms, including electrostatic interactions, hydrophobic interactions, and hydrogen bonding, reagents that disrupt these interactions can be used to effectively remove HCPs. To date, wash buffer solutions containing different additives have been tested for HCP removal [ 38–40 ]. Histidine hydrochloride has shown good HCP removal results, as it can form hydrogen bonds with proteins, disrupting hydrophobic and electrostatic interactions [ 40 ].
The separation of HCPs is attributed to charge–charge interactions. Most HCPs belong to acidic proteins, so AEX in flow-through mode is effective for HCP removal in the case of basic target proteins. In the flow-through mode, weak partitioning chromatography (WPC) has been proposed for the purification of mAbs using a relatively high pH to enhance HCP removal [ 41 ]. For the bind-and-elute mode, the pI differences between HCPs and target proteins can be exploited to remove parts of HCPs prior to protein elution, thus minimizing co-elution of HCPs with the target protein. Additionally, amino acids have been used in various chromatographic separations to enhance the clearance of HCP during mAb purification processes [ 42 , 43 ]. Our own practice showed the use of CEX in combination with additional wash steps and amino acid additives in the elution buffer to achieve effective HCP removal for a recombinant protein with a pI of 5.9 ( Fig. 7 ). The increase of wash buffer strength induces a partial loss of the target protein, but it proves effective in removing weakly bound HCPs. The addition of arginine in the elution buffer further reduces HCP content. The presence of arginine stabilizes the pH after protein elution, preventing the elution of more strongly bound HCPs. Therefore, by adding a wash step and optimizing the elution buffer, the HCP removal capability can be significantly enhanced.
The CEX chromatograms and quality results for different wash and elution conditions for a tag-free recombinant protein. (A) No wash, elution buffer: Tris-HAc. (B) Wash + elution buffer: Tris-HAc. (C) Wash + elution buffer: arginine. (D) The results for different wash and elution conditions.
Amino acids, which are the building blocks of proteins, differ in both charge and polarity, resulting in variations in the hydrophobicity between the target protein and HCP. HIC in both bind-and-elute mode and low salt flow-through mode have demonstrated great HCP removal capabilities [ 44 ]. The selectivity can be improved by controlling the type and concentration of salt, temperature, pH and ligands used in the HIC process. Figure S2 summarizes the effects of HIC in HCP removal for our selected platform projects. Both the bind-and-elute mode and the flow-through mode can achieve good HCP removal, with an average removal rate of over 90%.
Multimodal resins possess dual properties of ionic exchange and hydrophobicity, the combination of multiple interactions may provide better selectivity. Capto™ Adhere resin, harnessing both anion exchange and hydrophobic interaction functionalities, could achieve a clearance capability of up to 2–3 LRV to reduce HCP levels below 10 ppm [ 45 ]. Capto™ MMC resin, with both cation exchange and hydrophobic properties, is typically used in bind-and-elute mode and has demonstrated good HCP and impurity removal capabilities under optimal process conditions [ 46 ]. CHT™ resin, operating in flow-through mode, has also exhibited excellent competence in HCP removal [ 47 ]. However, achieving enhanced separation through multimodal chromatography requires comprehensive Design of Experiments study to explore the potential interaction of different factors.
Among all impurities related to the process, HCPs are usually of particular concern because they can affect the safety, efficacy and stability of the product. Recently, the development of high density and perfusion cell culture has introduced more challenges in the purification of increased level of HCPs and other impurities. Figure 8 recommends effective strategies for HCP removal at different stages of downstream processing. The purification should be optimized towards maximizing the overall removal capability of the process.
Effective strategies for HCP removal at different stages of downstream processing.
For complex proteins, the presence of a significant level of aggregates and impurities such as HCPs leads to lower yields. Therefore, maximizing recovery while maintaining product quality becomes a pivotal challenge for protein purification process development.
DF is used for the initial clarification of cell culture broth to protect chromatography columns. Selecting appropriate DF filter is key to reduce the protein loss due to adsorption. Uneven charges on a portion of the protein surface result in electrostatic interactions with the charged membrane, causing protein adsorption. Hence, investigation of the DF membrane with different materials improves the protein recovery rate and reduces loss from protein adsorption. Besides, partial adsorption can be mitigated by increasing the conductivity of the wash buffer. Nevertheless, special attention should be given to the adjustment of wash buffer salt concentration, as excessively high conductance is potentially unsuitable for the subsequent separation phase.
