Industrial production of monoclonal antibodies and therapeutic proteins by dialysis fermentation

A novel and powerful fermentation method is reported for the large-scale growth of mammalian cells and their secreted products. The system described illustrates many of the advantages of conventional batch fermentation processes but in addition has been shown to yield cell densities in excess of 1×10⁷ cells/ml with concomitant increase in product concentration.Abbreviations MAb Monoclonal Antibody - STR Stirred Tank Reactor - FBS Foetal Bovine Serum     

Linear pharmacokinetic parameters for monoclonal antibodies are similar within a species and across different pharmacological targets: A comparison between human,cynomolgus monkey and hFcRn Tg32 transgenic mouse using a population-modeling approach

The linear pharmacokinetics (PK) of therapeutic monoclonal antibodies (mAbs) can be considered a class property with values that are similar to endogenous IgG. Knowledge of these parameters across species could be used to avoid unnecessary in vivo PK studies and to enable early PK predictions and pharmacokinetic/pharmacodynamic (PK/PD) simulations. In this work, population-pharmacokinetic (popPK) modeling was used to determine a single set of ‘typical’ popPK parameters describing the linear PK of mAbs in human, cynomolgus monkey and transgenic mice expressing the human neonatal Fc receptor (hFcRn Tg32), using a rich dataset of 27 mAbs. Non-linear PK was excluded from the datasets and a 2-compartment model was applied to describe mAb disposition. Typical human popPK estimates compared well with data from comparator mAbs with linear PK in the clinic. Outliers with higher than typical clearance were found to have non-specific interactions in an affinity-capture self-interaction nanoparticle spectroscopy assay, offering a potential tool to screen out these mAbs at an early stage. Translational strategies were investigated for prediction of human linear PK of mAbs, including use of typical human popPK parameters and allometric exponents from cynomolgus monkey and Tg32 mouse. Each method gave good prediction of human PK with parameters predicted within 2-fold. These strategies offer alternative options to the use of cynomolgus monkeys for human PK predictions of linear mAbs, based on in silico methods (typical human popPK parameters) or using a rodent species (Tg32 mouse), and call into question the value of completing extensive in vivo preclinical PK to inform linear mAb PK.     

Bracketed generic inactivation of rodent retroviruses by low pH treatment for monoclonal antibodies and recombinant proteins

Viral safety is a predominant concern for monoclonal antibodies (mAbs) and other recombinant proteins (RPs) with pharmaceutical applications. Certain commercial purification modules, such as nanofiltration and low-pH inactivation, have been observed to reliably clear greater than 4 log(10) of large enveloped viruses, including endogenous retrovirus. The concept of "bracketed generic clearance" has been proposed for these steps if it could be prospectively demonstrated that viral log(10) reduction value (LRV) is not impacted by operating parameters that can vary, within a reasonable range, between commercial processes. In the case of low-pH inactivation, a common step in mAb purification processes employed after protein A affinity chromatography, these parameters would include pH, time and temperature of incubation, the content of salts, protein concentration, aggregates, impurities, model protein pI, and buffer composition. In this report, we define bracketed generic clearance conditions, using a prospectively defined bracket/matrix approach, where low-pH inactivation consistently achieves >or=4.6 log(10) clearance of xenotropic murine leukemia virus (X-MLV), a model for rodent endogenous retrovirus. The mechanism of retrovirus inactivation by low-pH treatment was also investigated.     

Quantification of IgG monoclonal antibody clearance in tissues

Monoclonal antibodies are an important therapeutic entity, and knowledge of antibody pharmacokinetics has steadily increased over the years. Despite this effort, little is known about the extent of IgG antibody degradation in different tissues of the body. While studies have been published identifying sites of degradation with the use of residualizing and non-residualizing radiolabels, quantitative tissue clearances have not yet been derived. Here, we show that in physiologically-based pharmacokinetic (PBPK) models we can combine mouse data of Indium-111 and Iodine-125 labeled antibodies with prior physiologic knowledge to determine tissue-specific intrinsic clearances. Unspecific total tissue clearance (mL/day) in the mouse was estimated to be: liver = 4.75; brain = 0.02; gut = 0.40; heart = 0.07; kidney = 0.97; lung = 0.20; muscle = 3.02; skin = 3.89; spleen = 0.45; rest of body = 2.16. The highest catabolic activity (per g tissue) was in spleen for an FcRn wild-type antibody, but shifts to the liver for an antibody with reduced FcRn affinity. In the model developed, this shift can be explained by the liver having a greater FcRn-mediated protection capacity than the spleen. The quantification of tissue intrinsic clearances and FcRn salvage capacity increases our understanding of quantitative processes that drive the therapeutic responses of antibodies. This knowledge is critical, for instance to estimate the non-specific cellular uptake and degradation of antibodies used for targeted delivery of payloads.     

