Case study and application of process analytical technology (PAT) towards bioprocessing: II. Use of ultra-performance liquid chromatography (UPLC) for making real-time pooling decisions for process chromatography

Process analytical technology (PAT) has been gaining a lot of momentum in the biopharmaceutical community due to the potential for continuous real-time quality assurance resulting in improved operational control and compliance. Two of the key goals that have been outlined for PAT are "variability is managed by the process" and "product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions". Recently, we have been examining the feasibility of applying different analytical tools for designing PAT applications for bioprocessing. We have previously shown that a commercially available online high performance liquid chromatography (HPLC) system can be used for analysis that can facilitate real-time decisions for column pooling based on product quality attributes (Rathore et al., 2008). In this article we test the feasibility of using a commercially available ultra- performance liquid chromatography (UPLC) system for real-time pooling of process chromatography columns. It is demonstrated that the UPLC system offers a feasible approach and meets the requirements of a PAT application. While the application presented here is of a reversed phase assay, the approach and the hardware can be easily applied to other modes of liquid chromatography.     

Large scale demonstration of a process analytical technology application in bioprocessing: Use of on‐line high performance liquid chromatography for making real time pooling decisions for process chromatography

Process Analytical Technology (PAT) has been gaining a lot of momentum in the biopharmaceutical community because of the potential for continuous real time quality assurance resulting in improved operational control and compliance. In previous publications, we have demonstrated feasibility of applications involving use of high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC) for real‐time pooling of process chromatography column. In this article we follow a similar approach to perform lab studies and create a model for a chromatography step of a different modality (hydrophobic interaction chromatography). It is seen that the predictions of the model compare well to actual experimental data, demonstrating the usefulness of the approach across the different modes of chromatography. Also, use of online HPLC when the step is scaled up to pilot scale (a 2294 fold scale‐up from a 3.4 mL column in the lab to a 7.8 L column in the pilot plant) and eventually to manufacturing scale (a 45930 fold scale‐up from a 3.4 mL column in the lab to a 158 L column in the manufacturing plant) is examined. Overall, the results confirm that for the application under consideration, online‐HPLC offers a feasible approach for analysis that can facilitate real‐time decisions for column pooling based on product quality attributes. The observations demonstrate that the proposed analytical scheme allows us to meet two of the key goals that have been outlined for PAT, i.e., “variability is managed by the process” and “product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions”. The application presented here can be extended to other modes of process chromatography and/or HPLC analysis. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Mechanistic modeling based process analytical technology implementation for pooling in hydrophobic interaction chromatography

A major challenge in chromatography purification of therapeutic proteins is batch-to-batch variability with respect to impurity levels and product concentration in the feed. Mechanistic model can enable process analytical technology (PAT) implementation by predicting impact of such variations and thereby improving the robustness of the resulting process and controls. This article presents one such application of mechanistic model of hydrophobic interaction chromatography (HIC) as a PAT tool for making robust pooling decisions to enable clearance of aggregates for a monoclonal antibody (mAb) therapeutic. Model predictions were performed before the actual chromatography experiments to facilitate feedforward control. The approach has been successfully demonstrated for four different feeds with varying aggregate levels (3.84%–5.54%) and feed concentration (0.6 mg/mL–1 mg/mL). The resulting pool consistently yielded a product with 1.32 ± 0.03% aggregate vs. a target of 1.5%. A comparison of the traditional approach involving column fractionation with the proposed approach indicates that the proposed approach results in achievement of satisfactory product purity (98.68 ± 0.03% for mechanistic model based PAT controlled pooling vs. 98.64 ± 0.16% for offline column fractionation based pooling). © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2758, 2019.     

