Cell Culture Broth Research Articles

Primary clarification of mammalian cell culture fluid using enhanced sedimentation on inclined surfaces inside the single-use disposable Sudhin BioSettler150.

The first downstream processing step in the purification of a biopharmaceutical protein secreted into mammalian cell culture fluid is the primary clarification of the culture fluid. As cell densities in the fed-batch and increasingly more common perfusion bioreactors have increased over last two decades through intensified upstream bioreactor production processes, the traditional primary clarification unit operations of centrifugation and/or microfiltration become more challenging with issues like frequent desludging, cell disruption due to shear damage and quick fouling of membranes. We have developed a novel compact cell settler device exploiting the enhanced sedimentation on inclined surfaces and demonstrated that this settler device can be adapted easily to remove and contain cells or cell clumps from the clarified supernatant collected via the top effluent of the settler. In this work, we present high product recovery results during primary clarification of mammalian cell culture supernatant using our novel single-use disposable BioSettler150 while processing about 10 L of cell culture broth within short processing times of about 4 h.

A Single-Step Method for Harvesting Influenza Viral Particles from MDCK Cell Culture Supernatant with High Yield and Effective Impurity Removal

Influenza vaccines, which are recommended by the World Health Organization (WHO), are the most effective preventive measure against influenza virus infection. Madin–Darby canine kidney (MDCK) cell culture is an emerging technology used to produce influenza vaccines. One challenge when purifying influenza vaccines using this cell culture system is to efficiently remove impurities, especially host cell double-stranded DNA (dsDNA) and host cell proteins (HCPs), for safety assurance. In this study, we optimized ion-exchange chromatography methods to harvest influenza viruses from an MDCK cell culture broth, the first step in influenza vaccine purification. Bind/elute was chosen as the mode of operation for simplicity. The anion-exchange Q chromatography method was able to efficiently remove dsDNA and HCPs, but the recovery rate for influenza viruses was low. However, the cation-exchange SP process was able to simultaneously achieve high dsDNA and HCP removal and high influenza virus recovery. For the SP process to work, the clarified cell culture broth needed to be diluted to reduce its ionic strength, and the optimal dilution rate was determined to be 1:2 with purified water. The SP process yielded a virus recovery rate exceeding 90%, as measured using a hemagglutination units (HAUs) assay, with removal efficiencies over 97% for HCPs and over 99% for dsDNA. Furthermore, the general applicability of the SP chromatography method was demonstrated with seven strains of influenza viruses recommended for seasonal influenza vaccine production, including H1N1, H3N2, B (Victoria), and B (Yamagata) strains, indicating that the SP process could be utilized as a platform process. The SP process developed in this study showed four advantages: (1) simple operation, (2) a high recovery rate for influenza viruses, (3) a high removal rate for major impurities, and (4) general applicability.

A Convenient Self-Removing Affinity Tag Method for the Simple Purification of Tagless Recombinant Proteins.

In this work, we describe a novel self-cleaving affinity tag technology based on a highly modified split-intein cleaving element. In this system, which has recently been commercialized by Protein Capture Science, LLC under the name iCapTagTM , the N-terminal segment of an engineered split intein is covalently immobilized onto a capture resin, while the smaller C-terminal intein segment is fused to the N-terminus of the desired target protein. The tagged target can then be expressed in an appropriate expression system, without concern for premature intein cleaving. During the purification, strong binding between the intein segments effectively captures the tagged target onto the capture resin while simultaneously generating a cleaving-competent intein complex. After unwanted impurities are washed from the resin, cleavage of the target protein is initiated by a shift of the buffer pH from 8.5 to 6.2. As a result, the highly purified tagless target protein is released from the column in the elution step. Alternately, the resin beads can be added directly to cell culture broth or lysate, allowing capture, purification and cleavage of the tagless target protein using a column-free format. These methods result in highly pure tagless target protein in a single step, and can thereby accelerate characterization and functional studies. In this work we demonstrate the single step purification of streptokinase, a fibrinolytic agent, and an engineered recombinant human hemoglobin 1.1 (rHb1.1). © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression of high-titer protein tagged with the Nostoc punctiforme (Npu) DnaE split-intein on the N-terminus Basic Protocol 2: Purification of high-titer protein using the Nostoc punctiforme (Npu) DnaE split-intein purification platform Alternate Protocol 1: Expression of low-titer protein tagged with the Nostoc punctiforme (Npu) DnaE split-intein on the N-terminus Alternate Protocol 2: Purification of low-titer protein using the Nostoc punctiforme (Npu) DnaE split-intein purification platform.

