Host Cell DNA Research Articles

Mixed Matrix PVDF Microfiltration Membranes with In-situ Synthesized Polyethyleneimine Particles as a Platform for Flow Through, High Capacity, Weak Base and Salt Tolerant Anion Exchange Membrane Adsorbers for Downstream Bioprocessing

The rapid growth of the monoclonal antibody (mAb) therapeutic market has led to a need for improved downstream bioprocessing, including mAb capture and release from bioreactor harvests followed by product purification and polishing. Membrane chromatography (MC) is poised to displace resin-based column chromatography in mAb product polishing. To remove negatively charged product impurities (e.g., host cell proteins, DNA, viruses, and endotoxins), anion exchange (AEX) membrane adsorbers offer higher process rates and eliminate the risks of cross contamination as they can be deployed as single use separation devices, which are increasingly being utilized in downstream bioprocessing to increase manufacturing speed and decrease production cost. Here, we describe a one-pot and single step phase inversion casting process for the preparation of a family of mixed matrix polyvinylidene fluoride (PVDF) microfiltration (MF) membranes with in-situ synthesized polyethyleneimine (PEI) microparticles that can serve as flow through, high capacity, and salt tolerant weak base AEX membrane adsorbers. These new membranes have a high content of weak base (WB) groups (primary and secondary amines) by virtue of the utilization of bis(2-chloroethyl)amine hydrochloride (BCAH) as crosslinker in the in-situ synthesis of PEI microparticles in the dope dispersions prior to membrane casting. By varying the concentration of PEI and BCAH in the casting dispersions, we successfully utilized nonsolvent induced phase separation (NIPS) with isopropyl alcohol as the nonsolvent in the membrane coagulation bath to prepare a mixed matrix PVDF-PEI MF membrane with (i) a relatively uniform and open microstructure in which the PEI particles completely cover the PVDF spherulites present at the membrane surface and cross section, (ii) high water flux (>1000 liters/m2/hr. at 2 bar) and (iii) a high loading of BCAH crosslinked PEI microparticles (51.0 wt.%) containing weak base primary, secondary, and tertiary amine groups. The protein binding measurements show that this new mixed matrix PVDF-PEI MF membrane can serve as a flow through, high capacity, and salt tolerant WB AEX membrane adsorber with a bovine serum albumin (BSA) dynamic binding capacity of ∼60 mg BSA per mL of membrane in a 50 mM TRIS buffer containing 100 mM of NaCl (15 mS/cm) at 10% breakthrough and flow rate of 10 MV/min. The overall results of this study indicate that our mixed matrix PVDF-PEI MF membranes with in-situ synthesized and BCAH crosslinked PEI microparticles have a promising potential to serve as a platform for the design, preparation, and scale up of a new generation of flow through, high capacity, salt tolerant, WB AEX membrane adsorber materials for downstream bioprocessing.

Highly selective split intein method for efficient separation and purification of recombinant therapeutic proteins from mammalian cell culture fluid

Biologics and vaccines have been successfully developed over the last few decades to treat many diseases. Each of these drugs must be highly purified for clinical use. Monoclonal antibodies (mAbs), the dominant therapeutic modality on the market, can be easily purified using the standard Protein A affinity platform. However, no generally applicable affinity platforms are available for the manufacture of other therapeutic proteins for clinical use. Thus, multicolumn chromatography processes for widely being used for product purification. These processes demand significant optimization to meet desired product quality attributes, where each step also decreases final yields. In this work, we demonstrate the novel self-removing iCapTagTM affinity tag, which provides a new platform for capturing, concentrating, and purifying recombinant proteins. Importantly, this system provides a tagless target protein, which is suitable for research and clinical use, where the only requirement for tag removal is a small change in buffer pH. No additional proteins, reagents or cofactors are required. We also present case studies demonstrating the use of iCapTagTM for highly efficient purification of untagged interferon alpha 2b, the ML39 single chain variable fragment (scFv), and the receptor binding domain (RBD) of SARS-CoV-2 spike protein. These proteins were expressed and secreted by Expi293 cells with the self-removing tag fused to their N-terminus. We were able to obtain highly pure (> 99%) tagless protein in a single purification step with high clearance of host cell DNA, tagged precursor, higher and lower molecular weight impurities. Based on these preliminary results, we propose the iCapTagTM as a universal capture platform for diverse classes of recombinant therapeutic proteins.