Model-based monitoring of industrial reversed phase chromatography to predict insulin variants

Downstream processing in the manufacturing biopharmaceutical industry is a multistep process separating the desired product from process- and product-related impurities. However, removing product-related impurities, such as product variants, without compromising the product yield or prolonging the process time due to extensive quality control analytics, remains a major challenge. Here, we show how mechanistic model-based monitoring, based on analytical quality control data, can predict product variants by modeling their chromatographic separation during product polishing with reversed phase chromatography. The system was described by a kinetic dispersive model with a modified Langmuir isotherm. Solely quality control analytical data on product and product variant concentrations were used to calibrate the model. This model-based monitoring approach was developed for an insulin purification process. Industrial materials were used in the separation of insulin and two insulin variants, one eluting at the product peak front and one eluting at the product peak tail. The model, fitted to analytical data, used one component to simulate each protein, or two components when a peak displayed a shoulder. This monitoring approach allowed the prediction of the elution patterns of insulin and both insulin variants. The results indicate the potential of using model-based monitoring in downstream polishing at industrial scale to take pooling decisions.     

Case study and application of process analytical technology (PAT) towards bioprocessing: Use of tryptophan fluorescence as at‐line tool for making pooling decisions for process chromatography

Process analytical technology (PAT) has been gaining momentum in the biopharmaceutical community due to the potential for continuous real time quality assurance resulting in improved operational control and compliance. Two imperatives for implementing any PAT tool are that “variability is managed by the process” and “product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions.” Recently, we have been examining the feasibility of applying different analytical tools to bioprocessing unit operations. We have previously demonstarted that commercially available online‐high performance liquid chromatography and ultra performance liquid chromatography systems can be used for analysis that can facilitate real‐time decisions for column pooling based on product quality attributes (Rathore et al., 2008 a,b). In this article, we review an at‐line tool that can be used for pooling of process chromatography columns. We have demonstrated that our tryptophan fluorescence method offers a feasible approach and meets the requirements of a PAT application. It is significantly faster than the alternative of fractionation, offline analysis followed by pooling. Although the method as presented here is not an online method, this technique may offer better resolution for certain applications and may be a more optimal approach as it is very conducive to implementation in a manufacturing environment. This technique is also amenable to be used as an online tool via front face fluorescence measurements done concurrently with product concentration determination by UV. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Purification of lignin peroxidase isoforms from <Emphasis Type="Italic">Streptomyces viridosporus</Emphasis> T7A by hydrophobic based chromatographies

Five lignin peroxidases ALiP-P3 isoenzymes from Streptomyces viridosporus T7A were separated by hydrophobic interaction chromatography. The most abundant isoenzyme was purified by reversed phase chromatography and it was analyzed by mass spectrometry.     

An integrated scale-down plant for optimal recombinant enzyme production by Pichia pastoris

An integrated bioprocess was created in a scale-down production plant by developing a two-stage enzyme production process with Pichia pastoris, containing a cell-breeding reactor and a production reactor in combination with a three-stage downstream process. To harvest the secreted enzymes, a disc separator and a cross-flow microfiltration clear the broth from the cells. Purification with hydrophobic interaction chromatography removes other proteins, concentrates the product, and prepares the enzyme solution for lyophilization. Fully automated and broad observable multi-stage parallel process courses have been developed using industrial process control systems and at-line measurements for enzyme concentration and enzyme activity. Optimal process conditions were found by application of Design of Experiments (DoE) for the production process.     

High‐throughput process development of purification alternatives for the protein avidin

With an increased number of applications in the field of the avidin‐biotin technology, the resulting demand for highly‐purified protein avidin has drawn our attention to the purification process of avidin that naturally occurs in chicken egg white. The high‐throughput process development (HTPD) methodology was exploited, in order to evaluate purification process alternatives to commonly used ion‐exchange chromatography. In a high‐throughput format, process parameters for aqueous two‐phase extraction, selective precipitation with salts and polyethylene glycol, and hydrophobic interaction and mixed‐mode column chromatography experiments were performed. The HTPD strategy was complemented by a high‐throughput tandem high‐performance liquid chromatography assay for protein quantification. Suitable conditions for the separation of avidin from the major impurities ovalbumin, ovomucoid, ovotransferrin, and lysozyme were identified in the screening experiments. By combination of polyethylene glycol precipitation with subsequent resolubilization and separation in a polyethylene glycol/sulfate/sodium chloride two‐phase system an avidin purity of 77% was obtained with a yield >90% while at the same time achieving a significant reduction of the process volume. The two‐phase extraction and precipitation results were largely confirmed in larger scale with scale‐up factors of 230 and 133, respectively. Seamless processing of the avidin enriched bottom phase was found feasible by using mixed‐mode chromatography. By gradient elution a final avidin purity of at least 97% and yield >90% was obtained in the elution pool. The presented identification of a new and beneficial alternative for the purification of the high value protein thus represents a successful implementation of HTPD for an industrially relevant purification task. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:957–973, 2015     