Single‐use centrifugal separator enables intensification of the clarification process in biomanufacturing of recombinant proteins

AbstractBACKGROUNDThe need to produce more biologics at lower costs and hence intensify bioprocessing has significantly improved upstream productivity (cell culture titers ≥3 g L−1 and cell densities ≥20 × 106 cells mL−1), shifting the economic and production bottleneck to downstream manufacturing. The first downstream unit operation is the clarification of the cell culture broth to remove various insoluble (such as cells and cell debris) and soluble (such as host cell DNA and proteins) contaminants, producing a feed stream suitable for further downstream purification. The high cell‐density cultures obtained in modern intensified bioprocessing have made legacy single‐use clarification trains (based primarily on depth filtration of the cell culture broth) ineffective and impractical, significantly increasing the economic and material burden of the clarification process.RESULTSWe have developed and characterized approaches that enable intensification of the clarification operation, by increasing process capacities while reducing material requirements and footprint, for single‐use manufacturing facilities. These approaches are based on primary clarification using the Alfa Laval CultureOne™ centrifuge (a continuous solid discharge disc‐stack centrifuge with a single‐use flow path) followed by secondary clarification using single‐use disposable depth filters. We show that these intensified clarification trains are suitable for processing high cell‐density cultures (6–12% packed cell volume) with significantly high yields (≥90%) and impurity clearance, along with reduced operating costs (30–60% savings).CONCLUSIONIncorporating a single‐use continuous discharge centrifuge into the clarification train enables clarification of high cell‐density cultures in single‐use manufacturing facilities and enhances process economic and environmental sustainability. © 2023 Society of Chemical Industry (SCI).

Antagonistic activity and application of Bacillus velezensis strain Bv-303 against rice bacterial-blight disease caused by Xanthomonas oryzae pv. oryzae

In this study, a new Bacillus velezensis strain Bv-303 was identified and its biocontrol effect against rice bacterial-blight (BB) disease caused by Xanthomonas oryzae pv. oryzae (Xoo) was investigated. Cell-free supernatant (CFS) of strain Bv-303 under different growth conditions were prepared to test the antagonistic activity and stability against Xoo by the Oxford-cup method in vitro. The antibacterial effect of strain Bv-303 to BB disease in rice were further analyzed in vivo by spraying the cell-culture broth (CCB), CFS and cell-suspension water (CSW), respectively, on the rice leaves inoculated with Xoo. Additionally, rice seeds germination rate and seedling growth under the strain Bv-303 CCB treatment were tested. The results showed that the strain Bv-303 CFS significantly inhibited Xoo growth by 85.7%‒88.0% in vitro, which was also stable under extreme environment conditions such as heat, acid, alkali and ultraviolet light. As tested in vivo, spraying the CCB, CFS or CSW of strain Bv-303 on the Xoo-infected leaves enhanced rice plant resistance to BB disease, with CCB showing the highest increase (62.7%) in disease-resistance. Notably, CCB does not have negative effects on rice seed germination and seedling growth. Therefore, strain Bv-303 has great potential for biocontrol of the rice BB disease.

Near-infrared spectroscopy based monitoring of all 20 amino acids in mammalian cell culture broth