Design, Synthesis, and in vitro Evaluation of Derivatives of Quinoxaline-2- One as a Myeloperoxidase Modulator Using in silico Methods

Background: In some pathological situations, the overproduction of oxidising agents also results in oxidative damage to host cell proteins and DNA, which induces abnormal expression of inflammatory cytokines and chemokines. A recently discovered biomarker of inflammation is myeloperoxidase. Various inflammatory conditions cause the release of this enzyme into the extracellular environment. Objective: Our study aimed to design, synthesize, and in vitro evaluate derivatives of quinoxaline- 2-one as a myeloperoxidase modulator using in silico methods. Methods: A series of quinoxaline-2-one derivatives was synthesized and characterized by various analytical techniques. Further, to confirm and explore the molecular mechanism, an in silico docking study against the myeloperoxidase enzyme was performed (PDB ID: 1DNU). Results: The compounds Q1, Q2, and Q5 showed better antioxidant activity in the DPPH assay, whereas the nitric oxide scavenging assay showed the compounds Q2, Q4, and Q5 had significant activity when compared to the standard IC50 value (28.8 μg/ml). Besides, the anti-inflammatory studies showed the compounds Q1, Q3, and Q5 had better inhibition (89.79%) when compared to the standard drug aceclofenac (85.37%) at 1000 μg/ml concentration. The top three ligands for myeloperoxidase (PDB ID: 1DNU) with the highest scores in activity were found as Q2, Q1, and Q5, with scores of -13.2838, -12.5841, and -11.6906 Kcal/mol, respectively. The compounds were efficiently bound to the myeloperoxidase active site with arene-arene, arene-cation, and hydrogen bonding interactions. Conclusion: By introducing the various heterocyclic rings and deactivating and activating groups, we may produce a newer class of candidates for many infectious diseases. Thus, from the computational studies carried out, we may obtain hints for optimising the molecular selectivity of the quinoxaline-2-one derivatives to provide help in the design of new compounds for effective myeloperoxidase enzyme modulators. However, further pharmacokinetics, pharmacodynamics, preclinical, and clinical studies permit the design of the new agents without undesirable interactions.

Peptide ligands for the affinity purification of adenovirus from HEK293 and vero cell lysates

Adenovirus (AdVs) is the viral vector of choice in vaccines and oncolytic applications owing to its high transduction activity and inherent immunogenicity. For decades, AdV isolation has relied on ultracentrifugation and ion-exchange chromatography, which are not suitable to large-scale production and struggle to deliver sufficient purity. Immunoaffinity chromatography resins of recent introduction feature high binding capacity and selectivity, but mandate harsh elution conditions (pH 3.0), afford low yield (< 20%), and provide limited reusability. Seeking a more efficient and affordable alternative, this study introduces the first peptide affinity ligands for AdV purification. The peptides were identified via combinatorial selection and in silico design to target hexons, the most abundant proteins in the adenoviral capsid. Selected peptide ligands AEFFIWNA and TNDGPDYSSPLTGSG were conjugated on chromatographic resins and utilized to purify AdV serotype 5 from HEK293 and Vero cell lysates. The peptide-functionalized resins feature high binding capacity (> 1010 active virions per mL at the residence time of 2 min), provide high yield (> 50%) and up to 100-fold reduction of host cell proteins and DNA. Notably, the peptide ligands enable gentle elution conditions (pH 8) that prevent the “shedding” of penton and fiber proteins, thus affording intact adenovirus particles with high cell-transduction activity. The study of the peptide ligands by surface plasmon resonance and molecular docking and dynamics simulations confirmed the selective targeting of hexon proteins and elucidated the molecular-level mechanisms underlying binding and release. Collectively, these results demonstrate the strong promise of peptide ligands presented herein for the affinity purification of AdVs from cell lysates.

Clear insight into complex multimodal resins and impurities to overcome recombinant protein purification challenges: A review.

Increasing attention has been paid to the purity of therapeutic proteins imposing extensive costs and challenges to the downstream processing of biopharmaceuticals. One of the efforts, that has been exerted to overcome such limitations, was developing multimodal or mixed-mode chromatography (MMC) resins for launching selective, orthogonal, non-affinity purification platforms. Despite relatively extensive usage of MMC resins, their real potential and fulfillment have not been extensively reviewed yet. In this work, the explanation of practical and key aspects of downstream processing of recombinant proteins with or without MMC resins was debated, as being useful for further purification process development. This review has been written as a step-by-step guide to deconvolute both inherent protein purification and MMC complexities. Here, after complete elucidation of the potential of MMC resins, the effects of frequently used additives (mobile phase modifiers) and their possible interactions during the purification process, the critical characteristics of common product-related impurities (e.g., aggregates, charge variants, fragments), host-related impurities (e.g., host cell protein and DNA) and process related impurities (e.g., endotoxin, and viruses) with solved or unsolved challenges of traditional and MMC resins have been discussed. Such collective experiences which are reported in this study could be considered as an applied guide for developing successful downstream processing in challenging conditions by providing a clear insight into complex MMC resins and impurities.

