Underestimated Ischemic Heart Disease in Major Adverse Cardiovascular Events after Septicemia Discharge.

Background and Objectives: Sepsis increases cardiovascular disease and causes death. Ischemic heart disease (IHD) without acute myocardial infarction has been discussed less, and the relationship between risk factors and IHD in septicemia survivors within six months is worthy of in-depth study. Our study demonstrated the incidence of IHD and the possible risk factors for IHD in septicemia patients within six months. Materials and Methods: An inpatient dataset of the Taiwanese Longitudinal Health Insurance Database between 2001 and 2003 was used. The events were defined as rehospitalization of stroke and IHD after discharge or death within six months after the first septicemia hospitalization. The relative factors of major adverse cardiovascular events (MACEs) and IHD were identified by multivariate Cox proportional regression. Results: There were 4323 septicemia survivors and 404 (9.3%) IHD. New-onset atrial fibrillation had a hazard ratio (HR) of 1.705 (95% confidence interval (C.I.): 1.156-2.516) for MACEs and carried a 184% risk with HR 2.836 (95% C.I.: 1.725-4.665) for IHD by adjusted area and other risk factors. Conclusions: This study explored advanced-aged patients who experienced more severe septicemia with new-onset atrial fibrillation, which increases the incidence of IHD in MACEs within six months of septicemia. Therefore, healthcare providers must identify patients with a higher IHD risk and modify risk factors beforehand.

Identifying the differences in mechanisms of mycophenolic acid controlling fucose content of glycoproteins expressed in different CHO cell lines.

In the biopharmaceutical industry, glycosylation is a critical quality attribute that can modulate the efficacy of a therapeutic glycoprotein. Obtaining a consistent glycoform profile is desired because molecular function can be defined by its carbohydrate structures. Specifically, the fucose content of oligosaccharides in glycoproteins is one of the most important attributes that can significantly affect antibody-dependent cellular cytotoxicity (ADCC) activity. It is therefore important to understand the fucosylation pathway and be able to control fucosylation at the desired level to match predecessor materials in late stage and biosimilar programs. Several strategies were explored in this study and mycophenolic acid (MPA) was able to finely modulate the fucose content with the least undesired side effects. However, the response was significantly different between CHO cell lines of different lineages. Further experiments were then performed for a deeper understanding of the mechanism of fucosylation in different CHO cell lines. Results indicated that changes in the intracellular nucleotide involved in fucosylation pathway after MPA treatment are the main cause of the differences in fucosylation level response in different CHO cell lines. Differences in MPA metabolism in the various CHO cell lines directly resulted in different levels of afucosylation measured in antibodies produced by the CHO cell lines. Biotechnol. Bioeng. 2016;113: 2367-2376. © 2016 Wiley Periodicals, Inc.

Toward Computationally Designed Self-Reporting Biosensors Using Leave-One-Out Green Fluorescent Protein.

Leave-one-out green fluorescent protein (LOOn-GFP) is a circularly permuted and truncated GFP lacking the nth ß-strand element. LOO7-GFP derived from the wild-type sequence (LOO7-WT) folds and reconstitutes fluorescence upon addition of ß-strand 7 (S7) as an exogenous peptide. Computational protein design may be used to modify the sequence of LOO7-GFP to fit a different peptide sequence, while retaining the reconstitution activity. Here we present a computationally designed leave-one-out GFP in which wild-type strand 7 has been replaced by a 12-residue peptide (HA) from the H5 antigenic region of the Thailand strain of H5N1 influenza virus hemagglutinin. The DEEdesign software was used to generate a sequence library with mutations at 13 positions around the peptide, coding for approximately 3 × 10(5) sequence combinations. The library was coexpressed with the HA peptide in E. coli and colonies were screened for in vivo fluorescence. Glowing colonies were sequenced, and one (LOO7-HA4) with 7 mutations was purified and characterized. LOO7-HA4 folds, fluoresces in vivo and in vitro, and binds HA. However, binding results in a decrease in fluorescence instead of the expected increase, caused by the peptide-induced dissociation of a novel, glowing oligomeric complex instead of the reconstitution of the native structure. Efforts to improve binding and recover reconstitution using in vitro evolution produced colonies that glowed brighter and matured faster. Two of these were characterized. One lost all affinity for the HA peptide but glowed more brightly in the unbound oligomeric state. The other increased in affinity to the HA peptide but still did not reconstitute the fully folded state. Despite failing to fold completely, peptide binding by computational design was observed and was improved by directed evolution. The ratio of HA to S7 binding increased from 0.0 for the wild-type sequence (no binding) to 0.01 after computational design (weak binding) and to 0.48 (comparable binding) after in vitro evolution. The novel oligomeric state is composed of an open barrel.