Analysis of viral clearance unit operations for monoclonal antibodies

Demonstration of viral clearance is a critical step in assuring the safety of biotechnology products. We generated a viral clearance database that contains product information, unit operation process parameters, and viral clearance data from monoclonal antibody and antibody‐related regulatory submissions to FDA. Here we present a broad overview of the database and resulting analyses. We report that the diversity of model viruses tested expands as products transition to late‐phase. We also present averages and ranges of viral clearance results by Protein A and ion exchange chromatography steps, low pH chemical inactivation, and virus filtration, focusing on retro‐ and parvoviruses. For most unit operations, an average log reduction value (LRV, a measure of clearance power) for retrovirus of >4 log₁₀ were measured. Cases where clearance data fell outside of the anticipated range (i.e., outliers) were rationally explained. Lastly, a historical analysis did not find evidence of any improvement trend in viral clearance over time. The data collectively suggest that many unit operations in general can reliably clear viruses. Biotechnol. Bioeng. 2010;106: 238–246. Published 2010 Wiley Periodicals, Inc.     

Utility of a human FcRn transgenic mouse model in drug discovery for early assessment and prediction of human pharmacokinetics of monoclonal antibodies

Therapeutic antibodies continue to develop as an emerging drug class, with a need for preclinical tools to better predict in vivo characteristics. Transgenic mice expressing human neonatal Fc receptor (hFcRn) have potential as a preclinical pharmacokinetic (PK) model to project human PK of monoclonal antibodies (mAbs). Using a panel of 27 mAbs with a broad PK range, we sought to characterize and establish utility of this preclinical animal model and provide guidance for its application in drug development of mAbs. This set of mAbs was administered to both hemizygous and homozygous hFcRn transgenic mice (Tg32) at a single intravenous dose, and PK parameters were derived. Higher hFcRn protein tissue expression was confirmed by liquid chromatography-high resolution tandem mass spectrometry in Tg32 homozygous versus hemizygous mice. Clearance (CL) was calculated using non-compartmental analysis and correlations were assessed to historical data in wild-type mouse, non-human primate (NHP), and human. Results show that mAb CL in hFcRn Tg32 homozygous mouse correlate with human (r² = 0.83, r = 0.91, p < 0.01) better than NHP (r² = 0.67, r = 0.82, p < 0.01) for this dataset. Applying simple allometric scaling using an empirically derived best-fit exponent of 0.93 enabled the prediction of human CL from the Tg32 homozygous mouse within 2-fold error for 100% of mAbs tested. Implementing the Tg32 homozygous mouse model in discovery and preclinical drug development to predict human CL may result in an overall decreased usage of monkeys for PK studies, enhancement of the early selection of lead molecules, and ultimately a decrease in the time for a drug candidate to reach the clinic.     

Comparison and critical analysis of robotized technology for monoclonal antibody high-throughput production

We have previously demonstrated how to transform the conventional method of hybridoma production and screening into a fast, high-throughput technology. Nevertheless, there were still open questions related to automated procedures and immunization protocols that we address now by comparing the hybridoma production work-flow in automated and manually executed processes. In addition, since the animals' antibody responses to single or multiple antigen challenge affect monoclonal antibody throughput, different immunization and fusion strategies were tested. Specifically, the results obtained with multiplexing (multiple target antigens injected into a single animal) and single antigen immunization followed by splenocyte pooling immediately before fusion were compared with conventional methods. The results presented here demonstrate that the optimal protocol consists of automated somatic-cell fusion and hybridoma dilution followed by manual plating of hybridoma cells. Additionally, more specific and productive hybridoma clones were obtained with multiplexed immunization in a single animal with respect to the splenocyte pooling from single antigen immunized animals. However, in terms of overall antibody yield, the conventional method consisting of single immunization for each single animal assured ten times more specific hybridoma cell lines than the strategy based on the multiple antigen immunization followed by separate fusion step. In conclusion, the most productive approach for recovering a large number of suitable antibodies relies on single antigen immunization followed by automated fusion and dilution steps and manual plating.     