In AC, strong binding of protein on the resin may make the elution of protein difficult, leading to low protein recovery. Previous studies have shown that the presence of high ionic strength may promote hydrophobic interactions between protein and Protein A, therefore requires lower pH for elution [ 48 ]. Additionally, fusion proteins generally exhibit weaker stability compared with mAbs, especially under low pH conditions, which pose challenges for elution at lower pH. By using arginine buffer or other additives, elution can be achieved at high pH while ensuring high recovery rate. HIC is often associated with low yield due to unfavorable solvent conditions, or denaturation of target protein on the hydrophobic surface. Various methods have been explored to improve the recovery rate of HIC. In HIC, bind-and-elute mode frequently led to overly strong protein binding, posing challenges in protein elution. Arginine and hexylene glycol can weaken the interactions between proteins and the resin matrix, thereby improving the recovery rate and purity of various proteins [ 49 ].
While striving for high purity by addressing issues such as the removal of aggregates and HCPs, part of the protein product is inevitably lost. Based on the data from our platform, protein loss primarily occurs during depth filtration and various chromatography steps. For depth filtration, selection of appropriate membranes and optimization of wash buffers are the main measures to address the protein loss. For chromatography, the addition of excipients in the product intermediate and process buffer is an effective strategy to improve the recovery by modulating the binding affinity between proteins and resins.
The development of therapeutic protein drugs is a multifaceted systemic engineering endeavor that involves diverse disciplines. In comparison to the substantial advancements seen in upstream processing areas such as molecular design and cell line engineering, there have been relatively fewer reports on the development of protein downstream purification methods. As we have discussed in this review, the diversity of BsAbs, fusion proteins, and recombinant proteins presents a significant challenge in developing effective and economical purification processes.
Chromatography techniques play a pivotal role in protein purification; however, the development of resins lags behind the demand arising from antibody technology advancement. Affinity resins and multimodal resins represent the most promising chromatographic media for addressing challenges in protein purification. Both types of resins exhibit stronger selectivity for impurities, enabling better differentiation between different proteins and process-related impurities. In contrast, progress in the development of filtration steps has been slow-paced, making ideal impurity removal based on filtration processes difficult to achieve. Nevertheless, the combination of resins and filtration processes in product design may offer a promising direction for breakthroughs in consumables. Such products would merge the efficiency of filtration processes with the selectivity of chromatographic processes, potentially enhancing the efficiency and economy of purification significantly.
In addition to anticipating suppliers to provide improved purification consumables, efficient experimental design is another pursuit in the development of purification processes. The design of protein purification processes is heavily based on empirical knowledge and a large number of trial-and-error experiments, often resulting in suboptimal overall processes and low utilization of raw materials. Addressing this issue primarily involves reducing the number of experiments and increasing experimental efficiency. Rational experimental design is the basis for reducing the number of experiments, requiring sufficient understanding of the target protein. Computational biology represents a future trend that leverages computer models and machine-learning algorithms to predict the properties of target proteins, identify correlations and optimize purification process conditions, thereby reduces trial-and-error experiments. This has the potential to fundamentally enhance the accuracy and efficiency of purification process development. On the other hand, the introduction of high-throughput screening technology has greatly improved the efficiency of protein purification. High-throughput screening accelerates the evaluation, optimization and selection of purification processes by parallel processing a large number of samples. The combination of computer-aided design and high-throughput screening is an excellent option for future purification process development, which is worth looking forward to.
In summary, this overview demonstrates that challenges still exist in seeking to improve the development of bioprocess purification, while also highlights the significant progress that has been made. Through the development of rational processes, we can address the majority of challenges to meet regulatory requirements. With the continuous advancement of technology, the exploration of more efficient, accurate and environmentally friendly protein purification methods will continue to drive the development of this field.
Supplementary_data_tbad028, acknowledgements.
The authors thank Weijuan Shen, Qianqian Zhu, Zhaoxin Liu, Shuyu Zhou, Weiwei Gu and Peilei Liu for helping generate data shown in the case studies in this article.
Shuo Tang, GenScript ProBio Biotechnology Co., Ltd, Nanjing, Jiangsu 21100, P.R. China.
Jiaoli Tao, GenScript ProBio Biotechnology Co., Ltd, Nanjing, Jiangsu 21100, P.R. China.
Ying Li, GenScript ProBio Biotechnology Co., Ltd, Nanjing, Jiangsu 21100, P.R. China.