Bombyx mori nucleopolyhedrovirus LEF-2 disrupts the cell cycle in the G2/M phase by triggering a host cell DNA damage response.

It is a common strategy for viruses to block the host cell cycle to favour their DNA replication. Baculovirus, being a double-stranded DNA virus, can arrest the cell cycle in the G2/M phase to facilitate its replication. However, the key viral genes and mechanisms crucial for inducing cell cycle arrest remain poorly understood. Here, we initially examined the impacts of several Bombyx mori nucleopolyhedrovirus (BmNPV) DNA replication-associated genes: ie1, lef-1, lef-2, lef-3, lef-4, odv-ec27 and dbp. We assessed their effects on both the host cells' DNA replication and cell cycle. Our findings reveal that when the lef-2 gene was overexpressed, it led to a significant increase in the number of cells in the G2/M phase and a reduction in the number of cells in the S phase. Furthermore, we discovered that the LEF-2 protein is located in the virogenic stroma and confirmed its involvement in viral DNA replication. Additionally, by employing interference and overexpression experiments, we found that LEF-2 influences host cell DNA replication and blocks the cell cycle in the G2/M phase by regulating the expression of CyclinB and CDK1. Finally, we found that BmNPV lef-2 triggered a DNA damage response in the host cell, and inhibiting this response removed the cell cycle block caused by BmNPV LEF-2. Thus, our findings indicate that the BmNPV lef-2 gene plays a crucial role in viral DNA replication and can regulate host cell cycle processes. This study furthers our understanding of baculovirus-host cell interactions and provides new insight into the molecular mechanisms of antiviral research.

Stable and reusable calcium-responsive biopolymer for affinity precipitation of therapeutic antibodies.

Affinity precipitation is an attractive method for protein purification due to its many advantages, including the rapid capture of target proteins, simple processing, high specificity, and ease of scale-up. We previously reported a robust antibody purification method using Ca2+-dependent precipitation of ZZ-hCSQ2, a fusion protein of human calsequestrin 2, and the antibody-binding protein ZZ. However, the stability of this fusion protein was not sufficiently high for industrial use because the antibody recovery yield decreased to 60% after being reused 10 times. To identify a more stable calsequestrin (CSQ), we calculated Rosetta energy values for the folding stabilities of various CSQ homologs and selected human CSQ1 (hCSQ1) with lowest energy value (-992.6) as the new CSQ platform. We also identified that the linker sequence between ZZ and CSQ was vulnerable to proteases and alkaline pH by N-terminal protein sequencing. Therefore, we changed the linker to four asparagine (4N) sequences, which were shorter and less flexible than the previous glycine-rich linker. The new version of ZZ-CSQ, ZZ-4N-hCSQ1, was stable in a protease-containing conditioned medium obtained from the cultured Chinese hamster ovary cell or high pH condition (0.1M sodium hydroxide) for more than 5 days and could be reused at least 25 times for antibody purification without loss of recovery yield. The antibodies purified by ZZ-4N-hCSQ1 precipitation also showed greater purity (~33.6-fold lower host cell DNA and ~6.4-fold lower host cell protein) than those purified by protein A chromatography. These data suggest that ZZ-4N-hCSQ1 precipitation is more efficient and can achieve cost-effectiveness of up to 12.5-fold cheaper than previous antibody purification methods and can lower the production costs of therapeutic antibodies.

In-Depth Comparison of Adeno-Associated Virus Containing Fractions after CsCl Ultracentrifugation Gradient Separation.

