Virus clearance by activated carbon for therapeutic monoclonal antibody purification.

In our previous study, activated carbon (AC) was employed in the purification process of therapeutic monoclonal antibody (mAb) as a replacement for Protein A affinity chromatography. In addition, we established an innovative column-free flow-through purification process using AC filter. In these investigations, the effective clearance of impurities (high-molecular-weight species, low-molecular-weight species, host cell proteins, and DNA) was observed compared to the conventional Protein A platform purification process. In this study, virus removal capability of our established AC process (AC filter) was investigated using two model viruses, Murine Leukemia Virus (MuLV) and Minute Virus of Mice (MVM) with the combination of two filtration methods (single-pass filtration and re-circulation filtration) using three kinds of mAbs. We found effective clearance of both MuLV and MVM (>3 log reduction factor, LRF) in all mAbs. Not only filtration method but also re-circulation duration didn't affect LRF, and >3 LRF of virus removal could be achieved by only single-pass filtration. From these results, it is expected that AC will be a promising candidate for the virus removal unit operation for mAb purification processes.

A rapid 2AB-UHPLC method based on magnetic beads extraction for N-glycan analysis of recombinant monoclonal antibody.

N-glycosylation is one of the major post-translational modifications, with significant effects on the mechanism of action, the efficacy, and the safety of antibody drugs or glycoproteins. With the growing application of therapeutic antibodies, routinely monitoring N-glycosylation becomes increasingly important during cell culture process development and quality control. However, the current pretreatment methods for N-glycan analysis are time- and labor-consuming. The purification procedure of enzymatically released glycans could also partly affect the accuracy of results due to its complexity. In this study, a rapid ultra-high performance liquid chromatography method based on magnetic bead extraction and 2-AB fluorescent labeling was developed and compared against three popular pretreatment methods for N-glycan profiling (two were solid phase extraction and the other was acetone precipitation). The method's repeatability results showed that magnetic bead extraction has higher precision (% relative standard deviation (RSD), 0.121.06%) than solid phase extraction (SPE) (%RSD, 0.38-8.02%) and acetone precipitation (%RSD, 0.42-8.58%). This robust pretreatment method also maximized the retention of some low abundance oligosaccharides, and may thus provide a rapid and high-throughput workflow option for N-Glycan analysis in the biopharmaceutical industry.

Sodium caprylate wash during Protein A chromatography as an effective means for removing protease(s) responsible for target antibody fragmentation.

For recombinant proteins produced in Chinese hamster ovary (CHO) cells, fragmentation is a common phenomenon that results in generation of product-related low-molecular-weight (LMW) species. Recently while purifying a bispecific antibody (bsAb), we observed that the target protein experienced cleavage at a couple of potential sites, leading to truncated products. Further studies suggest that the cleavage can likely be attributed to residual CHO cell protease activity. In order to maximally remove potential protease(s) that contribute fragmentation, we optimized Protein A chromatography by adding sodium caprylate (SC) to the wash buffer. Upon optimization, fragmentation of Protein A eluate happened to a much lesser degree as compared to that of eluate from unoptimized process, and the increased sample stability is in accordance with significantly reduced host cell protein (HCP) level. Taken together, the data suggest that SC wash during Protein A chromatography is an effective means for removing HCPs including endogenous protease(s) that are responsible for target antibody fragmentation.

Comparison of Bivalirudin Versus Heparin for Maintenance Systemic Anticoagulation During Adult and Pediatric Extracorporeal Membrane Oxygenation.