The biopharmaceutical industry extensively employs Chinese hamster ovary (CHO) cell culture for monoclonal antibody production. Amino acids represent an essential source of nutrients in all CHO cell culture media, and their concentration is known to significantly impact cell viability, titre, and monoclonal antibody critical quality attributes. In this study, a robust Fourier transform near-infrared spectroscopy (FT-NIR) based quantification method has been developed for of all 20 amino acids (0–24 mM), as well as concentrations of glucose (0–6.7 mg mL−1), lactate (0–2.7 mg mL−1), and trastuzumab (0–2.5 mg mL−1) in the CHO cell culture. Near infra-red absorbance spectrum in the range of 4000–11,000 cm−1 were acquired, and spectra pre-processing through smoothening and derivatives were employed to enhance key characteristic signals. High-performance liquid chromatography with pre-column derivatization was used as the orthogonal analytical tool for quantification. Principal component analysis and partial least squares regression were employed for region selection and calibration model development, respectively. The results demonstrate that a good calibration statistic with the acceptable coefficient of determinations for both calibration (Rc2 = 0.94–0.99) and prediction (Rp2 = 0.83–0.98) could be achieved, along with high RPD values (>3) for all components except alanine (2.4). The external validation study also exhibited a satisfactory outcome (REV2 = 0.89–0.99, RMSE = 0.04–1.04), validating the model's ability to predict the concentrations of the respective species. The calibration models were successfully applied for at-line monitoring of two perfusion runs on a 10 L scale. To our knowledge, this is the first application where NIR spectroscopy-based measurement of all 20 amino acids in mammalian cell culture samples has been demonstrated. The proposed tool can play a critical role as biopharma manufacturers implement continuous processing as well as for facilitating process analytical technology-based control of mammalian cell culture processes.

Membrane Chromatography-Based Downstream Processing for Cell-Culture Produced Influenza Vaccines.

New influenza strains are constantly emerging, causing seasonal epidemics and raising concerns to the risk of a new global pandemic. Since vaccination is an effective method to prevent the spread of the disease and reduce its severity, the development of robust bioprocesses for producing pandemic influenza vaccines is exceptionally important. Herein, a membrane chromatography-based downstream processing platform with a demonstrated industrial application potential was established. Cell culture-derived influenza virus H1N1/A/PR/8/34 was harvested from benchtop bioreactor cultures. For the clarification of the cell culture broth, a depth filtration was selected as an alternative to centrifugation. After inactivation, an anion exchange chromatography membrane was used for viral capture and further processing. Additionally, two pandemic influenza virus strains, the H7N9 subtype of the A/Anhui/1/2013 and H3N2/A/Hong Kong/8/64, were successfully processed through similar downstream process steps establishing optimized process parameters. Overall, 41.3–62.5% viral recovery was achieved, with the removal of 86.3–96.5% host cell DNA and 95.5–99.7% of proteins. The proposed membrane chromatography purification is a scalable and generic method for the processing of different influenza strains and is a promising alternative to the current industrial purification of influenza vaccines based on ultracentrifugation methodologies.

Purification Process of a Recombinant Human Follicle Stimulating Hormone Biosimilar (Primapur®) to Yield a Pharmaceutical Product with High Batch-to-Batch Consistency.

Recombinant human follicle stimulating hormone (r-hFSH) is widely used for infertility treatment and is subject to the development of biosimilars. There are different purification strategies that can yield r-hFSH of pharmaceutical quality from Chinese hamster ovary cell culture broth. We developed a purification process for r-hFSH centered on immunoaffinity chromatography with single-domain recombinant camelid antibodies. The resulting downstream process is simple and devoid of ultrafiltration operations. Studies on chromatography resin resource and ligand leakage showed that the immunoaffinity matrix employed was suitable for industrial use and stable for at least 40 full chromatography cycles, and the leaked single-domain antibody ligand was completely removed by subsequent purification steps. All chromatography resins employed withstood the same 40 cycles of use without significant changes in separation efficiency and product binding capacity. The resulting industrial purification process yielded batches of r-hFSH with consistent levels of purity and bioactivity.

Optimized process operations reduce product retention and column clogging in ATF-based perfusion cell cultures.

Product retention in hollow fibers is a common issue in ATF-based cell culture system. In this study, the effects of four major process factors on product (therapeutic antibody/recombinant protein) retention were investigated using Chinese hamster ovary cell. Hollow fibers made of polysulfone presented a product retention rate from 15% ± 8 to 43% ± 18% higher than those made of polyether sulfone varying with specific processes. Higher harvest flowrate and ATF exchange rate increased product retention by 13% ± 10% and up to 31% ± 13%, respectively. Hollow fibers with larger pore sizes (0.65 μm) appeared to have increased product retention by 38% ± 7% compared with smaller ones (0.2 μm) in this study. Further investigation revealed that the effects of pore size on retention could be correlated to the particle size distribution in the cell culture broth. A hollow fiber with a larger pore size (>0.5 μm) may reduce protein retention when small particles (approximately 0.01-0.2 μm in diameter) are dominant in the culture. However, if majority of the particles are larger than 0.2 μm in diameter, hollow fiber with smaller pore sizes (0.2 μm) could be a solution to reducing product retention. Alternatively, process optimization may modulate particle size distribution towards reduced production retention with selected ATF hollow fibers. This study for the first time highlights the importance of matching proper pore sizes of hollow fibers with the cell culture particles distribution and offers methods to reducing product retention and ATF column clogging in perfusion cell cultures. KEY POINTS: The material of ATF column could impact product retention during perfusion culture. Higher harvest flowrate and ATF exchange rate increased product retention. Matching culture particle size and ATF pore size is critical for retention modulation.