Improving computational efficiency and tractability of protein design using a piecemeal approach. A strategy for parallel and distributed protein design.

MOTIVATION: Accuracy in protein design requires a fine-grained rotamer search, multiple backbone conformations, and a detailed energy function, creating a burden in runtime and memory requirements. A design task may be split into manageable pieces in both three-dimensional space and in the rotamer search space to produce small, fast jobs that are easily distributed. However, these jobs must overlap, presenting a problem in resolving conflicting solutions in the overlap regions. RESULTS: Piecemeal design, in which the design space is split into overlapping regions and rotamer search spaces, accelerates the design process whether jobs are run in series or in parallel. Large jobs that cannot fit in memory were made possible by splitting. Accepting the consensus amino acid selection in conflict regions led to non-optimal choices. Instead, conflicts were resolved using a second pass, in which the split regions were re-combined and designed as one, producing results that were closer to optimal with a minimal increase in runtime over the consensus strategy. Splitting the search space at the rotamer level instead of at the amino acid level further improved the efficiency by reducing the search space in the second pass. AVAILABILITY AND IMPLEMENTATION: Programs for splitting protein design expressions are available at www.bioinfo.rpi.edu/tools/piecemeal.html CONTACT: bystrc@rpi.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Advanced process monitoring and feedback control to enhance cell culture process production and robustness.

It is a common practice in biotherapeutic manufacturing to define a fixed-volume feed strategy for nutrient feeds, based on historical cell demand. However, once the feed volumes are defined, they are inflexible to batch-to-batch variations in cell growth and physiology and can lead to inconsistent productivity and product quality. In an effort to control critical quality attributes and to apply process analytical technology (PAT), a fully automated cell culture feedback control system has been explored in three different applications. The first study illustrates that frequent monitoring and automatically controlling the complex feed based on a surrogate (glutamate) level improved protein production. More importantly, the resulting feed strategy was translated into a manufacturing-friendly manual feed strategy without impact on product quality. The second study demonstrates the improved process robustness of an automated feed strategy based on online bio-capacitance measurements for cell growth. In the third study, glucose and lactate concentrations were measured online and were used to automatically control the glucose feed, which in turn changed lactate metabolism. These studies suggest that the auto-feedback control system has the potential to significantly increase productivity and improve robustness in manufacturing, with the goal of ensuring process performance and product quality consistency.

Development of bioreactor scale-down model using orthogonal projections to latent structures method and CO2 supplementation.

Scale-down model qualification is an important step for developing a large-scale cell culture process to enhance process understanding and support process characterization studies. Traditionally, only harvest data are used to show consistency between small-scale and large-scale bioreactor performance, allowing attributes that are dynamic over the cell culture period to be overlooked. A novel statistical method, orthogonal projections to latent structures (OPLS) analysis, can be utilized to compare time-course cell culture data across scales. Here we describe an example where OPLS is used to identify gaps between small-scale and large-scale bioreactor performances. In this case, differences in the partial pressure of carbon dioxide (pCO2) and lactate profiles were observed between small- and large-scale bioreactors, which were linked to differences in the product-quality attributes fragments and galactosylation. An improved small-scale model was developed, leading to improved consistency in the process performance and product qualities across scales and qualification of the scale-down model for regulatory submissions. This new statistical approach can provide valuable insights into process understanding and process scale-up.

23ME-00610, a genetically informed, first-in-class antibody targeting CD200R1 to enhance antitumor T cell function.

Immune checkpoint inhibition (ICI) has revolutionized cancer treatment; however, only a subset of patients benefit long term. Therefore, methods for identification of novel checkpoint targets and development of therapeutic interventions against them remain a critical challenge. Analysis of human genetics has the potential to inform more successful drug target discovery. We used genome-wide association studies of the 23andMe genetic and health survey database to identify an immuno-oncology signature in which genetic variants are associated with opposing effects on risk for cancer and immune diseases. This signature identified multiple pathway genes mapping to the immune checkpoint comprising CD200, its receptor CD200R1, and the downstream adapter protein DOK2. We confirmed that CD200R1 is elevated on tumor-infiltrating immune cells isolated from cancer patients compared to the matching peripheral blood mononuclear cells. We developed a humanized, effectorless IgG1 antibody (23ME-00610) that bound human CD200R1 with high affinity (KD <0.1 nM), blocked CD200 binding, and inhibited recruitment of DOK2. 23ME-00610 induced T-cell cytokine production and enhanced T cell-mediated tumor cell killing in vitro. Blockade of the CD200:CD200R1 immune checkpoint inhibited tumor growth and engaged immune activation pathways in an S91 tumor cell model of melanoma in mice.