OBJECTIVES: To provide a comparative analysis of conventional heparin-versus bivalirudin-based systemic anticoagulation in adult and pediatric patients supported on extracorporeal membrane oxygenation. DESIGN: Retrospective chart review study of adult and pediatric patients receiving extracorporeal membrane oxygenation from January 1, 2014, to October 1, 2019. SETTING: A large, high-volume tertiary referral adult and pediatric extracorporeal membrane oxygenation center. PATIENTS: Four hundred twenty-four individuals requiring extracorporeal membrane oxygenation support and systemically anticoagulated with either unfractionated heparin (223 adult and 65 pediatric patients) or bivalirudin (110 adult and 24 pediatric patients) were included. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Digital data abstraction was used to retrospectively collect patient details. The majority of both groups were cannulated centrally (67%), and the extracorporeal membrane oxygenation type was predominantly venoarterial (84%). The adult bivalirudin group had a greater occurrence of heparin-induced thrombocytopenia (12% vs 1%; p < 0.01) and was more likely to require postcardiotomy extracorporeal membrane oxygenation (36% vs 55%; p < 0.01). There were no statistical differences between the groups in regards to age, sex, and extracorporeal membrane oxygenation initiation location. The main finding was a reduced mortality in the adult bivalirudin group (odds ratio, 0.39; p < 0.01), whereas no difference was noted in the pediatric group. A significant reduction in the composite transfusion requirement in the first 24 hours was noted in the pediatric bivaluridin group with an odds ratio of 0.28 (p = 0.02). Groups did not differ in regard to laboratories per day, anticoagulant dose adjustments, or ischemic complications. CONCLUSIONS: When compared with heparin-based systemic anticoagulation, bivalirudin demonstrated feasibility and safety as established by the absence of increases in identifiable adverse outcomes while manifesting substantial improvements in hospital mortality in adult patients. Further studies are necessary to corroborate these findings and further elucidate the role of bivalirudin during extracorporeal membrane oxygenation support.

Biosimilar recombinant follitropin alfa preparations versus the reference product (Gonal-F®) in couples undergoing assisted reproductive technology treatment: a systematic review and meta-analysis.

BACKGROUND: Live birth has increasingly been identified as the standard clinical approach to measure the success of medically assisted reproduction (MAR). However, previous analyses comparing biosimilar preparations of follitropin alfa versus the reference product (GONAL-f®, Merck KGaA, Darmstadt, Germany or GONAL-f® RFF; EMD Serono, Inc., Rockland, MA), have had insufficient power to detect differences in clinically meaningful outcomes such as live birth. METHODS: Medline, Embase, the Cochrane Library, Web of Science and clinical trial registries were searched for randomised controlled trials (RCTs) and conference abstracts comparing biosimilar follitropin alfa versus the reference product in controlled ovarian stimulation (COS) cycles published before 31 October 2020. Only studies in humans and publications in English were included. Retrieved studies were screened independently by two authors based on titles and abstracts, and then by full text. INCLUSION CRITERIA: RCTs comparing follitropin alfa biosimilar preparations with the reference product in infertile patients of any age, with any type of infertility for any duration, undergoing COS for the purposes of MAR treatment (including frozen cycles). The primary outcome was live birth. Combined data for biosimilar preparations were analysed using a fixed-effects model. RESULTS: From 292 unique records identified, 17 studies were included in the systematic review, representing five unique RCTs that were included in the meta-analysis. Rates of live birth (RR = 0.83, 95% CI 0.71, 0.97; 4 RCTs, n = 1881, I2 = 0%), clinical pregnancy (RR = 0.82, 95% CI 0.72, 0.94; 4 RCTs, n = 2222, I2 = 0%) and ongoing pregnancy (RR = 0.81, 95% CI 0.68, 0.96; 4 RCTs, n = 1232, I2 = 0%) were significantly lower with biosimilar preparations versus the reference product. Rates of cumulative live birth and cumulative clinical pregnancy were also significantly lower with biosimilars versus the reference product. There was high risk of publication bias. CONCLUSIONS: This meta-analysis included data from RCTs evaluating the efficacy and safety of the biosimilar follitropin alfa preparations and demonstrated lower probability of live birth and pregnancy (ongoing and clinical) in couples treated with biosimilar preparations compared with the reference product. This study provides more insight into the differences between biosimilar r-hFSH preparations and the reference product than previously reported. TRIAL REGISTRATION: Registration number: CRD42019121992 .

