Biotechnology Progress最新文献

{"title":"In silico mediated development of orthogonally selective mAb downstream processes for the removal of process-related impurities","authors":"Dongyoun Jang,&nbsp;Mario A. Gutierrez-Diaz,&nbsp;Scott H. Altern,&nbsp;Hendri Tjandra,&nbsp;Steven M. Cramer","doi":"10.1002/btpr.70097","DOIUrl":"10.1002/btpr.70097","url":null,"abstract":"<p>Continued advancements in recombinant CHO expression of therapeutic mAbs have led to improved productivity but have also increased the HCP burden on the downstream purification process. In this work, we developed an in silico mediated workflow to facilitate the rapid development of non-protein A three-step processes for the effective removal of HCPs from a CHO-derived mAb therapeutic. Null CCF and pure mAb retention patterns were generated using linear gradient screens on a set of strategically selected resins, membrane adsorbers, and novel adsorbents. HCP characterization of key fractions was then carried out using RPLC “HCP fingerprinting” and the resulting retention database was processed using an in silico tool to generate a list of all possible three-step sequences subject to design constraints. Top-ranked processes generated by the tool were then evaluated and refined at the bench scale to produce several successful processes consisting of bind-elute capture followed by either a bind-elute and flowthrough step (91.4 ppm HCP with a cumulative product yield of 78.7%) or two flowthrough steps with no salt (96.1 ppm HCP with a cumulative yield of 81.4%).</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":"42 2","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145761963","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Artificial intelligence and machine learning-assisted digital applications for biopharmaceutical manufacturing","authors":"Shyam Panjwani,&nbsp;Hao Wei,&nbsp;John Mason","doi":"10.1002/btpr.70089","DOIUrl":"10.1002/btpr.70089","url":null,"abstract":"<p>Artificial intelligence and automation are no longer just buzzwords in the biopharmaceutical industry. The manufacturing of a class of biologics, comprising monoclonal antibodies, cell therapies, and gene therapies, is far more complex than that of traditional small molecule drugs. Therefore, applications based on artificial intelligence are essential for successfully manufacturing this new class of biologics more quickly and more economically. Some biologics manufacturers, academic researchers, and young entrepreneurs have already begun implementing artificial intelligence-based applications to increase operational efficiency, enhance process understanding, improve process monitoring, and achieve better regulatory compliance. Regulatory guidance from health agencies on the use of artificial intelligence and machine learning is acting as a catalyst in the adoption process of these new technologies by the biopharmaceutical industry. Research in artificial intelligence and machine learning has also advanced significantly in the last decade. At the same time, new cloud technologies have made the development and deployment of machine learning applications much easier. Several examples of artificial intelligence and machine learning applications in monoclonal antibodies manufacturing already exist. Cell and gene therapy, which present the future of medicine, will also benefit from this new technology. Overall, advancements in this domain will essentially help better serve patients' needs.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":"42 2","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13055133/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145480580","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enhancing CHO cell recombinant protein production using a perfusion-directed host evolution approach","authors":"Peter Amaya,&nbsp;Rajesh K. Mistry,&nbsp;Susie Sou,&nbsp;Sonja Hess,&nbsp;Bijay Khanal,&nbsp;Zayla Schaeffer,&nbsp;Jie Zhu,&nbsp;Lina Chakrabarti","doi":"10.1002/btpr.70093","DOIUrl":"10.1002/btpr.70093","url":null,"abstract":"<p>Clonally derived cell lines generated from Chinese hamster ovary (CHO) cells encounter numerous stressors when cultured in high-intensity perfusion bioreactors leading to poor process performance. To circumvent this, the ability of CHO cells to adapt to different culture environments was exploited. Here host cells were selected in the presence of physical and chemical stressors associated with a perfusion environment by culturing at a high cell density in a perfusion bioreactor for 30 days. Following recovery and expansion, the performance of the resulting perfusion-evolved host was evaluated using stable transfectant pools and clones expressing biotherapeutics of different formats. Cell lines generated from the perfusion host outperformed the parental host at several fundamental stages of the clone selection process. Perfusion host-derived pools showed elevations in productivity, cell-specific productivity, end-of-run viability, and reduced lactate production in fed-batch culture. Use of the perfusion host for cell line generation resulted in an increased frequency of high-producing clones. Moreover, the perfusion host-derived clones demonstrated 30% higher productivity and improved mannose profile in the perfusion environment compared to the clones from the parental host. Furthermore, a comparative proteomic analysis between the two host types revealed unique regulatory networks that allowed us to gain insights into the underlying molecular processes influencing production performance. Taken together, the results suggest that the perfusion host may not only increase the efficiency of the cell line development process but may also serve as an efficient tool for improvement in production capability in the perfusion platform.