Recombinant adeno-associated viruses (rAAVs) play a pivotal role in the treatment of genetic diseases. However, current production and purification processes yield AAV-based preparations that often contain unwanted empty, partially filled or damaged viral particles and impurities, including residual host cell DNA and proteins, plasmid DNA, and viral aggregates. To precisely understand the composition of AAV preparations, we systematically compared four different single-stranded AAV (ssAAV) and self-complementary (scAAV) fractions extracted from the CsCl ultracentrifugation gradient using established methods (transduction efficiency, analytical ultracentrifugation (AUC), quantitative and digital droplet PCR (qPCR and ddPCR), transmission electron microscopy (TEM) and enzyme-linked immunosorbent assay (ELISA)) alongside newer techniques (multiplex ddPCR, multi-angle light-scattering coupled to size-exclusion chromatography (SEC-MALS), multi-angle dynamic light scattering (MADLS), and high-throughput sequencing (HTS)). Suboptimal particle separation within the fractions resulted in unexpectedly similar infectivity levels. No single technique could simultaneously provide comprehensive insights in the presence of both bioactive particles and contaminants. Notably, multiplex ddPCR revealed distinct vector genome fragmentation patterns, differing between ssAAV and scAAV. This highlights the urgent need for innovative analytical and production approaches to optimize AAV vector production and enhance therapeutic outcomes.

Single-use chromatographic clarification to eliminate endonuclease treatment in production of recombinant adeno-associated viral vectors

In current manufacturing processes for recombinant adeno-associated viral vectors (rAAV), endonuclease treatment is used prior to depth filtration to break down host cell DNA that is released from cells post-lysis. Without this treatment, traditional depth filters exhibit negligible clearance of host cell DNA, a key process-related impurity. In this work, we evaluate single-use chromatographic clarification using Harvest RCTM filters (3 M) to process rAAV8 and rAAV9 HEK293 cell culture lysate with and without endonuclease pretreatment. We demonstrate endonuclease-free clarification for both serotypes with yield >90 % and up to 3 log reduction of host cell DNA, from >104 ng/mL to <50 ng/mL, matching or exceeding the filtrate quality in traditional depth filtration with 100 U/mL of endonuclease added during lysis. The addition of 100 mM of salt in the harvest buffer is also shown to improve filtration throughput, prevent filter clogging, and limit rAVV binding to the filter device. Finally, a mechanistic cake-fiber fouling model is shown to be a good representation of the Harvest RCTM filtration mechanism. The endonuclease-free clarification approach enables cost savings of USD 100,000 per 500 L batch, equivalent to the cost of cGMP-grade endonuclease at this scale. The benefits of endonuclease-free clarification further include easier raw material sourcing as well as eliminating the need for a residual endonuclease assay in rAAV manufacturing processes.

Purification of recombinantly produced somatostatin-28 comparing hydrochloric acid and polyethyleneimine as E. coli extraction aids

Peptides are used for diagnostics, therapeutics, and as antimicrobial agents. Most peptides are produced by chemical synthesis, but recombinant production has recently become an attractive alternative due to the advantages of high titers, less toxic waste and correct folding of tertiary structure. Somatostatin-28 is a peptide hormone that regulates the endocrine system, cell proliferation and inhibits the release of numerous secondary hormones in human body. It is composed of 28 amino acids and has one disulfide bond, which makes it to an optimal model peptide for a whole downstream purification process. We produced the peptide in the periplasm of E. coli using the CASPON™ technology, an affinity fusion technology system that enables high soluble expression of recombinant proteins and cleaves the fusion tag with a circularly permuted human caspase-2. Furthermore, purification of the products is straight forward using an established platform process. Two different case studies for downstream purification are presented, starting with either hydrochloric acid or polyethyleneimine as an extraction aid. After release of affinity-tagged somatostatin-28 out of E. coli's periplasm, several purification steps were performed, delivering a pure peptide solution after the final polishing step. The process was monitored by reversed-phase high-performance liquid chromatography as well as mass spectrometry to determine the yield and correct disulfide bond formation. Monitoring of impurities like host cell proteins, DNA and endotoxins after each downstream unit confirmed effective removal for both purification pathways.

Investigating Early HIV Infection: Utilizing Artificial Cell Membranes for Capturing Unintegrated Viral RNA

The high mutation rate of HIV poses a significant challenge for research and treatment development. Obtaining unaltered viral genetic material before integration into the host cell's DNA would be a valuable tool for studying early infection stages and developing more effective therapies. This study explores the feasibility of using artificial cell membranes containing human immunodeficiency virus (HIV) receptors (CD4 and co-receptor) to capture unintegrated viral RNA. We discuss the potential advantages and limitations of this approach compared to existing methods for studying HIV infection.

Antiviral activity of adenoviral vector expressing human interferon lambda-4 against influenza virus.