Production of high-quality SARS-CoV-2 antigens: Impact of bioprocess and storage on glycosylation, biophysical attributes, and ELISA serologic tests performance.

Serological assays are valuable tools to study SARS-CoV-2 spread and, importantly, to identify individuals that were already infected and would be potentially immune to a virus reinfection. SARS-CoV-2 Spike protein and its receptor binding domain (RBD) are the antigens with higher potential to develop SARS-CoV-2 serological assays. Moreover, structural studies of these antigens are key to understand the molecular basis for Spike interaction with angiotensin converting enzyme 2 receptor, hopefully enabling the development of COVID-19 therapeutics. Thus, it is urgent that significant amounts of this protein became available at the highest quality. In this study, we produced Spike and RBD in two human derived cell hosts: HEK293-E6 and Expi293F™. We evaluated the impact of different and scalable bioprocessing approaches on Spike and RBD production yields and, more importantly, on these antigens' quality attributes. Using negative and positive sera collected from human donors, we show an excellent performance of the produced antigens, assessed in serologic enzyme-linked immunosorbent assay (ELISA) tests, as denoted by the high specificity and sensitivity of the test. We show robust Spike productions with final yields of approx. 2 mg/L of culture that were maintained independently of the production scale or cell culture strategy. To the best of our knowledge, the final yield of 90 mg/L of culture obtained for RBD production, was the highest reported to date. An in-depth characterization of SARS-CoV-2 Spike and RBD proteins was performed, namely the antigen's oligomeric state, glycosylation profiles, and thermal stability during storage. The correlation of these quality attributes with ELISA performance show equivalent reactivity to SARS-CoV-2 positive serum, for all Spike and RBD produced, and for all storage conditions tested. Overall, we provide straightforward protocols to produce high-quality SARS-CoV-2 Spike and RBD antigens, that can be easily adapted to both academic and industrial settings; and integrate, for the first time, studies on the impact of bioprocess with an in-depth characterization of these proteins, correlating antigen's glycosylation and biophysical attributes to performance of COVID-19 serologic tests.

Preparation, stability and commutability of candidate reference materials for lactate dehydrogenase (LDH).

BACKGROUND: Lactate dehydrogenase (LDH) is a key enzyme that functions as a marker of cell damage. Its activity can be measured by a variety of laboratory methods. To eliminate inter-method bias and enhance equivalence among different measurement procedures, LDH detection needs to be standardized. METHODS: Optimized sequences coding for lactate dehydrogenase subunit A (LDH-A) and subunit B (LDH-B) were synthesized and cloned into the pRSFDuet vector, and then the constructed 6His-LDHA-pRSFDuet, 6His-LDHB-pRSFDuet, and 6His-LDHA-LDHB-pRSFDuet plasmids were transformed into Escherichia coli BL21 (DE3) for expression. The enzyme activity and specific activity of recombinant LDHs were detected. Electrophoresis of LDH isoenzymes was performed. The stability of recombinant LDHs and serum LDH was evaluated. Commutability of recombinant LDH-B was studied by the IFCC reference method and six routine methods. RESULTS: Three plasmids were constructed and three highly concentrated recombinant LDH isoenzymes were obtained. The specific activities of LDH-A, LDH-AB, and LDH-B were 18.08 U/mg, 21.74 U/mg, and 14.18 U/mg, respectively. There was a desirable linear correlation between the activities of recombinant LDH isoenzymes and their protein concentrations. Electrophoresis of LDH isoenzymes showed that the recombinant LDH-B corresponded to LDH1 and it demonstrated good stability at 4 °C and 25 °C for 5 weeks. LDH-B formulations in saline-bovine serum albumin solution and human serum matrix were commutable for six routine methods. CONCLUSION: Human recombinant LDH-B has great potential to become an improved and less expensive standard or reference material in external quality assessment for clinical LDH measurement.

Summary of the WHO hearing on the development of product-specific reference materials for coagulation factor VIII and factor IX products.