Endothelin-1 stimulates suppressor of cytokine signaling-3 gene expression in adipocytes.

Endothelin (ET)-1 and suppressor of cytokine signaling (SOCS)-3 were respectively found to regulate energy metabolism and hormone signaling in fat cells. Although ET-1 can also regulate the expression of SOCS-3-stimulating hormones, it is still unknown whether ET-1 regulates SOCS-3 gene expression. This study investigated the pathways involved in ET-1's modulation of SOCS-3 gene expression in 3T3-L1 adipocytes. ET-1 upregulated SOCS-3 mRNA and protein expression in dose- and time-dependent manners. The concentration of ET-1 that increased SOCS-3 mRNA levels by 250-400% was ∼100nM with 2-4h of treatment. Treatment with actinomycin D prevented ET-1-stimulated SOCS-3 mRNA expression, suggesting that the effect of ET-1 requires new mRNA synthesis. Pretreatment with the ET type A receptor (ET(A)R) antagonist, BQ-610, but not the ET type B receptor (ET(B)R) antagonist, BQ-788, prevented the stimulatory effect of ET-1 on SOCS-3 gene expression. The specific inhibitors of either MEK1 (U-0126 and PD-98059), JAK (AG-490), JNK (SP-600125), or PI3K (LY-294002 and wortmannin) reduced ET-1-increased levels of SOCS-3 mRNA and respectively inhibited ET-1-stimulated activities of MEK1, JAK, JNK, and PI3K. These results imply that the ET(A)R, ERK, JAK, JNK, and PI3K are functionally necessary for ET-1's stimulation of SOCS-3 gene expression. Moreover, ET-1 was observed to upregulate expressions of SOCS-1, -2, -3, -4, -5, and -6 mRNAs, but not SOCS-7 or cytokine-inducible SH2-containing protein-1 mRNAs. This suggests that ET-1 selectively affects particular types of SOCS family members. Changes in SOCS gene expressions induced by ET-1 may help explain the mechanism by which ET-1 modulates hormone signaling of adipocytes.

Females with Diabetes Mellitus Increased the Incidence of Premenstrual Syndrome.

Background: Premenstrual syndrome (PMS) is a multifactorial disorder caused by hormone and autonomic imbalance. In our study, hyperglycemia-induced insulin secretion increased progesterone secretion and progressive autonomic imbalance. The young patients with diabetes mellitus (DM) revealed hypo-parasympathetic function and hypersympathetic function compared with nondiabetic controls. Young female patients with DM with higher blood sugar and autonomic malfunction may be associated with PMS. However, there is a lack of evidence about DM in females related to PMS. We evaluated female patients with DM who subsequently followed PMS in a retrospective cohort study. Methods: We retrieved data from the National Health Insurance Research Database in Taiwan. Female patients with DM between 20 and 50 years old were assessed by the International Classification of Disease, 9 Revision, Clinical Modification (ICD-9-CM) disease code of 250. Patients who were DM-free females were fourfold matched to the control group by age and disease index date. The ICD-9-CM disease code of 625.4 identified the incidence of PMS followed by the index date as events. The possible risk factors associated with PMS were detected with a Cox proportional regression. Results: DM was a significant risk factor for PMS incidence with an adjusted hazard ratio of 1.683 (95% confidence interval: 1.104−2.124, p < 0.001) in females after adjusting for age, other comorbidities, season, urbanization status of patients and the hospital status of visiting. Conclusions: This study noted an association between DM and PMS in female patients. Healthcare providers and female patients with DM must be aware of possible complications of PMS, aggressive glycemic control, decreased hyperglycemia and autonomic dysfunction to prevent this bothersome disorder.

A rewired green fluorescent protein: folding and function in a nonsequential, noncircular GFP permutant.