Interferon lambda (IFNλ), classified as a type III IFN, is a representative cytokine that plays an important role in innate immunity along with type I IFN. IFNλ can elicit antiviral states by inducing peculiar sets of IFN-stimulated genes (ISGs). In this study, an adenoviral vector expression system with a tetracycline operator system was used to express human IFNλ4 in cells and mice. The formation of recombinant adenovirus (rAd-huIFNλ4) was confirmed using immunohistochemistry assays and transmission electron microscopy. Its purity was verified by quantifying host cell DNA and host cell proteins, as well as by confirming the absence of the replication-competent adenovirus. The transduction of rAd-huIFNλ4 induced ISGs and inhibited four subtypes of the influenza virus in both mouse-derived (LA-4) and human-derived cells (A549). The antiviral state was confirmed in BALB/c mice following intranasal inoculation with 109 PFU of rAd-huIFNλ4, which led to the inhibition of four subtypes of the influenza virus in mouse lungs, with reduced inflammatory lesions. These results imply that human IFNλ4 could induce antiviral status by modulating ISG expression in mice.

Structural implications of BK polyomavirus sequence variations in the major viral capsid protein Vp1 and large T-antigen: a computational study.

Type and rate of amino acid variations in BKPyV may provide important insights into BKPyV diversity in human populations and an important step toward defining determinants of BKPyV-specific immunity needed to protect vulnerable patients from BKPyV diseases. Our analysis of BKPyV sequences obtained from human specimens reveals an unexpectedly high genetic variability for this double-stranded DNA virus that strongly relies on host cell DNA replication machinery with its proof reading and error correction mechanisms. BKPyV variability and immune escape should be taken into account when designing further approaches to antivirals, monoclonal antibodies, and vaccines for patients at risk of BKPyV diseases.

Novel purification platform based on multimodal preparative scale separation of mAb fragments and aggregates

Monoclonal antibodies (mAbs) continue to dominate the biopharmaceutical industry. Certain mAbs are prone to fragmentation and clipping and in these cases, adequate removal of these species is critical during manufacturing. Fragments can be generated during fermentation, purification, storage, formulation, and administration. Their addition to the acidic charge-variant of the purified mAb has been reported to decrease stability and potency of the final product. However, contrary to mAb aggregation, manufacturers have not given much attention to removal of fragments and clipped species and as a result most conventional mAb platforms offer at best limited capabilities for their removal. In this study, we propose a novel purification platform that uses multimodal chromatography and achieves complete removal of a range of mAb fragments and clipped products (25–120 kDa). The utility of the platform has been successfully demonstrated for 2 IgG1s and 2 IgG4s. Further, adequate removal of the various host cell impurities such as host cell proteins (<10 ppm) and host cell DNA (<5 ppb) has been achieved. Finally, the platform was able to deliver adequate removal of high molecular weight impurities (<1 %) and a 30 % clearance of the acidic charge variant. The proposed single step has been shown to deliver what the polishing chromatography and intermediate purification chromatography steps deliver in a traditional mAb platform.

Factors associated with unquantifiable total HIV-1 DNA in peripheral blood in persons living with HIV: An observational study

BackgroundThe human immunodeficiency virus type 1 (HIV-1) cannot be eradicated even with suppressive antiretroviral therapy because its retrotranscribed genome integrates into the DNA of host cells, creating a long-term reservoir. Quantification of total HIV-1 DNA in peripheral blood is a biomarker of this reservoir that can predict progression of the infection, treatment response, and HIV-1-related complications. A deeper understanding of the reservoir may help develop a cures. ObjectiveThis study aimed to characterize persons living with HIV-1 (PLWH) with unquantifiable total HIV-1 DNA in blood (below the quantification threshold) and identify associated factors. MethodsWe have conducted a retrospective observational study. During the study period, all PLWH who had total leukocyte-associated HIV-1 DNA measured by quantitative PCR were included. We have isolated a population of participants with HIV-1 DNA levels below the quantification threshold (40 copies/106 leukocytes). ResultsOut of 1094 patients analysed, 62 had unquantifiable and 1032 quantifiable HIV-1 DNA levels in blood. We have found that those with unquantifiable HIV-1 DNA had a higher CD4 T cell nadir (p = 0.006) and a lower viral load zenith (p < 0.001). Multivariate analyses showed that initiation of treatment in primary infection was the only protective factor against HIV-1 DNA quantifiability, the odds of HIV-1 DNA quantifiability decreased by 82% in those treated within 30 days of infection, after controlling for other factors. ConclusionOur research highlights the importance of an early start of anti-retroviral therapy to limit the size of the HIV-1 reservoir, as receiving treatment during primary infection was found as the only protective factor against quantifiability of HIV-1 DNA in blood.