In recent years, several modified recombinant factor (F) VIII and FIX therapeutics with extended half-life have been licensed internationally for the treatment of haemophilia. Safe and effective use of these products requires monitoring of factor activity in patient plasma. The potency of all FVIII and FIX products is currently assigned in International Units (IU) which anchors the relationship between potency labelling, dosing and clinical monitoring. However, varying degrees of discrepancies in factor activity assays are observed between and within the factor activity analytical methods (one-stage clotting and chromogenic), when measuring these modified products against plasma and plasma-derived (concentrate) International Standards (IS) or in-house reference standard traceable to the IS. Availability of product-specific reference reagents would mitigate assay discrepancies, facilitate independent testing of assay methods and reagents, and ensure long-term continuity of the IU related to each product. A hearing meeting was organised by the WHO to discuss the requirements for product-specific reference materials for these products and whether these reference materials should be produced by the WHO. Advantages and disadvantages of product-specific reference materials were identified and discussed.

A CFD model for predicting protein aggregation in low-pH virial inactivation for mAb production.

Significant amounts of soluble product aggregates were observed in the low-pH viral inactivation (VI) operation during an initial scale-up run for an immunoglobulin-G 4 (IgG4) monoclonal antibody (mAb IgG4-N1). Being earlier in development, a scale-down model did not exist, nor was it practical to use costly Protein A eluate (PAE) for testing the VI process at scale, thus, a computational fluid dynamics (CFD)-based high-molecular weight (HMW) prediction model was developed for troubleshooting and risk mitigation. It was previously reported that the IgG4-N1 molecules upon exposure to low pH tend to change into transient and partially unfolded monomers during VI acidification (i.e., VIA) and form aggregates after neutralization (i.e., VIN). Therefore, the CFD model reported here focuses on the VIA step. The model mimics the continuous addition of acid to PAE and tracks acid distribution during VIA. Based on the simulated low-pH zone (≤pH 3.3) profiles and PAE properties, the integrated low-pH zone (ILPZ) value was obtained to predict HMW level at the VI step. The simulations were performed to examine the operating parameters, such as agitation speed, acid addition rate, and protein concentration of PAE, of the pilot scale (50-200 L) runs. The conditions with predictions of no product aggregation risk were recommended to the real scale-up runs, resulted in 100% success rate of the consecutive 12 pilot-scale runs. This study demonstrated that the CFD-based HMW prediction model could be used as a tool to facilitate the scale up of the low-pH VI process directly from bench to pilot/production scale.

Impact of enzymatic reduction on bivalent bispecific antibody fragmentation and loss of product purity upon reoxidation.

Antibody disulfide bond (DSB) reduction during manufacturing processes is a widely observed phenomenon attributed to host cell reductases present in harvest cell culture fluid. Enzyme-induced antibody reduction leads to product fragments and aggregates that increase the impurity burden on the purification process. The impact of reduction on bivalent bispecific antibodies (BisAbs), which are increasingly entering the clinic, has yet to be investigated. We focused on the reduction and reoxidation properties of a homologous library of bivalent BisAb formats that possess additional single-chain Fv (scFv) fragments with engineered DSBs. Despite all BisAbs having similar susceptibilities to enzymatic reduction, fragmentation pathways were dependent on the scFv-fusion site. Reduced molecules were allowed to reoxidize with and without low pH viral inactivation treatment. Both reoxidation studies demonstrated that multiple, complex BisAb species formed as a result of DSB mispairing. Furthermore, aggregate levels increased for all molecules when no low pH treatment was applied. Combined, our results show that complex DSB mispairing occurs during downstream processes while aggregate formation is dependent on sample treatment. These results are applicable to other novel monoclonal antibody-like formats containing engineered DSBs, thus highlighting the need to prevent reduction of novel protein therapeutics to avoid diminished product quality during manufacturing.