This work was funded by GenScript ProBio Biotechnology Co., Ltd.
Shuo Tang, Jiaoli Tao and Ying Li are current employees of GenScript ProBio Biotechnology Co., Ltd.
Shuo Tang (Data curation-lead, Formal analysis-lead, Investigation-equal, Project administration-equal, Writing-original draft-lead, Writing-review and editing-equal), Jiaoli Tao (Data curation-supporting, Investigation-supporting, Writing-original draft-supporting) and Ying Li (Conceptualization-lead, Investigation-lead, Methodology-lead, Project administration-equal, Supervision-lead, Writing-review and editing-lead).
Ethics and consent statement.
No patient consent is required.
Not applicable.
Academia.edu no longer supports Internet Explorer.
To browse Academia.edu and the wider internet faster and more securely, please take a few seconds to upgrade your browser .
Enter the email address you signed up with and we'll email you a reset link.
Biotechnology and Bioengineering
Balaji Somasundaram
Biotechnology Progress
Nigel Titchener-hooker
International Journal for Research in Applied Science & Engineering Technology (IJRASET)
IJRASET Publication
The creation of therapeutic monoclonal antibodies has increased recently. mAbs have grown significantly in relevance, and these compounds are now more crucial than ever in the global biotechnological drug industry. For the treatment of various diseases, a lot of biotechnology companies are investing in the manufacture of monoclonal antibodies. The two main processes in the synthesis of these antibodies are bioreactor production and purification. This article goes into great detail on the current purification techniques utilized for these compounds. The fundamental monoclonal antibody recovery and purification procedures, including cell harvesting, Protein A affinity chromatography, and further polishing steps, are all enumerated.
International Journal of Pharmaceutics
Christopher Maycock
Shritama Ray
Monoclonal antibody (mAb) therapy is a form of immunotherapy that uses mAbs to bind mono-specifically to certain cells or proteins. This may then stimulate the patient's immune system to attack those cells. MAbs are currently used to treat medical conditions such as cancer, diabetes, arthritis, psoriasis, and Crohn’s Disease, but have the potential to treat countless diseases and disorders. In 2015, the mAb market was valued at $85.4 billion, and is expected to reach $138.6 billion by 2024.1 In manufacturing, mAbs are typically produced in suspension in a series of fed-batch bioreactors using genetically engineered cells originally obtained from Chinese Hamster Ovaries (CHO).2 In this proposal, two upstream bioreactor designs were analyzed for economic comparison given an annual production goal of 100 kg of mAb, with the first design culminating in a 20,000 L volume at low mAb titer and the second design culminating with a 2,000 L volume at high mAb titer. Following upstream mAb...
Patricia Rowicki
The emergence of monoclonal antibody (mAb) therapies has created a need for faster and more efficient bioprocess development strategies in order to meet timeline and material demands. In this work, a high‐throughput process development (HTPD) strategy implementing several high‐throughput chromatography purification techniques is described. Namely, batch incubations are used to scout feasible operating conditions, miniature columns are then used to determine separation of impurities, and, finally, a limited number of lab scale columns are tested to confirm the conditions identified using high‐throughput techniques and to provide a path toward large scale processing. This multistep approach builds upon previous HTPD work by combining, in a unique sequential fashion, the flexibility and throughput of batch incubations with the increased separation characteristics for the packed bed format of miniature columns. Additionally, in order to assess the applicability of using miniature column...
Ana Azevedo
The commercial potential of monoclonal antibodies (mAbs) has been continuously increasing during the last years alongside with the number of approved mAb-based drugs and clinical trials. Despite their effectiveness and safety, the general access to this class of biopharmaceuticals is barred by high selling prices. Downstream processing is now considered the bottleneck in the manufacturing of mAbs. Therefore, the design of novel and economic operations and their implementation in the current technology platforms constitutes a pressing need. This review provides an insight into the current state-of-the-art in mAbs purification, focusing on multimodal chromatography as one of the viable options to upgrade the established purification train.
jonathan Coffman
Muthu Kumar
Acta Scientific Medical Sciences
Burak Erkal
Loading Preview
Sorry, preview is currently unavailable. You can download the paper by clicking the button above.