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":"42 2","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13055126/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145628444","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A case study showing the role of hydrophobicity variants and other enriched mAb proteoforms on filterability through a virus filter with productivity improvement measures","authors":"Solomon Isu,&nbsp;Derek Silva,&nbsp;Melissa Holstein,&nbsp;Angela Lewandowski,&nbsp;Kristina Cunningham,&nbsp;Adam Sokolnicki,&nbsp;Bala Raghunath","doi":"10.1002/btpr.70101","DOIUrl":"10.1002/btpr.70101","url":null,"abstract":"<p>A rapid assessment of manufacturability for drug candidates is crucial for advancing a prospective biotherapeutic from a candidate to a bulk drug substance. A lot-to-lot approach to manufacturability is adopted where each biologic batch is assessed for manufacturability as a bulk, unfractionated pool. Manufacturers may explore a more granular approach, independently enriching and evaluating the filterability of antibody variants within each lot, especially within the confines of relative hydrophobicity and surface charge. This study examined the use of bind-and-elute chromatography to alter the proportions of monoclonal antibody (mAb) proteoforms in eluate sub-pools from a mixed-mode chromatography resin-packed column. Filterability of each sub-pool through a virus-retaining filter was subsequently examined. Circular dichroism and Fourier transform infrared spectroscopy were performed for each sub-pool to probe for higher-order structure differences between mAb variants enriched therein. Bioanalytical techniques were also used to assess colloidal stability, surface hydrophobicity, surface charge, and size differences. Results showed that basic charge variants, high-mannose glycovariants, high relative hydrophobicity proteoforms, and high-molecular-weight species were enriched in the last-eluting (terminal) sub-pools. The first sub-pool and the final sub-pool showed the most fouling propensity on VPro virus filters. Circular dichroism showed that enriched proteoforms in the last sub-pool possessed a higher percentage of bends. Most secondary structures did not vary significantly between sub-pools. Diffusion interaction parameter was highly negative across all sub-pools and the bulk unfractionated pool. These results provide a design space for identifying and depleting problematic mAb variants before the crucial virus filtration step.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":"42 2","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13055118/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145997225","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a co-culture of Ureibacillus thermosphaericus and Cupriavidus taiwanensis for inhibitors removal from hemicellulose prehydrolysate","authors":"Mariem Theiri,&nbsp;Mariya Marinova,&nbsp;Hassan Chadjaa,&nbsp;Mario Jolicoeur","doi":"10.1002/btpr.70107","DOIUrl":"10.1002/btpr.70107","url":null,"abstract":"<p>For biofuels production, hemicellulose pre-hydrolysate is considered an attractive feedstock rich in fermentable sugars. The lignocellulosic biomass comprises, along with sugars, several inhibitors that can hamper its efficient conversion. In this work, mixed cultures of <i>Ureibacillus thermosphaericus</i> and <i>Cupriavidus taiwanensis</i> were used for the first time to detoxify the pre-hydrolysate. The nutrient source was first optimized in synthetic media with mono-cultures to detoxify phenolic compounds, and a medium containing inorganic salts was selected. Afterwards, the efficiency of phenolic degradation was compared in a single-compound solution and in a mixture. The simultaneous co-culture showed the highest degradation efficiency (90% at 2.8 g/L of phenolic compounds). Finally, the detoxification of a raw pre-hydrolysate was conducted, and a maximum degradation of 14% of the phenolics was obtained using sequential inoculation of <i>Ureibacillus thermosphaericus</i> followed by <i>Cupriavidus taiwanensis</i> addition.