Multifungicide resistance profiles and biocontrol in Lasiodiplodia theobromae from mango fields

Stem-end rot (SER) caused by Lasiodiplodia theobromae is a major postharvest disease of mango. To better understand the occurrence of multifungicides resistance in field populations, the sensitivity to five fungicides was investigated in 224 L. theobromae isolates collected from the mango growing region of Hainan Province in 2014–2018. The biocontrol activity of Bacillus amyloliquefaciens HBR-2 against resistant phenotypes of L. theobromae was evaluated using in vitro and in vivo tests. Our studies have established the baseline sensitivity of L. theobromae to difenoconazole, prochloraz and iprodione. Most isolates tested were sensitive to difenoconazole, prochloraz and iprodione. The overall resistance frequencies for difenoconazole, prochloraz and iprodione were 24.11, 15.18 and 1.34%, respectively. In addition, the overall resistance frequencies of L. theobromae for carbendazim and pyraclostrobin were 71.43% and 57.14%, respectively. In Hainan, 16 fungicide-resistant phenotypes were detected: 29.02, 60.27, 7.14, and 0.45% of the isolates were resistant to one, two, three, and four fungicides, respectively. The novel isolate B. amyloliquefaciens HBR-2 was found to have strong antagonistic activity against different resistant phenotypes of L. theobromae. The cell culture broth was the most effective treatment for controlling mango fruit rot in vivo. Multifungicide-resistant isolates of L. theobromae were widespread in Hainan. Biocontrol could be a valuable additional tool to manage the SER of mango fruit in the future. These findings would be useful in designing and implementing fungicide resistance management spray programs for the control of SER in mango.

Elucidation and Identification of an Antifungal Compound from Pseudomonas aeruginosa DA3.1 Isolated from Soil in Vietnam

Background: Fusarium sp. and Rhizoctonia sp. fungi have been always threats to short-term crops. In Vietnam, corn and soybean suffer serious losses annually. Therefore, it is necessary to utilize an environmentally friendly antifungal compound that is highly effective against phytopathogenic fungi. Pseudomonas sp. is a popular soil bacterial strain and well known for its high antifungal activity. Objectives: This study was carried out to evaluate and assess the antifungal activity of a local bacterial strain namely DA3.1 that was later identified as Pseudomonas aeruginosa. This would be strong scientific evidence to develop an environmentally friendly biocide from a local microorganism strain for commercial use. Methods: The antifungal compound was purified from ethyl acetate extraction of deproteinized cell culture broth by a silica gel column (CH2Cl2/MeOH (0% - 10% MeOH)). The purity of the isolated compound was determined by HPLC, and its molecular structure was elucidated using spectroscopic experiments including one-dimensional (1D) (1H NMR, 13C NMR, DEPT) and two-dimensional (2D) (HMBC and HSQC) spectra. The activity of the purified compound against Fusarium sp. and Rhizoctonia sp. fungi was measured using the PDA-disk diffusion method, and its growth-promoting ability was evaluated using the seed germination test of corn and soybean. Results: The results showed that the antifungal compound produced by Pseudomonas aeruginosa DA3.1 had a retention factor (Rf) of 0.86 on thin layer chromatography (TLC). Based on the evidence of spectral data including proton nuclear magnetic resonance (1H NMR), carbon nuclear magnetic resonance (13C NMR), distortionless enhancement by polarization transfer (DEPT), heteronuclear multiple bond correlation (HMBC), and heteronuclear single quantum coherence (HSQC), the chemical structure was elucidated as phenazine-1-carboxylic. The purified compound showed inhibitory activity against F. oxysporum and R. solani and exhibited the ability of the germination of corn and soybean seeds. The results revealed the benefit of native P. aeruginosa DA3.1 and phenazine-1-carboxylic acid for use as a biocontrol agent, as well as a plant growth promoter. Conclusions: The antifungal compound isolated from local Pseudomonas DA3.1 was identified as phenazine-1-carboxylic acid that posed high antifungal activity and was a plant germination booster.