Journal of Membrane Science
Eva Rosenberg , S. Hepbildikler , Wolfgang Kuhne
Journal of Clinical Medicine
sanjeevani ghone
Gregory Zarbis-Papastoitsis , marco cacciuttolo
xionghui li
Journal of Pharmaceutical and Biomedical Analysis
Evelin Farsang
American Journal of Biochemistry and Biotechnology
Ali Demirci
Biotechnology Journal
Chia-Yun Sun
Alex Chatel
Journal of biotechnology
Boris Napadensky , Andrew Tustian
Darrin Tisdale
Chemical Engineering Science: X
Ruud van Beckhoven
IRJET Journal
abiy berhanu
Separations
Amarande Murisier
Journal of Chromatography a
Daniel Bracewell
Journal of Chromatography A
Maria Chiara Zorzoli
Biologicals
Anja ter Avest
Biotechnology and applied biochemistry
Martin Vanderlaan
Kerstin Thurow
Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.
Nature Biotechnology volume 42 , pages 1343–1349 ( 2024 ) Cite this article
Metrics details
The proper use of spike-in normalization in ChIP-seq improves sensitivity for detecting genome-wide changes between conditions, but improper use is common, calling some biological conclusions into question. A survey of public datasets generates guidelines for implementation of spike-in normalization for future ChIP-seq experiments.
This is a preview of subscription content, access via your institution
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
24,99 € / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
195,33 € per year
only 16,28 € per issue
Buy this article
Prices may be subject to local taxes which are calculated during checkout
Data generated in this study (Fig. 1b , Fig. 4 , and Supplementary Figs. 2–6 and 8–14 ) are at GSE273915 . Public data used to generate Fig. 1a are from GSE60104 . UCSC Genome Browser sessions are available for Fig. 1b , Fig. 4 human (target) data and Fig. 4 yeast (spike-in) data .
Code is available on a Github repository.
Orlando, D. A. et al. Cell Rep. 9 , 1163–1170 (2014).
Article CAS PubMed Google Scholar
Bonhoure, N. et al. Genome Res. 24 , 1157–1168 (2014).
Article CAS PubMed PubMed Central Google Scholar
Meers, M. P., Bryson, T. D., Henikoff, J. G. & Henikoff, S. eLife 8 , e46314 (2019).
Article PubMed PubMed Central Google Scholar
Chen, K. et al. Mol. Cell. Biol. 36 , 662–667 (2015).
Article PubMed Google Scholar
Jiang, L. et al. Genome Res. 21 , 1543–1551 (2011).
Skene, P. J. & Henikoff, S. eLife 6 , e21856 (2017).
Wooten, M., Takushi, B., Ahmad, K. & Henikoff, S. Sci. Adv. 9 , eadg3257 (2023).
Terekhanova, N. V. et al. Nature 623 , 432–441 (2023).
Javasky, E. et al. Genome Res. 28 , 1455–1466 (2018).
Guertin, M. J., Cullen, A. E., Markowetz, F. & Holding, A. N. Nucleic Acids Res. 46 , e75 (2018).
Egan, B. et al. PLoS One 11 , e0166438 (2016).
Ma, Z. et al. eLife 7 , e35368 (2018).
Wang, Z. et al. Nat. Genet. 54 , 295–305 (2022).
Yano, S. et al. Nat. Commun. 13 , 4440 (2022).
Nakato, R. et al. Nat. Commun. 14 , 5647 (2023).
Greulich, F., Wierer, M., Mechtidou, A., Gonzalez-Garcia, O. & Uhlenhaut, N. H. Cell Rep. 34 , 108742 (2021).
Okabe, A. et al. Nat. Genet. 52 , 919–930 (2020).
Kent, W. J. et al. Genome Res. 12 , 996–1006 (2002).
Landt, S. G. et al. Genome Res. 22 , 1813–1831 (2012).
Cherry, J. M. et al. Nucleic Acids Res. 26 , 73–79 (1998).
Love, M. I., Huber, W. & Anders, S. Genome Biol. 15 , 550 (2014).
Grzybowski, A. T., Chen, Z. & Ruthenburg, A. J. Mol. Cell 58 , 886–899 (2015).
Vale-Silva, L. A., Markowitz, T. E. & Hochwagen, A. BMC Genom. 20 , 54 (2019).
Article Google Scholar
Download references
Research reported in this publication was supported in part by US National Institutes of Health/National Institute of Mental Health grant R01MH127077 (A.G. and C.B.) and National Science Foundation grant 2003358 (A.G.). We thank I. Simon for providing conceptual guidance and feedback on the manuscript, R. Wachs for helping with illustrations and the Pillus lab at UCSD for providing S. cerevisiae cells and guidance on yeast culture and ChIP-seq.