Detection of two high molecular weight hydrophobic forms of the human estrogen receptor

The human estrogen receptor gene encodes a single protein of molecular weight 65,000 daltons. However, using a sensitive and rapid technique of high-performance hydrophobic interaction chromatography we have detected two distinct estrogen receptor species both of which are high molecular weight proteins (ca. 60A) as determined by high-performance size-exclusion chromatography. These are detected either in the presence or absence of sodium molybdate; rechromatography of individual isoform indicates that the two protein complexes have independent hydrophobic contact points. Consistent elution patterns of the two receptor species indicates they are formed selectively. We conclude that different post-translational modifications of the estrogen receptor protein could allow their specific interaction with non-receptor components resulting in the formation of two distinct high molecular weight complexes which would be rapidly resolved by high-performance hydrophobic interaction chromatography.     

Digital twin of a continuous chromatography process for mAb purification: Design and model-based control

Model-based design of integrated continuous train coupled with online process analytical technology (PAT) tool can be a potent facilitator for monitoring and control of Critical Quality Attributes (CQAs) in real time. Charge variants are product related variants and are often regarded as CQAs as they may impact potency and efficacy of drug. Robust pooling decision is required for achieving uniform charge variant composition for mAbs as baseline separation between closely related variants is rarely achieved in process scale chromatography. In this study, we propose a digital twin of a continuous chromatography process, integrated with an online HPLC-PAT tool for delivering real time pooling decisions to achieve uniform charge variant composition. The integrated downstream process comprised continuous multicolumn capture protein A chromatography, viral inactivation in coiled flow inverter reactor (CFIR), and multicolumn CEX polishing step. An online HPLC was connected to the harvest tank before protein A chromatography. Both empirical and mechanistic modeling have been considered. The model states were updated in real time using online HPLC charge variant data for prediction of the initial and final cut point for CEX eluate, according to which the process chromatography was directed to switch from collection to waste to achieve the desired charge variant composition in the CEX pool. Two case studies were carried out to demonstrate this control strategy. In the first case study, the continuous train was run for initially 14 h for harvest of fixed charge variant composition as feed. In the second case study, charge variant composition was dynamically changed by introducing forced perturbation to mimic the deviations that may be encountered during perfusion cell culture. The control strategy was successfully implemented for more than ±5% variability in the acidic variants of the feed with its composition in the range of acidic (13%–17%), main (18%–23%), and basic (59%–68%) variants. Both the case studies yielded CEX pool of uniform distribution of acidic, main and basic profiles in the range of 15 ± 0.8, 31 ± 0.3, and 53 ± 0.5%, respectively, in the case of empirical modeling and 15 ± 0.5, 31 ± 0.3, and 53 ± 0.3%, respectively, in the case of mechanistic modeling. In both cases, process yield for main species was >85% and the use of online HPLC early in the purification train helped in making quicker decision for pooling of CEX eluate. The results thus successfully demonstrate the technical feasibility of creating digital twins of bioprocess operations and their utility for process control.     