Establishment and evaluation of detection methods for process-specific residual host cell protein and residual host cell DNA in biological preparation.

To establish accurate detection methods of process-specific Escherichia coli residual host cell protein (HCP) and residual host cell DNA (rcDNA) in recombinant biological preparations. Taking the purification process of GLP expressed by E. coli as a specific-process model, the HCP of empty E. coli was intercepted to immunize mice and rabbits. Using IgG from immunized rabbits as the coating antibody and mouse immune serum as the second sandwich antibody, a process-specific enzyme-linked immunosorbent assay(ELISA) for E. coli HCP was established. Targeting the 16S gene of E. coli, ddPCR was used to obtain the absolute copies of rcDNA in samples. Non-process-specific commercial ELISA kit and the process-specific ELISA established in this study were used to detect the HCP in GLP preparation. About 62% of HCPs, which should be process-specific HCPs, could not be detected by the non-process-specific commercial ELISA kit. The sensitivity of established ELISA can reach 338 pg/mL. The rcDNA could be absolutely quantitated by ddPCR, for the copies of rcDNA in three multiple diluted samples showed a reduced gradient. While the copies of rcDNA in three multiple diluted samples could not be distinguished by the qPCR. Process-specific ELISA has high sensitivity in detecting process-specific E. coli HCP. The absolutely quantitative ddPCR has much higher accuracy than the relatively quantitative qPCR, it is a nucleic acid quantitative method that is expected to replace qPCR in the future.

What goes into developing an in-house method for quantitation of residual host cell DNA?

What goes into developing an in-house method for quantitation of residual host cell DNA?

Recombinant AAV Purification.

Purification of rAAV is a crucial unit operation of the AAV production process. It enables the capture of AAV and removal of contaminants such as host cell proteins, host cell DNA, and other cell culture-related impurities. Here we describe the purification of rAAV produced in insect cells Sf9/rBEV by immuno-affinity capture chromatography. The method is fully scale-amenable unlike other traditional purification methods based on ultracentrifugation. The method reported herein has two main steps: (1) the clarification of cell lysate by depth filtration and (2) the selective capture and single-step purification of AAV via immune-affinity chromatography. This purification method has been successfully implemented to purify the majority of wild-type AAV serotypes.

The beneficial impact of kosmotropic salts on the resolution and selectivity of Protein A chromatography

During the manufacturing of therapeutic antibodies, effective Protein A chromatography as initial column step is crucial to simplify the remaining purification effort for subsequent polishing steps. This is particularly relevant for molecules with high impurity content so that desired product purity can be attained. The present study demonstrates beneficial effects on impurity removal when applying kosmotropic salts, e.g., sodium sulfate or sodium chloride, in the elution phase. Initially, a screen using negative linear pH gradient elution evaluated the impact of the kosmotropic salts in comparison to no additive and chaotropic urea using three mAbs and three common resins. Retaining acceptable yield, the kosmotropic salts improved resolution of monomer and impurities and reduced the contents of process-related host cell proteins and DNA as well as of product-related low and high molecular weight forms, despite some resin- and mAb-dependent variations. Moreover, a decrease in hydrolytic activity measured by a new assay for polysorbase activity was observed. In contrast, urea was hardly effective. The findings served to establish optimized step elution conditions with 0.25 M of sodium sulfate for a challenging mAb with complex format (bispecific 2 + 1 CrossMab) displaying high relative hydrophobicity and impurity levels. With yield and purity both in the range of 90 %, the contents of all impurity components were reduced, e.g., low molecular weight forms by two-fold and polysorbase activity by four-fold. The study indicates the potential of kosmotropic salts to establish efficient and comprehensive impurity separation by Protein A for facilitated downstream processing and economic manufacturing of complex antibodies.

Application and Technical Challenges in Design, Cloning, and Transfer of Large DNA.

In the field of synthetic biology, rapid advancements in DNA assembly and editing have made it possible to manipulate large DNA, even entire genomes. These advancements have facilitated the introduction of long metabolic pathways, the creation of large-scale disease models, and the design and assembly of synthetic mega-chromosomes. Generally, the introduction of large DNA in host cells encompasses three critical steps: design-cloning-transfer. This review provides a comprehensive overview of the three key steps involved in large DNA transfer to advance the field of synthetic genomics and large DNA engineering.