Continuous capture of recombinant antibodies by ZnCl 2 precipitation without polyethylene glycol.

The capture of recombinant antibodies from cell culture broth is the first critical step of downstream processing. We were able to develop a precipitation‐based method for the capture and purification of monoclonal antibodies based on divalent cations, namely ZnCl2. Traditional precipitation processes have to deal with high dilution factors especially for resolubilization and higher viscosity due to the use of PEG as precipitation or co‐precipitation agent. By the use of the crosslinking nature of divalent cations without the use of PEG, we kept viscosity from the supernatant and resolubilization dilution factors very low. This is especially beneficial for the solid–liquid separation for the harvest and wash of the precipitate in continuous mode. For this harvest and wash, we used tangential flow filtration that benefits a lot from low viscosity solutions, which minimizes the membrane fouling. With this precipitation based on ZnCl2, we were able to implement a very lean and efficient process. We demonstrated precipitation studies with three different antibodies, Adalimumab, Trastuzumab, and Denosumab, and a continuous capture case study using tangential flow filtration for precipitate recovery. In this study, we achieved yields of 70%.

Multivariate Optimization of the Refolding Process of an Incorrectly Folded Fc-Fusion Protein in a Cell Culture Broth.

Protein misfolding is a common problem in large-scale production of recombinant proteins, which can significantly reduce the yield of the process. In this work, we aimed at treating a cell culture broth containing high levels (>45%) of incorrectly folded Fc-fusion proteins by a simple redox buffer system in order to increase the proportion of the protein with correct conformation. Multi-variable process optimization was firstly conducted at a small scale (25 mL), employing an experimental design methodology. After identifying the key variables using a resolution IV Fractional Factorial Design (FFD), the process was then optimized by the Central Composite Design (CCD). The optimal conditions for the refolding reaction were 340 mM Tris-base, 6.0 mM L-cysteine, 0.5 mM L-cystine, a buffer pH of 9.0, a reaction temperature of 8.5ºC and a reaction time of 24 h. Based on the treatment conditions obtained at a small scale, the process was further scaled up to 4500- L. The misfolded content was always less than 20%. The reaction can proceed well in the absence of chemical additives, such as chaotropic agents, aggregation suppressors, stabilizers and chelators. The refolding process increases the fraction of active protein in the original broth reducing the burden on downstream purification steps markedly.

Achieving Maximal Production of Fusaricidins from Paenibacillus kribbensis CU01 via Continuous Fermentation.

In this study, we investigated the potential of Paenibacillus kribbensis CU01 in producing fusaricidin, a strong antifungal substance, via optimization of metal ions and carbon and nitrogen source, and continuous fermentation. In the cultivation of a 2-l batch, maximal production of fusaricidins (581 mg l-1) was achieved in a modified M9 medium containing metal ions, 10 g l-1 glucose, and 1 g l-1 ammonium chloride. Most of glucose was consumed at a rate of 0.74 g l-1 h-1 within 24 h and fusaricidin production began 15 h after batch cultivation. Continuous fermentation was performed using a 7-l fermenter with 2-l working volume of modified M9 medium containing 10 g l-1 glucose, 1 × 10-3 M FeSO4, and 1 × 10-6 M MnCl2. After 24 h of the start of cultivation, fresh M9 medium was continuously supplied at a flow rate of 2.5 ml min-1, and simultaneously, the same amount of cell culture broth was removed. In a continuous system, the highest fusaricidin concentration (579 mg l-1) was obtained using a dilution rate of 0.075 h-1 with an average productivity of 10.4 mg l-1 h-1 for 24 to 72 h of incubation. Based on these results, it was found that fusaricidin production using P. kribbens CU01 strain increased by at least 28 times the values reported in previous studies.