Control of antibody high and low molecular weight species by depth filtration-based cell culture harvesting

Depth filtration-based harvesting is widely used in mAb manufacturing to remove cell and process-related impurities. However, it has not been studied on control of product-related impurities, which are very critical for product quality. In this article, we studied the interactions of depth filter with high and low molecular weight species (HMWs and LMWs) for their direct removal from cell culture. The process parameters (filter, loading, temperature, and flux) were evaluated for adsorption of HMWs and LMWs by depth filters. The adsorption is significantly dependent on filter media and loading capacity and is mainly on the basis of hydrophobic interaction during harvesting. The HMW and LMW species were characterized as HMW1, HMW2, LMW1, and LMW2. The increasing binding from LMW2 to LMW1, HMW1, and HMW2 is correlated with their increasing hydrophobicity score. Adsorption using enriched HMW sample demonstrated similar total protein binding capacity (36–40 g/m²) between depth filters D0HC and X0HC. However, X0HC has stronger HMW binding than D0HC (71% vs 43% of bound protein), indicating more hydrophobic interaction in X0HC. HMW2 DBC on X0HC reached 12 g/m², similar to protein binding on hydrophobic interaction membrane adsorbers. Further study showed LMW can induce HMW formation. This study provides a critical understanding of HMW and LMW interaction with depth filters. The strategy of HMW and LMW control by depth filtration-based harvesting was implemented successfully in mAb manufacturing.     

Practical approaches for overcoming challenges in heightened characterization of antibody-drug conjugates with new methodologies and ultrahigh-resolution mass spectrometry

Antibody-drug conjugation strategies are continuously evolving as researchers work to improve the safety and efficacy of the molecules. However, as a part of process and product development, confirmation of the resulting innovative structures requires new, specialized mass spectrometry (MS) approaches and methods, as compared to those already established for antibody-drug conjugates (ADCs) and the heightened characterization practices used for monoclonal antibodies (mAbs), in order to accurately elucidate the resulting conjugate forms, which can sometimes have labile chemical bonds and more extreme chemical properties like hydrophobic patches. Here, we discuss practical approaches for characterization of ADCs using new methodologies and ultrahigh-resolution MS, and provide specific examples of these approaches. Denaturing conditions of typical liquid chromatography (LC)/MS analyses impede the successful detection of intact, 4-chain ADCs generated via cysteine site-directed chemistry approaches where hinge region disulfide bonds are partially reduced. However, this class of ADCs is detected intact reliably under non-denaturing size-exclusion chromatography/MS conditions, also referred to as native MS. For ADCs with acid labile linkers such as one used for conjugation of calicheamicin, careful selection of mobile phase composition is critical to the retention of intact linker-payload during LC/MS analysis. Increasing the pH of the mobile phase prevented cleavage of a labile bond in the linker moiety, and resulted in retention of the intact linker-payload. In-source fragmentation also was observed with typical electrospray ionization (ESI) source parameters during intact ADC mass analysis for a particular surface-accessible linker-payload moiety conjugated to the heavy chain C-terminal tag, LLQGA (via transglutaminase chemistry). Optimization of additional ESI source parameters such as cone voltages, gas pressures and ion transfer parameters led to minimal fragmentation and optimal sensitivity. Ultrahigh-resolution (UHR) MS, combined with reversed phase-ultrahigh performance (RP-UHP)LC and use of the FabRICATOR® enzyme, provides a highly resolving, antibody subunit-domain mapping method that allows rapid confirmation of integrity and the extent of conjugation. For some ADCs, the hydrophobic nature of the linker-payload hinders chromatographic separation of the modified subunit/domains or causes very late elution/poor recovery. As an alternative to the traditionally used C₄ UHPLC column chemistry, a diphenyl column resulted in the complete recovery of modified subunit/domains. For ADCs based on maleimide chemistry, control of pH during proteolytic digestion is critical to minimize ring-opening. The optimum pH to balance digestion efficiency and one that does not cause ring opening needed to be established for successful peptide mapping.     

Purification of carbonic anhydrase isoenzymes by high-performance affinity chromatography and hydrophobic interaction chromatography

Isoenzymes of carbonic anhydrase were purified by a combination of affinity chromatography and hydrophobic interaction chromatography. Immobilization of sulfonamides on an epoxy-activated support provided a stationary phase for affinity chromatography which was stable to hydrolysis by carbonic anhydrase. A first purification step allowed the isolation of enzymes directly from homogenates of human erythrocytes and rat stomach. Without any further preparation, except the addition of ammonium sulfate to the eluate from affinity chromatography, the isoenzymes could be separated by hydrophobic interaction chromatography with very high recovery of protein and retention of enzymatic activity.     