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":"42 2","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13055127/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145997216","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A size-exclusion chromatography fingerprinting workflow for the development of flow-through polishing operations for mAbs derived from continuous precipitation processes","authors":"Mario A. Gutierrez-Diaz,&nbsp;Scott H. Altern,&nbsp;Todd M. Przybycien,&nbsp;Steven M. Cramer","doi":"10.1002/btpr.70104","DOIUrl":"10.1002/btpr.70104","url":null,"abstract":"<p>In this work we present a workflow for developing two-step, flow-through polishing processes for monoclonal antibodies (mAbs). The approach is demonstrated using redissolved precipitates from three CHO-derived mAbs generated by a continuous, PEG/ZnCl₂-mediated precipitation capture process. Size Exclusion Chromatography (SEC) fingerprinting and a percent SEC clearance (PSC) metric are developed to enable simultaneous quantification of monomer yield and impurity removal during high-throughput screening and scale-down column studies. Batch slurry plate screens are used to evaluate multimodal anion exchange (MMA) resins and an activated carbon composite adsorber under varying pH and ionic strengths, assessing partition coefficients and PSC values against both low-molecular-weight (LMW) and high-molecular-weight (HMW) impurities. Top candidates were then assessed in single-column, higher-loading flow-through experiments using the redissolved precipitates as feeds. Activated carbon emerged as a highly effective first polishing step for LMW impurity removal under acidic, low-conductivity conditions, while MMA resins provided complementary LMW and HMW clearances in a subsequent flow-through step. The two-step processes achieved overall mAb recoveries of 80%–87%, reduced HMW species from &gt;1.7% down to 1.1%, and decreased host-cell protein levels from &gt;10,000 to &lt;40 ppm for all three mAbs. SEC fingerprints showed the ability to identify orthogonal impurity removal opportunities between the two polishing materials, validating the screening methodology for a process devoid of bind-elute processing steps. This work demonstrates that SEC-based impurity profiling and PSC metrics can guide the development of flow-through polishing processes and offer a useful intensification strategy to alleviate DSP bottlenecks and reduce reliance on affinity capture.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":"42 2","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145997255","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enhanced lentiviral vector recovery and separation using arginine hydrochloride with CIM QA monoliths","authors":"Andrew J. Kocot,&nbsp;Shivani Kulkarni,&nbsp;Ronit Ghosh,&nbsp;Scott H. Altern,&nbsp;Jonathan S. Dordick,&nbsp;Todd M. Przybycien,&nbsp;Steven M. Cramer","doi":"10.1002/btpr.70102","DOIUrl":"10.1002/btpr.70102","url":null,"abstract":"<p>Lentiviral vectors (LVVs) offer distinct advantages including large payload capacity and stable transduction of non-dividing cells, making them well-suited for ex vivo modification of stem or immune cells. However, chromatographic purification of LVVs is hindered by low recoveries, co-elution of product related impurities, and vector instability. In this study, we evaluated arginine hydrochloride (ArgHCl) as an alternative eluent to sodium chloride using CIMmultus™ QA (CIM QA) monoliths. Screening of solution conditions identified that phosphate buffer concentrations between 100–200 mM enhanced infective particle stability. During anion exchange screening experiments using a CIM QA 96-well plate, ArgHCl improved both infectious particle and p24 recoveries, which was corroborated in linear gradient elution (LGE) experiments using the monolith in a disk format. Furthermore, fractions eluted with ArgHCl exhibited improved colloidal stability compared to those eluted with NaCl. Increasing the ArgHCl gradient endpoint concentration to 1.5 M ArgHCl yielded infectious particle recoveries of 71%. Analysis of gradient fractions with nanoflow cytometry revealed a two-peak elution profile, with the more strongly retained peak enriched in vesicular stomatitis virus glycoprotein (VSV-G) positive particles, corresponding to greater infectious particle recoveries. ArgHCl also improved the chromatographic resolution between the initial impurity peak and the secondary peak, enabling improved separation. These findings support the use of ArgHCl and phosphate buffers to enhance recovery during the purification of LVVs using AEX chromatography and highlight the utility of nanoflow cytometry for rapid detection of product-related impurities.