The sequential order of secondary structural elements in proteins affects the folding and activity to an unknown extent. To test the dependence on sequential connectivity, we reconnected secondary structural elements by their solvent-exposed ends, permuting their sequential order, called "rewiring". This new protein design strategy changes the topology of the backbone without changing the core side chain packing arrangement. While circular and noncircular permutations have been observed in protein structures that are not related by sequence homology, to date no one has attempted to rationally design and construct a protein with a sequence that is noncircularly permuted while conserving three-dimensional structure. Herein, we show that green fluorescent protein can be rewired, still functionally fold, and exhibit wild-type fluorescence excitation and emission spectra.

Discovery and investigation of misincorporation of serine at asparagine positions in recombinant proteins expressed in Chinese hamster ovary cells.

Misincorporation of amino acids in proteins expressed in Escherichia coli has been well documented but not in proteins expressed in mammalian cells under normal recombinant protein production conditions. Here we report for the first time that Ser can be incorporated at Asn positions in proteins expressed in Chinese hamster ovary cells. This misincorporation was discovered as a result of intact mass measurement, peptide mapping analysis, and tandem mass spectroscopy sequencing. Our analyses showed that the substitution was not related to specific protein molecules or DNA codons and was not site-specific. We believe that the incorporation of Ser at sites coded for Asn was due to mischarging of tRNA(Asn) rather than to codon misreading. The rationale for substitution of Asn by Ser and not by other amino acids is also discussed. Further investigation indicated that the substitution was due to the starvation for Asn in the cell culture medium and that the substitution could be limited by using the Asn-rich feed. These observations demonstrate that the quality of expressed proteins should be closely monitored when altering cell culture conditions.

Control of misincorporation of serine for asparagine during antibody production using CHO cells.

A recombinant monoclonal antibody produced by Chinese hamster ovary (CHO) cell fed-batch culture was found to have amino acid sequence misincorporation upon analysis by intact mass and peptide mapping mass spectrometry. A detailed analysis revealed multiple sites for asparagine were being randomly substituted by serine, pointing to mistranslation as the likely source. Results from time-course analysis of cell culture suggest that misincorporation was occurring midway through the fed-batch process and was correlated to asparagine reduction to below detectable levels in the culture. Separate shake flask experiments were carried out that confirmed starvation of asparagine and not excess of serine in the medium as the root cause of the phenomenon. Reduction in serine concentration under asparagine starvation conditions helped reduce extent of misincorporation. Supplementation with glutamine also helped reduce extent of misincorporation. Maintenance of asparagine at low levels in 2 L bench-scale culture via controlled supplementation of asparagine-containing feed eliminated the occurrence of misincorporation. This strategy was implemented in a clinical manufacturing process and scaled up successfully to the 200 and 2,000 L bioreactor scales.

Complementation and reconstitution of fluorescence from circularly permuted and truncated green fluorescent protein.

Green fluorescent protein (GFP) has been used as a proof of concept for a novel "leave-one-out" biosensor design in which a protein that has a segment omitted from the middle of the sequence by circular permutation and truncation binds the missing peptide and reconstitutes its function. Three variants of GFP have been synthesized that are each missing one of the 11 beta-strands from its beta-barrel structure, and in two of the variants, adding the omitted peptide sequence in trans reconstitutes fluorescence. Detailed biochemical analysis indicates that GFP with beta-strand 7 "left out" (t7SPm) exists in a partially unfolded state. The apo form t7SPm binds the free beta-strand 7 peptide with a dissociation constant of approximately 0.5 microM and folds into the native state of GFP, resulting in fluorescence recovery. Folding of t7SPm, both with and without the peptide ligand, is at least a three-state process and has a rate comparable to that of the full-length and unpermuted GFP. The conserved kinetic properties strongly suggest that the rate-limiting steps in the folding pathway have not been altered by circular permutation and truncation in t7SPm. This study shows that structural and functional reconstitution of GFP can occur with a segment omitted from the middle of the chain, and that the unbound form is in a partially unfolded state.

Activation of MET promotes resistance to sorafenib in hepatocellular carcinoma cells via the AKT/ERK1/2-EGR1 pathway.