Authors and affiliations.
Department of Bioengineering, Jacobs School of Engineering, University of California San Diego, La Jolla, CA, USA
Lauren A. Patel
Department of Medicine, Division of Endocrinology & Metabolism, University of California San Diego, La Jolla, CA, USA
Lauren A. Patel & Christopher Benner
Department of Medicine, Division of Genomics & Precision Medicine, University of California San Diego, La Jolla, CA, USA
Lauren A. Patel, Yuwei Cao & Alon Goren
Bioinformatics and Systems Biology Graduate Program, University of California San Diego, La Jolla, CA, USA
HudsonAlpha Institute for Biotechnology, Huntsville, AL, USA
Eric M. Mendenhall
You can also search for this author in PubMed Google Scholar
Correspondence to Christopher Benner or Alon Goren .
Competing interests.
L.A.P., Y.C., C.B. and A.G. are inventors on related patent applications.
Peer review information.
Nature Biotechnology thanks the anonymous reviewers for their contribution to the peer review of this work.
Supplementary information.
Descriptions of Supplementary Tables 1–5, Supplementary Figs. 1–15, Table 1 and Methods.
Supplementary Tables 2–5.
Reprints and permissions
Cite this article.
Patel, L.A., Cao, Y., Mendenhall, E.M. et al. The Wild West of spike-in normalization. Nat Biotechnol 42 , 1343–1349 (2024). https://doi.org/10.1038/s41587-024-02377-y
Download citation
Published : 13 September 2024
Issue Date : September 2024
DOI : https://doi.org/10.1038/s41587-024-02377-y
Anyone you share the following link with will be able to read this content:
Sorry, a shareable link is not currently available for this article.
Provided by the Springer Nature SharedIt content-sharing initiative
Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.
COMMENTS
Owing to a rapidly increasing market demand for therapeutic proteins, for example, monoclonal antibodies (mAbs), the industry is facing the challenge of efficiently manufacturing large-product amounts [1].After recent process-intensification improvements in the upstream processing (USP), the current bottleneck of production processes shifts to in the purification of protein products in the ...
Explore the latest full-text research PDFs, articles, conference papers, preprints and more on DOWNSTREAM PROCESSING. Find methods information, sources, references or conduct a literature review ...
Downstream process: toward cost/energy effectiveness. Ramesh Kumar, ... Alak Kumar Ghosh, in Handbook of Biofuels, 2022. Abstract. Downstream processing (DSP) involves multistaged unit operations after upstream processes that improve the quality of the final product in terms of concentration and purity. One of the most important objectives of the DSP, in addition to maximizing product recovery ...
A variety of downstream processing technological advances have led to a paradigm shift in how therapeutic antibodies are developed and manufactured. A key driver of change has been the increased adoption of single-use technologies for process development and manufacturing. ... Feature papers represent the most advanced research with significant ...
Downstream processing plays an important role in the supply of pharmaceuticals and, in particular, biopharmaceuticals, where a large proportion of the production costs are attributable to the recovery and purification processes (Straathof, 2011) although this may have shifted to upstream processes in the case of cell and gene therapy ...
Unit operations and their assembly to downstream processing (DSP) routes for carbohydrate products are the focus of the current study (Hatti-Kaul, 2010; Illanes et al., 2016; Moreno et al., 2014a). Functional carbohydrates can be classified structurally into monosaccharides, disaccharides, oligosaccharides and polysaccharides.
Development of efficient and economical downstream processing strategies has been applied in industrial manufacture of enzymes because of their important role not only in food and agricultural industries but also used in textile, paper and pharmaceutical industries, scientific research, etc (Roque et al., 2004).
Therefore, an urgent need for process intensification in downstream processing (DSP) has been identified to produce biopharmaceuticals more efficiently. The DSP currently accounts for the majority of production costs of pharmaceutically relevant proteins. This short review gathers essential research over the past 3 years that addresses novel ...
Downstream processing (DSP) is engaged in the separation and refinement of mixtures of components. In its simplest definition, DSP encompasses a tool box of separation techniques designed to achieve mass transfer phenomena, converting mixtures of substances into subsets of mixtures or fractions [].At its onset, industrial DSP of proteins considered many of the traditional chemical unit ...