High-Throughput Process Development for Recombinant Human Serum Albumin Separation from Pichia pastoris Broth with Mixed-Mode Chromatography

Due to the shortage of blood donation and safe issues of blood-derived pathogens, recombinant human serum albumin (rHSA) expressed by Pichia pastoris is a potential substitute for plasma-derived HSA (pHSA). In this work, four commercialized mixed-mode resins (Capto adhere, Capto MMC, MX-Trp-650M, and Nuvia cPrime) and two ion-exchange resins (Q Sepharose FF and SP Sepharose FF) were used for rHSA separation from Pichia pastoris cell culture broth, and loading and elution conditions were optimized via a high-throughput process using microtiter filter plates. The results indicated that resins with cation-exchange groups had better adsorption performance. The loading conditions at pH 4.0 and dilution rate of 1.0 were suitable for SP Sepharose FF, and no dilution worked for mixed-mode resins. The optimal elution pH was determined to be pH 7.0 for SP Sepharose FF, pH 8.0 for MX-Trp-650M and Nuvia cPrime, and pH 9.0 for Capto MMC. Moreover, rHSA separation from Pichia pastoris broth was performed on column unde...

Connected flow-through chromatography processes as continuous downstream processing of proteins

Continuous manufacturing is expected to increase the productivity of protein drug production. However, it is not easy to build the continuous process especially for downstream processing as many unit operations (chromatography and membrane filtration) are involved. An operation method known as flow-through chromatography (FTC) is considered to be an efficient method for separating two components as the flow is continuous. In FTC, a target protein is eluted from the chromatography column without adsorption whereas contaminants are strongly bound. Since at least two different modes of chromatography are needed in order to remove contaminants, two FTC columns have to be connected in order to build the continuous process. This is not an easy task since the mobile phase properties (pH, salt, buffer ions) are different for the two columns. In this paper, we investigated how connected FTC columns can remove impurities efficiently from the cell culture broth containing monoclonal antibody. It was found that the sequence (activated carbon - anion exchange chromatography - cation exchange chromatography) is most efficient when the mobile phase pH and conductivity were properly chosen.

Cloning, Expression and Purification of Recombinant Forms of Full Length and Extracellular Domain EBOV Glycoprotein within Mammalian Cell-Lines

Background: Full length Ebolavirus glycoprotein (GP) intersperses the outer most lipid membrane to form spikes, where it mediates virus-host cell interaction. A secretory form of GP (sGP) is also produced by all 5 known Ebolavirus species. These attributes make GP an ideal target for research and development (R and D) of Ebolavirus and possibly pan-filovirus targeted Rapid Diagnostic Tests (RDTs), bio-therapeutics and vaccines. Prior cloning of recombinant Zaire ebolavirus (EBOV) GP has majorly used insect (baculovirus ) expression systems. We report the cloning, expression and purification of the full length and extracellular domain (ECD) forms of recombinant EBOV GP in mammalian cell-lines. Methods and results: 2034 and 1956 base-pair (bp) coding DNA sequences corresponding to the 669 and 643 amino acids (aa) residues of full length and ECD forms of EBOV GP were sub-cloned into the pTGE plasmids. Recombinant pTGE-plasmids were used to transfect 293-6E HEK mammalian cells grown in serum-free FreeStyleTM 293 Expression Medium. Cell lysates and or culture supernatants were used to obtain purified protein, followed by analysis on SDS-PAGE and Western blot. Purified full length GP was detected as membrane bound protein in cell lysates with estimated molecular weight of ~100 kDa (Cal.M.W.~71.67 kDa) on Western blot; and 0.02 mg GP (Concentration: 0.2 mg/mL, Purity: ~50%) derived. On the contrary, ECD GP was detected in supernatants of cell culture broth with estimated molecular weights of ~116 kDa based on SDS-PAGE and Western blot; and 1.6 mg (Concentration: 0.4 mg/ml, Purity: ~70%) of GP_ECD was obtained. Conclusion: Within mammalian cells, recombinant full length EBOV GP is predominantly expressed as transmembrane protein (tGP), while ECD GP is eluted into the culture medium. Both recombinant forms of GP are critical for the R and D of rapid diagnostic tests (RDTs).