Prevalence of Toxoplasma gondii parasite in captive Mexican jaguars determined by recombinant surface antigens (SAG1) and dense granular antigens (GRA1 and GRA7) in ELISA-based serodiagnosis.

Toxoplasma gondii is an obligate intracellular protozoan parasite that infects almost all warm-blooded animals, including humans, causing serious public health problems. In this study, the seroprevalence of T. gondii in captive jaguars in 10 Mexican zoos was determined using single and mixtures of recombinant surface antigens (SAG1) and dense granular antigens (GRA1 and GRA7) in immunoglobulin G (IgG) enzyme-linked immunosorbent assays (ELISAs). Their efficacy was compared with the tachyzoite lysate antigen. All recombinant antigens were characterised by high sensitivity (92.5-97.5%); the specificity of the IgG ELISAs was variable (83.3-91.6%). Mixtures of the two recombinant proteins were generally more reactive than single antigens. GRA7 + SAG1 showed the highest sensitivity (97.5%) and specificity (91.6%), almost perfect agreement (96.2%), and a kappa value of 0.89. An area under the curve value of 0.998 represented a highly accurate test with a cutoff value of 0.8. The seroprevalence of anti-T. gondii IgG antibodies in the single and mixed recombinant antigen ELISAs was 75.0-76.9%. This study shows that GRA7 + SAG1 can be successfully used to diagnose T. gondii infection in jaguars for effective monitoring of prevalence and for devising control methods and prevention strategies against toxoplasmosis.

The Effect of Fluctuating Temperature on the Stability of Turoctocog Alfa for Hemophilia A.

BACKGROUND AND OBJECTIVE: Factor VIII (FVIII) is indicated for the prevention or treatment of bleeding in patients with hemophilia A. FVIII product stability under high and fluctuating temperatures is important, particularly for patients who reside in, or travel to, regions with high ambient temperatures, as they may remove their product from the refrigerator and return it, unused, multiple times. We evaluated the effect of variable temperature storage conditions, including up to 40 °C, on the stability of the recombinant FVIII product, turoctocog alfa. METHODS: Turoctocog alfa dry powder stability was assessed when moved between storage conditions of 5 °C (ambient humidity) and 40 °C (75% relative humidity) multiple times over a 2-month period, followed by long-term storage at 40 °C for 3 months and 5 °C for 1 month. Three product strengths (250, 1500, and 3000 IU), including the lowest and highest doses, were evaluated. Stability assessments included potency, purity, oxidized forms, high molecular weight protein (HMWP), and water content. RESULTS: Overall, the three doses of turoctocog alfa tested remained stable under varying temperature conditions, without any potency or purity impairment, nor were any major increases in oxidized forms, HMWP, or water content observed. All results were within shelf-life specification limits. CONCLUSION: The results demonstrated that turoctocog alfa can be subjected to variable storage conditions, including cycling between 5 °C and ≤ 40 °C, and subsequent storage for 3 months up to 40 °C, without loss of stability. This suggests that turoctocog alfa may offer greater product storage flexibility for patients in everyday practice, with a potential reduction in wastage.

A development of high-throughput HPLC method for the polysorbate 80 quantitation in protein therapeutic products.

A quantitative analysis of polysorbate 80 is an essential part of a characterization of therapeutic protein products. There are many methods to perform such type of analysis, including spectrophotometry and HPLC. However, there is no high-throughput method with high sensitivity and accuracy. In our work, we optimized conditions of polysorbate 80 hydrolysis to reduce the sample preparation time and developed a new isocratic reversed phase HPLC method for the quantification of oleic acid, the principal product of the hydrolysis. The validation of developed method shows that it is characterized by the repeatability less than 2.7% with the LOD about 25 ng (125 ppb), LOQ about 100 ng (500 ppb) within the analytical range of 0.025 to 1.5 mg/ml polysorbate 80 concentrations, accuracy between 92and108% and high precision (coefficient of variation less than3.2%). The total time needed for the analysis was about 1 h. The method could be routinely used for the quality control during therapeutic proteins manufacturing.