Case study and application of process analytical technology (PAT) towards bioprocessing: use of on-line high-performance liquid chromatography (HPLC) for making real-time pooling decisions for process chromatography

Process analytical technology (PAT) has been gaining a lot of momentum in the biopharmaceutical community due to the potential for continuous real time quality assurance resulting in improved operational control and compliance. This paper presents a PAT application for one of the most commonly used unit operation in bioprocessing, namely liquid chromatography. Feasibility of using a commercially available online-high performance liquid chromatography (HPLC) system for real-time pooling of process chromatography column is examined. Further, experimental data from the feasibility studies are modeled and predictions of the model are compared to actual experimental data. It is found that indeed for the application under consideration, the online-HPLC offers a feasible approach for analysis that can facilitate real-time decisions for column pooling based on product quality attributes. It is shown that implementing this analytical scheme allows us to meet two of the key goals that have been outlined for PAT, that is, "variability is managed by the process" and "product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions." Finally, the implications of implementing such a PAT application in a manufacturing environment are discussed. The application presented here can be extended to other modes of process chromatography and/or HPLC analysis.     

Methods for measuring hydrophobicity of microorganisms

Hydrophobicity of three bacteria and four fungi has been determined by contact angle measurements, hydrophobic interaction chromatography, phase distribution, adhesion test and salt aggregation test. Good agreement among the five methodes is found frot the very hydrophobic (Moniliella pollinis) and the very hydrophilic (Saccharomyces cerevisiae and Acetobacter aceti) microorganisms. Evaluation of the hydrophobicity of the others (Enterobacter aerogenes, Klebsiella oxytoca, Saccharomyces carlsbergensis, Kluyveromyces fragilis) varies according to the method used. Comparison of the results and discussion of the meaning of the parameters involved indicate that the water contact angle is a significant measure of cell hydrophobicity. Due to certain limitations combining this method with hydrophobic interaction chromatography and with the test of adhesion to polystyrene is recommended.     

测序级胰蛋白酶的制备工艺与质量检测简

目的：制备高纯度、酶解效率高、酶切位点专一的测序级胰蛋白酶,应用于蛋白组学研究的蛋白质鉴定与分析中。方法：取实验室自制的猪胰蛋白酶粗酶,经硼氢化钠、甲醛还原甲基化修饰抑制胰蛋白酶自水解,采用高效液相色谱仪15RPC反相柱纯化,收集对应的甲基化胰蛋白酶峰组分,冷冻干燥;甲基化修饰的胰蛋白酶进一步经甲苯磺酰苯丙氨酰氯甲酮（TPCK）修饰,以抑制糜蛋白酶等非特异性酶切活性,并经反相色谱柱再纯化,获得终产物即质谱测序级胰蛋白酶;自制的测序级胰蛋白酶经SDS-PAGE、HPLC反相色谱分析、酶比活力测定,并应用于胶内蛋白质酶切质谱鉴定氨基酸序列等,检测其纯度、酶水解效率及酶切位点特异性。结果：自制甲基化TPCK修饰的测序级胰蛋白酶纯度大于95％,酶比活力为200U/mgP（TAME）以上,质谱分析酶切特异性好;且酶的制备工艺流程稳定,可应用于测序级胰蛋白酶产品的生产与开发中。结论：制备的测序级胰蛋白酶纯度高、酶解效率优、酶切特异性强,可广泛应用于实验室中蛋白质和肽段测序鉴定、HPLC肽段谱图分析等蛋白组学研究分析中。     

Multi‐dimensional fractionation and characterization of crude protein mixtures: Toward establishment of a database of protein purification process development parameters