Sorafenib is an oral multikinase inhibitor that has become an established therapeutic approach in advanced hepatocellular carcinoma (HCC). However, the benefit of sorafenib in clinical therapy is often affected by drug resistance. Therefore, it is important to explore the mechanisms underlying sorafenib resistance and to develop individualized therapeutic strategies for coping with this problem. In this study, we found that addition of HGF to sorafenib-treated HCC cells activated MET and re-stimulated the downstream AKT and ERK1/2 pathways. Thereby, restored sorafenib-treated HCC cells proliferation, migration and invasion ability, and rescued cells from apoptosis. In addition, we found that HGF treatment of HCC cells induced early growth response protein (EGR1) expression, which is involved in sorafenib resistance. Importantly, the HGF rescued effect in sorafenib-treated HCC cells could be abrogated by inhibiting MET activation with PHA-665752 or by downregulating EGR1 expression with small interfering RNA (siRNA). Therefore, inhibition of the HGF/MET pathway may improve response to sorafenib in HCC, and combination therapy should be further investigated.

Computational design of a modular protein sense-response system.

Sensing and responding to signals is a fundamental ability of living systems, but despite substantial progress in the computational design of new protein structures, there is no general approach for engineering arbitrary new protein sensors. Here, we describe a generalizable computational strategy for designing sensor-actuator proteins by building binding sites de novo into heterodimeric protein-protein interfaces and coupling ligand sensing to modular actuation through split reporters. Using this approach, we designed protein sensors that respond to farnesyl pyrophosphate, a metabolic intermediate in the production of valuable compounds. The sensors are functional in vitro and in cells, and the crystal structure of the engineered binding site closely matches the design model. Our computational design strategy opens broad avenues to link biological outputs to new signals.

Concentrated fed-batch cell culture increases manufacturing capacity without additional volumetric capacity.

Biomanufacturing factories of the future are transitioning from large, single-product facilities toward smaller, multi-product, flexible facilities. Flexible capacity allows companies to adapt to ever-changing pipeline and market demands. Concentrated fed-batch (CFB) cell culture enables flexible manufacturing capacity with limited volumetric capacity; it intensifies cell culture titers such that the output of a smaller facility can rival that of a larger facility. We tested this hypothesis at bench scale by developing a feeding strategy for CFB and applying it to two cell lines. CFB improved cell line A output by 105% and cell line B output by 70% compared to traditional fed-batch (TFB) processes. CFB did not greatly change cell line A product quality, but it improved cell line B charge heterogeneity, suggesting that CFB has both process and product quality benefits. We projected CFB output gains in the context of a 2000-L small-scale facility, but the output was lower than that of a 15,000-L large-scale TFB facility. CFB's high cell mass also complicated operations, eroded volumetric productivity, and showed our current processes require significant improvements in specific productivity in order to realize their full potential and savings in manufacturing. Thus, improving specific productivity can resolve CFB's cost, scale-up, and operability challenges.

Application of high-throughput mini-bioreactor system for systematic scale-down modeling, process characterization, and control strategy development.

High-throughput systems and processes have typically been targeted for process development and optimization in the bioprocessing industry. For process characterization, bench scale bioreactors have been the system of choice. Due to the need for performing different process conditions for multiple process parameters, the process characterization studies typically span several months and are considered time and resource intensive. In this study, we have shown the application of a high-throughput mini-bioreactor system viz. the Advanced Microscale Bioreactor (ambr15(TM) ), to perform process characterization in less than a month and develop an input control strategy. As a pre-requisite to process characterization, a scale-down model was first developed in the ambr system (15 mL) using statistical multivariate analysis techniques that showed comparability with both manufacturing scale (15,000 L) and bench scale (5 L). Volumetric sparge rates were matched between ambr and manufacturing scale, and the ambr process matched the pCO2 profiles as well as several other process and product quality parameters. The scale-down model was used to perform the process characterization DoE study and product quality results were generated. Upon comparison with DoE data from the bench scale bioreactors, similar effects of process parameters on process yield and product quality were identified between the two systems. We used the ambr data for setting action limits for the critical controlled parameters (CCPs), which were comparable to those from bench scale bioreactor data. In other words, the current work shows that the ambr15(TM) system is capable of replacing the bench scale bioreactor system for routine process development and process characterization.

Cultivation of microplantlets derived from the marine red alga Agardhiella subulata in a stirred tank photobioreactor.