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":"42 2","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146028355","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Purification of antisense oligonucleotides using hydrophobic interaction chromatography","authors":"Robert S. Gronke,&nbsp;Jonas P. Immel-Brown,&nbsp;Sanjeev Jeyabalan,&nbsp;Patrick D. Banzon,&nbsp;Armin Delavari,&nbsp;Juan Cueva Tello,&nbsp;Ratnesh Joshi,&nbsp;Thi Ho","doi":"10.1002/btpr.70099","DOIUrl":"10.1002/btpr.70099","url":null,"abstract":"<p>Hydrophobic interaction chromatography (HIC) provides a powerful alternative impurity control method for antisense oligonucleotide purification relative to traditionally used anion exchange (AEX) and/or reverse phase methods. HIC is particularly effective in clearing process-related solvents and small molecules by ≥3 log₁₀ as well as failure sequences (sometimes called early eluting impurities (EEIs) by ≥90%). Additionally, HIC reduces harder to remove product-related impurities. These include branchmers (late eluting impurities (LEIs), oligonucleotides missing a single nucleotide (N-1 impurities), oligonucleotides lacking appropriate phosphorothioate sulfurization (P = O₁ impurity), and other synthesis-related impurities. To optimize the purification process, variables such as resin ligand, salt types, processing conditions, types of gradients, and loading ratios were systematically evaluated to achieve 90% yield and maximal impurity resolution. Loading the column at 32%–78% of its dynamic binding capacity (DBC), combined with stepwise wash and elution gradients, provided effective resolution of impurities in crude oligonucleotide mixtures. The desorption of the purified product was achieved in low lyotropic salt concentrations (typically ≤50 mM) using a stepwise gradient. This approach retained non-polar impurities such as LEIs within the column. When properly designed, HIC is an all-aqueous, scalable, cost effective and predictable purification process. It can be implemented as a stand-alone method or integrated into a dual-column process alongside orthogonal techniques, such as AEX, to achieve even higher levels of product purity.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":"42 2","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145833115","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enhancing mRNA vaccine production: Optimization of in vitro transcription for improved yield and purity","authors":"Kaixi Zhao,&nbsp;Jessica Raffaele,&nbsp;David A. Holland,&nbsp;Kristen Feibelman,&nbsp;Kathleen Lengel,&nbsp;Jon Shanter,&nbsp;Emily Wen","doi":"10.1002/btpr.70109","DOIUrl":"10.1002/btpr.70109","url":null,"abstract":"<p>In vitro transcription (IVT) is a powerful method to generate RNA which not only facilitates RNA research but also plays a key role in the development and manufacture of RNA-based vaccines. mRNA is produced via the IVT process with a DNA template that contains information for the target antigen. However, as many disease-causing viruses mutate quickly and the cost of raw materials is high for the IVT reaction, there is a need for a system to develop a cost-effective and efficient IVT process platform. In this paper, we showed how total nucleoside-5′-triphosphates (NTPs) input, Mg²⁺ concentration and NTP preparation methods can influence IVT reaction yield and purity level of the final RNA constructs of different lengths and sequences. We propose an IVT design that will result in high RNA yield, high RNA integrity and low double-stranded RNA (dsRNA) concentrations for multiple RNA sequences. The approach presented here could significantly contribute to the development of a cost-effective, easy-to-adopt IVT process platform for RNA manufacturing.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":"42 2","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146083907","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of gene editing in CHO cells using the Cas-CLOVER system","authors":"Tiffany McLamarrah,&nbsp;Efecan Aral,&nbsp;Michael Hoffman,&nbsp;Jennifer Tedstone,&nbsp;Thomas King,&nbsp;Jason Vitko,&nbsp;Maria João Sebastião,&nbsp;Jose M. Escandell,&nbsp;Mafalda M. Dias,&nbsp;Iona McCall,&nbsp;Daniel Machado,&nbsp;Victor Cairns,&nbsp;Christine DeMaria,&nbsp;John J. Scarcelli","doi":"10.1002/btpr.70108","DOIUrl":"10.1002/btpr.70108","url":null,"abstract":"<p>Recent advances in gene editing technologies have transformed the genetic engineering of Chinese hamster ovary (CHO) hosts, enabling the development of cell lines with improved stability and productivity. In this study, we employed the programmable nuclease (PN) Cas-CLOVER to precisely target the Glutamine synthetase (GS) locus in CHO cells. A total of 30 unique serum-free, suspension-adapted CHO-K1 candidate host cell lines were subjected to Cas-CLOVER-mediated gene editing, generating over one hundred potential GS knockout (GSKO) clones. A subset of the GSKO clones was subsequently validated using three orthogonal methods to confirm complete knockout of the GS gene in 98 clones. Randomly selected GSKO clones were utilized to produce standard monoclonal antibodies. The resulting pools demonstrated enhanced productivity, with up to a 14.5-fold increase in titer compared to their wild-type parental hosts. These findings highlight the potential of gene editing approaches to significantly improve recombinant protein production in CHO expression systems, offering valuable insights for biopharmaceutical manufacturing applications.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":"42 2","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13055124/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146140892","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}