A multi‐dimensional fractionation and characterization scheme was developed for fast acquisition of the relevant molecular properties for protein separation from crude biological feedstocks by ion‐exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and size‐exclusion chromatography. In this approach, the linear IEX isotherm parameters were estimated from multiple linear salt‐gradient IEX data, while the nonlinear IEX parameters as well as the HIC isotherm parameters were obtained by the inverse method under column overloading conditions. Collected chromatographic fractions were analyzed by gel electrophoresis for estimation of molecular mass, followed by mass spectrometry for protein identification. The usefulness of the generated molecular properties data for rational decision‐making during downstream process development was equally demonstrated. Monoclonal antibody purification from crude hybridoma cell culture supernatant was used as case study. The obtained chromatographic parameters only apply to the employed stationary phases and operating conditions, hence prior high throughput screening of different chromatographic resins and mobile phase conditions is still a prerequisite. Nevertheless, it provides a quick, knowledge‐based approach for rationally synthesizing purification cascades prior to more detailed process optimization and evaluation. Biotechnol. Bioeng. 2012; 109: 3070–3083. © 2012 Wiley Periodicals, Inc.     

Rapid purification of EGFP, EYFP, and ECFP with high yield and purity

Most current high throughput purification procedures for the green fluorescent protein (GFP) suffer from poor yields and low purity. An improved purification procedure that delivers highly pure protein (>95% homogeneity) in high yields (>70% of the initial fluorescent protein content) has been developed. The purification procedure requires only two steps: the cell lysate is heated to 60 degrees C for 4 min in ammonium sulfate and triethylamine, followed by hydrophobic interaction chromatography using isopropanol during the elution phase. The resulting pure product exhibits the same fluorescence profile as the crude sample. This procedure has been demonstrated on three commercial variants of GFP from Aequorea victoria, enhanced green, enhanced yellow, and enhanced cyan fluorescent protein (Becton-Dickinson). The yield and purity of material are superior to other recently described methods.     

Purification of a highly modified RNA-aptamer: Effect of complete denaturation during chromatography on product recovery and specific activity

To evaluate RNA-aptamers as potential drug candidates, efficient and scaleable purification protocols are needed. Because aptamers are highly structured and rigid molecules, denaturation during the purification process is a critical aspect to obtain a pure and active product. A two-step chromatographic procedure was developed to purify a synthetic anti-VEGF aptamer at the preparative scale. A reversed-phase chromatographic step was optimized with a highly hydrophobic ion pairing reagent, followed by ion-exchange chromatography in which heat and a chaotropic salt were used. Because of the presence of 2′-modified ribose, denaturation conditions had to be optimized in both chromatographic steps to achieve a fully active molecule.     

Application of Ultra-Centrifugation and Bench-Top <Superscript>19</Superscript>F NMR for Measuring Drug Phase Partitioning for the Ophthalmic Oil-in-Water Emulsion Products

Generic drug products are expected to have the same active pharmaceutical ingredient (API) (Q1) with the same content (Q2) and microstructure arrangement (Q3) as the innovator product. In complex oil-in-water emulsion drugs, the hydrophobic API is mainly formulated in oil droplets stabilized by surfactant and micelles composed of extra surfactant molecules. The API phase partition in oil and water (mainly micelle) is a critical quality attribute (CQA) of emulsion product in demonstrating physicochemical equivalence using difluprednate (DFPN) emulsion product Durezol® as a model, we developed a novel low-field benchtop NMR method to demonstrate its applicability in measuring DFPN phase partition for ophthalmic oil-in-water emulsion products. Low-field ¹⁹F spectra were collected for DFPN in formulation, in water phase and oil phase after separation from ultra-centrifugation. The NMR data showed the mass balance of DFPN before and after phase separation. The average water phase content of different Durezol® lots was 32 ± 3% with 1% variation from method reproducibility test. The partition results were 52 ± 2% for the in-house control products prepared in Q1/Q2 equivalence to Durezol® but by a different process. The significant difference in DFPN-phase partition between Durezol® and the in-house formulation demonstrated manufacture difference readily changed the API partition. The newly developed ultra-centrifugation and ¹⁹F NMR by benchtop instrument is a simple, robust, and sensitive analytical method for ophthalmic emulsion drug product development and control.