Macrophytic marine red algae are a diverse source of bioactive natural compounds. "Microplantlet" suspension cultures established from red algae are potential platforms for biosynthesis of these compounds, provided suitable bioreactor configurations for mass culture can be identified. The stirred tank bioreactor offers high rates of gas-liquid mass transfer, which may facilitate the delivery of the CO(2) in the aeration gas to the phototrophic microplantlet suspension culture. Therefore, the effects of impeller speed and CO(2) delivery on the long-term production of microplantlet biomass of the model red alga Agardhiella subulata was studied within a stirred tank photobioreactor equipped with a paddle blade impeller (D(i)/D(T) = 0.5). Nutrient medium replacement was required for sustained biomass production, and the biomass yield coefficient based on nitrate consumption was 1.08 +/- 0.09 g dry biomass per mmol N consumed. Biomass production went through two exponential phases of growth, followed by a CO(2) delivery limited growth phase. The CO(2)-limited growth phase was observed only if the specific growth rate in the second exponential phase of growth was at least 0.03 day(-)(1), the CO(2) delivery rate was less than 0.258 mmol CO(2) L(-)(1) culture h(-)(1), and the plantlet density was at least 10 g fresh mass L(-)(1). Increasing the aeration gas CO(2) partial pressure from 0.00035 to 0.0072 atm decreased the cultivation pH from 8.8 to 7.8, prolonged the second exponential phase of growth by increasing the CO(2) delivery rate, and also increased the photosynthetic oxygen evolution rate. Impeller speeds ranging from 60 to 250 rpm, which generated average shear rates of 2-10 s(-)(1), did not have a significant effect on biomass production rate. However, microplantlets cultivated in a stirred tank bioreactor ultimately assumed compact spherical shape, presumably to minimize exposure to hydrodynamic stress.

Dynamics of oxygen evolution and biomass production during cultivation of Agardhiella subulata microplantlets in a bubble-column photobioreactor under medium perfusion.

Cell and tissue cultures derived from macrophytic marine red algae are potential platforms for unique secondary metabolites. This work presents the first successful bioreactor cultivation study of an in vitro tissue culture derived from a macrophytic marine red alga. Specifically, the photosynthetic growth characteristics of a novel microplantlet suspension culture established from the macrophytic marine red alga Agardhiella subulata were studied. A bubble-column bioreactor with external illumination (43 microE m(-2) s(-1), 10:14 LD photoperiod), liquid medium perfusion, and 3800 ppm CO(2) in the aeration gas provided sufficient light and nutrient delivery for sustained growth of the microplantlet suspension at 24 degrees C and pH 8. Microplantlets, which consisted of shoot tissues of 3-5 mm length branching out from a common center, were not friable in a bubble-aerated suspension of about 1100 plantlets per liter. Since the microplantlet tissues were not friable, only batch and fed-batch cultivation modes were considered. Batch cultivation was phosphate-limited in ASP12 artificial seawater medium. However, cultivation at a medium perfusion rate of 20% per day avoided phosphate limitation and extended the growth phase to provide plantlet mass densities exceeding 14 g FW L(-1) (3.7 g DW L(-1)) after 50 days of cultivation if the suspension was not sampled. The specific oxygen evolution rate vs cultivation time profile possessed a significant pulse within the 14 days following inoculation and then leveled off at longer times. In recognition of this nonexponential growth pattern, a new photobioreactor growth model was developed that used the oxygen evolution rate vs time profile to predict the biomass growth curve in perfusion culture. Model predictions agreed reasonably with the measured growth curves.

Perfusion seed cultures improve biopharmaceutical fed-batch production capacity and product quality.

Volumetric productivity and product quality are two key performance indicators for any biopharmaceutical cell culture process. In this work, we showed proof-of-concept for improving both through the use of alternating tangential flow perfusion seed cultures coupled with high-seed fed-batch production cultures. First, we optimized the perfusion N-1 stage, the seed train bioreactor stage immediately prior to the production bioreactor stage, to minimize the consumption of perfusion media for one CHO cell line and then successfully applied the optimized perfusion process to a different CHO cell line. Exponential growth was observed throughout the N-1 duration, reaching >40 × 10(6) vc/mL at the end of the perfusion N-1 stage. The cultures were subsequently split into high-seed (10 × 10(6) vc/mL) fed-batch production cultures. This strategy significantly shortened the culture duration. The high-seed fed-batch production processes for cell lines A and B reached 5 g/L titer in 12 days, while their respective low-seed processes reached the same titer in 17 days. The shortened production culture duration potentially generates a 30% increase in manufacturing capacity while yielding comparable product quality. When perfusion N-1 and high-seed fed-batch production were applied to cell line C, higher levels of the active protein were obtained, compared to the low-seed process. This, combined with correspondingly lower levels of the inactive species, can enhance the overall process yield for the active species. Using three different CHO cell lines, we showed that perfusion seed cultures can optimize capacity utilization and improve process efficiency by increasing volumetric productivity while maintaining or improving product quality.