Assessment of methods for determination of glycan composition of erythropoietin.

Erythropoietin (EPO) is a monomeric, highly glycosylated, protein hormone (molecular size around 30-35 kD), produced mainly in adult kidneys, which acts principally on red blood cell progenitors and precursors to promote red cell production. Therapeutic EPO products are widely used biotherapeutics. They are mainly produced by recombinant DNA technology in mammalian cells and their biological activity is closely linked to the degree of N-glycan sialylation. Determination of the sialic acids' content and complexity by glycan mapping therefore appears critical to ensure the quality and efficacy of the EPO therapeutic products. The European Directorate for the Quality of Medicines & HealthCare organised a study (BSP144) under the aegis of the Biological Standardisation Programme to assess N-glycan mapping tests with the aim of incorporating a standard method into the European Pharmacopoeia monograph 'Erythropoietin concentrated solution' (1316). The use of a 'reagent panel' consisting of six EPO preparations with a range of iso-electric properties facilitated comparison between laboratories and methodologies. Based on the study results, a robust and repeatable HPAEC-PAD chromatographic method was identified and work to introduce it in the monograph as an example method has been initiated.

Elimination of ghost peaks by optimization of anion exchange chromatography method for determination of gamma-carboxyglutamic acid (Gla)-domainless impurity in recombinant activated clotting factor VII drug products.

Secreted recombinant activated clotting factor VII activated (rFVIIa) in cell culture media missing gamma-carboxyglutamic acid (Gla) domain as a result of failure in gamma-carboxylation or cell lysis is called Gla-domainless impurity which has less negative charge compared to native rFVIIa. Based on risk assessment, this type of impurity is considered as critical drug product quality attribute of rFVIIa and its quantitative analysis in product batches is a critical issue in quality control laboratories. Analysis of Gla-domainless impurity is accomplished by Strong Anion Exchange Chromatography (SAX) in recombinant factor VIIa using Tris and Bis-Tris propane salt buffers as equilibrating buffers and high concentration ammonium acetate as an eluent. Appearance of ghost peaks with notable intensity during elution time of Gla-domainless impurity caused distortion of the related peak and interference with robust and accurate quantification of this impurity. Subsequently, the ghost peak was analyzed by LC-ESI-MS to determine the structure which showed the m/z values at 905.27, 623.53 and 341.60 and 563.73. To find the source of these ghost peaks, quality of water, buffer salts and Chelex-100 together with ionic strength of mobile phase A (addition of 25 mM NaCl) were considered as affecting parameters and several experiments designed with DOE software to optimize the best condition of highest quality the method with lowest signal of ghost peak noises. By interpretation of DOE result, it is concluded that high grade water and buffer salt along with high quality Chelex-100 resins are important factors to achieve a method with lowest ghost peaks. However, addition of 25 mM NaCl to mobile phase A with either lower quality buffer salts or lower water grade yields high quality chromatogram peak with acceptable ghost peaks. LC/MS analysis indicates that macrostructures of Bis-Tris propane made up as a result of hydrogen bonds with each other or Tris molecules can be the source of ghost peaks.

Discovery and characterization of CHO host cell protease-induced fragmentation of a recombinant monoclonal antibody during production process development.