Process development in the Quality by Design paradigm: Modeling of Protein A chromatography resin fouling

Protein A chromatography is quite commonly used for capture of monoclonal antibodies from the clarified cell culture broths. Protein A resins are expensive and economic feasibility demands that the resin be reused for 50–300 cycles. Resin reuse is, however, accompanied by resin fouling, impacting both the binding and mass transfer characteristics of the resin. In the present study, we attempt to model the variations in binding and mass transfer characteristics of a commercially available Protein A resin, mAbSelect SuRe™, as a function of resin’s reuse. Simplified linear driving force modeling and kinetic modeling of Protein A chromatography step elution cycling data has been successfully used to predict resin performance up to 100 cycles based on fouling data up to 50 cycles. Fouling factor for Protein A resin has been empirically modeled as a function of binding and mass transfer characteristics of resin and the resin’s reuse using a combination of Buckingham’s π theorem and statistical analysis. The proposed empirical model enables reliable prediction of performance of Protein A resin as well as offers an improved understanding of the underlying mechanism behind the decline in resin performance during fouling.

Evaluation of recent Protein A stationary phase innovations for capture of biotherapeutics

We describe a comprehensive evaluation of 12 Protein A stationary phases for capture of biotherapeutics. We first examine the morphological properties of the stationary phases using a variety of orthogonal techniques including electron microscopy, particle sizing, pressure-flow behavior, and isocratic pulse response. A panel of nine proteins spanning a wide range of structures and biochemical properties was then used to assess equilibrium uptake, mass transport, dynamic binding capacity, and elution pH. Process performance and product quality were also examined under realistic bioprocess conditions using clarified mammalian cell culture broth. Equilibrium isotherms were found to be highly favorable, with equilibrium binding capacity for monoclonal and bispecific antibodies ranging from 47–100 mg/mL packed bed across all stationary phases tested. Effective pore diffusivities, De, were obtained by fitting the chromatography general rate model to breakthrough data. The fitted De values for monoclonal antibodies ranged from 1.1–5.7 × 10−8 cm2/s. The stationary phases had high dynamic binding capacities for the model proteins. The highest dynamic capacities for monoclonal and bispecific antibodies were seen with MabSelect SuRe pcc and MabSelect PrismA, which ranged from 58–74 mg/mL packed bed at 4 min residence times. Product capture using clarified cell culture broth as a feedstock showed high yields and elution pool volumes that ranged from 2–3 column volumes in most cases. Host cell protein, DNA, and aggregate levels in the elution pool were dependent on the specific nature of protein being purified, and levels were consistent between stationary phases. Lastly, we perform an analysis of bivariate correlations and discuss considerations for process design and optimization.

Protein A affinity chromatography of Chinese hamster ovary (CHO) cell culture broths containing biopharmaceutical monoclonal antibody (mAb): Experiments and mechanistic transport, binding and equilibrium modeling

Protein A-based affinity chromatography is a highly-efficient separation method to capture, purify and isolate biosimilar monoclonal antibodies (mAb) – an important medical product of biopharmaceutical industrial manufacturing. It is considered the most expensive step in purification downstream operations; therefore, its performance optimization offers a great cost saving in the overall production expenditure. The biochemical mixture-separating specific interaction experiments with Chinese hamster ovary (CHO) cell culture harvest, containing glycosylated extracellular immunoglobulins (Ig), were made using five different state-of-the-art commercial resins. Packing breakthrough curves were recorded at an array of prolonged residence times. A mathematical simulation model was developed, applied and validated in combination with non-linear regression algorithms on bed effluent concentrations to determine the previously-unknown binding properties of stationary phase materials. Apart from the columns' differential partitioning, the whole external system was also integrated. It was confirmed that internal pore diffusion is the global rate-limiting resistance of the compound retention process. Immobilizing substrate characteristics, obtained in this engineering study, are indispensable for the scale-up of the periodic counter-current control with mechanistic load, elution and wash reduction. Furthermore, unit's volumetric flow screening measurements revealed dynamic effect correlation to eluate quality parameters, like the presence of aggregates, the host cell-related impurities at supernatant's extended feeding, and titre. Numerical sensitivity outputs demonstrated the impacts of fluidics (e.g. axial dispersion coefficient), thermodynamics (Langmuir adsorption) and mass transfer fluxes.