The platform process most applied in biopharmaceutical downstream processing (DSP) is the systematic purification of mAbs and Fc fusion proteins. Shukla et al. 41 described the antibody platform process in several consecutive steps, which are consistent in process structure, but single unit operations are exchangeable. After cell harvest and ...
Downstream Processing . Leader Peptide . Oxidative Decarboxylation . Cancerous Cell . In Vitro Reconstitution . Modified Peptides . In Vitro Biosynthesis. Thioholgamides are ribosomally synthesized and post-translationally modified peptides (RiPPs) with potent activity against cancerous cell lines and an unprecedented structure.
However research and development of the concerned firms is working to get advanced ways to improve the downstream processing of biopharmaceuticals. The upcoming future trends are in silico-based methods: algorithmic (super structure optimisation, model-based hybrid approach) and non-algorithmic (heuristic approach based, experimental and ...
A variety of downstream processing technological advances have led to a paradigm shift in how therapeutic antibodies are developed and manufactured. A key driver of change has been the increased adoption of single-use technologies for process development and manufacturing. ... This is an active area for further research and development. 5 ...
Moreover, downstream processing steps now require the capacity to process 15-100 kg of mAb per batch, despite being traditionally designed to process 5-10 kg of mAb. The challenge in processing such large quantities of products is that the resin-based chromatography operations are limited by scale-up to a maximum column diameter of 2.0 to 2 ...
The current book gives an excellent insight into downstream processing technology and explains how to establish a successful strategy for an efficient recovery, isolation and purification of biosynthetic products. In addition to the overview of purification steps and unit operations, the authors provide practical information on capital and operating costs related to downstream processing.
Perspectives for an improved downstream processing of biologically produced diols, especially 1,3-propanediol are discussed based on our own experience and recent work. It is argued that separation technologies such as aqueous two-phase extraction with short chain alcohols, pervaporation, reverse osmosis, and in situ extractive or pervaporative ...
Downstream processing refers to the series of unit operations used to isolate, purify, and concentrate the product. Downstream processing often determines the economic feasibility of the process. The first operation is cell separation, which can be done by cross-flow microfiltration. When a microfilter or ultrafilter is combined in a semi ...
This chapter provides an overview of the chemical and biophysical properties of proteins and their main contaminants such as DNA and endotoxins. It discusses commonly used expression systems and the general structure of downstream processes needed to achieve the desired product purity. The chapter focuses on the production of recombinant ...
The article summarizes the primary challenges encountered during the downstream processing of proteins, and presents effective solutions and case studies to tackle each major challenge. ... With the advancement of genetic engineering technology, research in protein drugs achieved significant breakthroughs. The application of technologies such ...
Academia.edu is a platform for academics to share research papers. Overview of Upstream and Downstream Processing of Biopharmaceuticals ... fed-batch, continuous, perfusion • Downstream processing - Philosophy - Chromatography - Examples • Conclusions 2 What is a bioprocess? • Application of natural or genetically manipulated ...
This solvent-detergent 442 Chapter 11 - Downstream Processing f (S/D) treatment, originated at the New York Blood Center in the 1980s, is the most widespread method of viral inactivation in plasma fractionation processes and is used in the production of some recombinant proteins.
The low dry weight content of microalgae cultures (typically around 1 g L −1) makes harvesting and concentration an expensive unit operation of any biorefinery.Harvesting and dewatering are the main downstream processes required for any applications where the whole microalgae biomass can be used directly as the final product, such as for animal and fish feed or food supplements (e.g ...
The proper use of spike-in normalization in ChIP-seq improves sensitivity for detecting genome-wide changes between conditions, but improper use is common, calling some biological conclusions into ...
Organoid generation from intestinal resections. Segments of human colonic tissue were supplied by Donor Network West. Donor Network West received IRB approval of research, appropriate informed consent of all subjects contributing biological materials, and all other authorizations, consents, or permissions as necessary for the transfer and use of the biological materials for research at Genentech.
Research review paper. Downstream processing of proteins: Recent advances. Author links open overlay panel John R. Ogez a, James C. Hodgdon a, Marc P. Beal a, Stuart E ... DOWNSTREAM PROCESSING OF PROTEINS 471 CELL DISINTEGRATION Mechanical disintegration is the most common form of cell disruption used in industry today, although treatment with ...
Today's announcement adds a processing LOI with USSM that builds upon this, with a three-way strategic partnership (the "Agreement") exploring collaboration on domestic US processing solutions ...