Monoclonal antibody (mAb) fragmentation is a widespread issue of protein stability that needs to be carefully monitored for critical mAb quality control during the production process development. This study describes here the discovery and characterization of CHO host cell protease-induced fragmentation of a therapeutic mAb-X in the formulation samples from an early production process. The fragmentation was observed in the sodium dodecyl sulfate capillary electrophoresis (CE-SDS) analysis of mAb-X formulation samples incubated at elevated temperature. Size exclusion liquid chromatography (SEC-HPLC) was used to analyze and collect these cleaved fragments derived from mAb-X. Reversed phase liquid chromatography mass spectrometry (RP-LC-MS) and tandem mass (MS/MS) analysis demonstrated that the fragment was generated mainly due to the hinge region cleavage of mAb-X. The fragmentation rate was characterized in the mAb-X formulation samples at pH from 4.0 to 6.0 using CE-SDS and SDS-PAGE analysis. The percentage of the main fragment increased dramatically from 2.8% to 31.2% as pH decreased from 6.0 to 4.0 at 40 °C for 28 days, which indicated the fragmentation was highly pH-dependent. The SDS-PAGE analysis further verified the pH-dependent property of the framentation of mAb-X. Moreover, the fragmentation was characterized in the presence and absence of pepstatin A, an inhibitor of acidic proteases. Significant inhibition of mAb-X fragmentation was observed with the addition of pepstatin A to mAb-X formulation samples. These results suggested residual acidic host cell protease(s) in the formulation samples from an early production process caused the fragmentation of mAb-X. To prove evidence, we developed an optimized protein A chromatography to enhance the residual host cell protease(s) removal capability of mAb-X purification process and consequently eliminate the above described cleaved fragment of mAb-X, which further supported the hypothesis that the fragmentation of mAb-X was catalyzed by the residual host cell protease(s) in the formulation samples from the early production process. This case study reiterated that residual host cell protease is a critical quality attribute (CQA) that should be carefully controlled and evaluated to guarantee successful manufacture processes for mAb products.

Biopharmaceutical Industry Approaches to Facility Segregation for Viral Safety: An Effort from the Consortium on Adventitious Agent Contamination in Biomanufacturing.

Appropriate segregation within manufacturing facilities is required by regulators and utilized by manufacturers to ensure that the final product has not been contaminated with (a) adventitious viruses, (b) another pre-/postviral clearance fraction of the same product, or (c) another product processed in the same facility. However, there is no consensus on what constitutes appropriate facility segregation to minimize these risks. In part, this is due to the fact that a wide variety of manufacturing facilities and operational practices exist, including single-product and multiproduct manufacturing, using traditional segregation strategies with separate rooms for specific operations that may use stainless steel or disposable equipment to more modern ballroom-style operations that use mostly disposable equipment (i.e., pre- and postviral clearance manufacturing operations are not physically segregated by walls). Further, consensus is lacking around basic definitions and approaches related to facility segregation. For example, given that several unit operations provide assurance of virus clearance during downstream processing, how does one define pre- and postviral clearance and at which point(s) should a viral segregation barrier be introduced? What is a "functionally closed" system? How can interventions be conducted so that the system remains functionally closed? How can functionally closed systems be used to adequately isolate a product stream and ensure its safety? To address these issues, the member companies of the Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) have conducted a facility segregation project with the following goals: define "pre- and postviral clearance zones" and "pre- and postviral clearance materials"; define "functionally closed" manufacturing systems; and identify an array of facility segregation approaches that are used for the safe and effective production of recombinant biologics as well as plasma products. This article reflects the current thinking from this collaborative endeavor.LAY ABSTRACT: Operations in biopharmaceutical manufacturing are segregated to ensure that the final product has not been contaminated with adventitious viruses, another fraction of the same product, or with another product from within the same facility. Yet there is no consensus understanding of what appropriate facility segregation looks like. There are a wide variety of manufacturing facilities and operational practices. There are existing facilities with separate rooms and more modern approaches that use disposable equipment in an open ballroom without walls. There is also no agreement on basic definitions and approaches related to facility segregation approaches. For example, many would like to claim that their manufacturing process is functionally closed, yet exactly how a functionally closed system may be defined is not clear. To address this, the member companies of the Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) have conducted a project with the goal of defining important manufacturing terms relevant to designing an appropriately segregated facility and identifying different facility segregation approaches that are used for the safe and effective production of recombinant biologics as well as plasma products.

Multiproduct Resin Reuse for Clinical and Commercial Manufacturing-Methodology and Acceptance Criteria.

Chromatography resins used for purifying biopharmaceuticals are generally dedicated to a single product. In good manufacturing practice (GMP) facilities that manufacture a limited amount of any particular product, this practice can result in the resin being used for a fraction of its useful life. A methodology for extending resin reuse to multiple products is described. With this methodology, resin and column performance, product carryover, and cleaning effectiveness are continually monitored to ensure that product quality is not affected by multiproduct resin reuse (MRR). Resin and column performance is evaluated in terms of (a) system suitability parameters, such as peak-shape and transition, and height equivalent theoretical plate (HETP) data; (b) key operating parameters, such as flow rate, inlet pressure, and pressure drop across the column; and (c) process performance parameters, such as impurity profiles, product quality, and yield. Historical data are used to establish process capability limits (PCLs) for these parameters. Operation within the PCLs provides assurance that column integrity and binding capacity of the resin are not affected by MRR.Product carryover defined as the carryover of the previously processed product (A) into a dose of the subsequently processed product (B) (COA→B), should be acceptable from a predictive patient safety standpoint. A methodology for determining COA→B from first principles and setting acceptance limits for cleaning validation is described.Cleaning effectiveness is evaluated by performing a blank elution run after inter-campaign cleaning and prior to product changeover. The acceptance limits for product carryover (COA→B) are more stringent for MRR than for single-product resin reuse. Thus, the inter-campaign cleaning process should be robust enough to consistently meet the more stringent acceptance limits for MRR. Additionally, the analytical methods should be sensitive enough to adequately quantify the concentration of the previously processed product (A) and its degradants in the eluent.General considerations for designing small-scale chromatographic studies for process development are also described. These studies typically include process-cycling runs with multiple products followed by viral clearance studies with a panel of model viruses. Small-scale studies can be used to optimize cleaning parameters, predict resin performance and product quality, and estimate the number of multiproduct purification cycles that can be run without affecting product quality. The proposed methodology is intended to be broadly applicable; however, it is acknowledged that alternative approaches may be more appropriate for specific scenarios.LAY ABSTRACT: Chromatography resins used for purifying biopharmaceuticals are generally dedicated to a single product. In good manufacturing practice (GMP) facilities that make a limited amount of any particular product, this practice can result in the resin being used for a fraction of its useful life. A methodology for extending resin reuse to multiple products is described. With this methodology, resin and column performance, product carryover, and cleaning effectiveness are continually monitored to ensure that product quality is not affected by multiproduct resin reuse.General considerations for designing small-scale chromatographic studies for process development are described. These studies typically include process-cycling runs with multiple products followed by viral clearance studies with a panel of model viruses. Small-scale studies can be used to optimize cleaning parameters, predict resin performance and product quality, and estimate the number of multiproduct purification cycles that can be run without impacting product quality.The proposed methodology is intended to be broadly applicable; however, it is acknowledged that alternative approaches may be more appropriate for specific scenarios.

Heterologous recombinant expression of non-originator NISTmAb.

The successful development and regulatory approval of originator and biosimilar therapeutic proteins requires a systems approach to upstream and downstream processing as well as product characterization and quality control. Innovation in process design and control, product characterization strategies, and data integration represent an ecosystem whose concerted advancement may reduce time-to-market and further improve comparability and biosimilarity programs. The biopharmaceutical community has made great strides to this end, yet there currently exists no pre-competitive monoclonal antibody (mAb) expression platform for open innovation. Here, we describe the development and initial expression of an intended copy of the NISTmAb using three non-originator murine cell lines. It was found that, without optimization and in culture flasks, all three cell lines produce approximately 100 mg mAb per liter of culture. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, size-exclusion chromatography, nuclear magnetic resonance spectroscopy, intact mass spectrometry, and surface plasmon resonance were used to demonstrate that the products of all three cell lines embody quality attributes with a sufficient degree of sameness to the NISTmAb Reference Material 8671 to warrant further bioreactor studies, process improvements and optimization. The implications of the work with regard to pre-competitive innovation to support process design and feedback control, comparability and biosimilarity assessments, and process analytical technologies are discussed.