Impact of cell culture on recombinant monoclonal antibody product heterogeneity

Recombinant monoclonal antibodies are commonly expressed in mammalian cell culture and purified by several steps of filtration and chromatography. The resulting high purity bulk drug substance still contains product variants differing in properties such as charge and size. Posttranslational modifications and degradations occurring during cell culture are the major sources of heterogeneity in bulk drug substance of recombinant monoclonal antibodies. The focus of the current review is the impact of cell culture conditions on the types and levels of various modifications and degradations of recombinant monoclonal antibodies. Understanding the relationship between cell culture and product variants can help to make consistently safe and efficacious products. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1103–1112, 2016     

Dextrose-mediated aggregation of therapeutic monoclonal antibodies in human plasma: Implication of isoelectric precipitation of complement proteins

Many therapeutic monoclonal antibodies (mAbs) are clinically administered through intravenous infusion after mixing with a diluent, e.g., saline, 5% dextrose. Such a clinical setting increases the likelihood of interactions among mAb molecules, diluent, and plasma components, which may adversely affect product safety and efficacy. Avastin® (bevacizumab) and Herceptin® (trastuzumab), but not Remicade® (infliximab), were shown to undergo rapid aggregation upon dilution into 5% dextrose when mixed with human plasma in vitro; however, the biochemical pathways leading to the aggregation were not clearly defined. Here, we show that dextrose-mediated aggregation of Avastin or Herceptin in plasma involves isoelectric precipitation of complement proteins. Using mass spectrometry, we found that dextrose-induced insoluble aggregates were composed of mAb itself and multiple abundant plasma proteins, namely complement proteins C3, C4, factor H, fibronectin, and apolipoprotein. These plasma proteins, which are characterized by an isoelectronic point of 5.5–6.7, lost solubility at the resulting pH in the mixture with formulated Avastin (pH 6.2) and Herceptin (pH 6.0). Notably, switching formulation buffers for Avastin (pH 6.2) and Remicade (pH 7.2) reversed their aggregation profiles. Avastin formed little, if any, insoluble aggregates in dextrose-plasma upon raising the buffer pH to 7.2 or above. Furthermore, dextrose induced pH-dependent precipitation of plasma proteins, with massive insoluble aggregates being detected at pH 6.5–6.8. These data show that isoelectric precipitation of complement proteins is a prerequisite of dextrose-induced aggregation of mAb in human plasma. This finding highlights the importance of assessing the compatibility of a therapeutic mAb with diluent and human plasma during product development.     

Suitability of a generic virus safety evaluation for monoclonal antibody investigational new drug applications

Biologics produced from CHO cell lines with endogenous virus DNA can produce retrovirus-like particles in cell culture at high titers, and other adventitious viruses can find their way through raw materials into the process to make a product. Therefore, it is the industry standard to have controls to avoid introduction of viruses into the production process, to test for the presence of viral particles in unclarified cell culture, and to develop purification procedures to ensure that manufacturing processes are robust for viral clearance. Data have been accumulated over the past four decades on unit operations that can inactivate and clear adventitious virus and provide a high degree of assurance for patient safety. During clinical development, biological products are traditionally tested at process set points for viral clearance. However, the widespread implementation of platform production processes to produce highly similar IgG antibodies for many indications makes it possible to leverage historical data and knowledge from representative molecules to allow for better understanding and control of virus safety. More recently, individualized viral clearance studies are becoming the rate-limiting step in getting new antibody molecules to clinic, particularly in Phase 0 and eIND situations. Here, we explore considerations for application of a generic platform virus clearance strategy that can be applied for relevant investigational antibodies within defined operational parameters in order to increase speed to the clinic and reduce validation costs while providing a better understanding and assurance of process virus safety.     

Dextrose-mediated aggregation of therapeutic monoclonal antibodies in human plasma: Implication of isoelectric precipitation of complement proteins

Many therapeutic monoclonal antibodies (mAbs) are clinically administered through intravenous infusion after mixing with a diluent, e.g., saline, 5% dextrose. Such a clinical setting increases the likelihood of interactions among mAb molecules, diluent, and plasma components, which may adversely affect product safety and efficacy. Avastin® (bevacizumab) and Herceptin® (trastuzumab), but not Remicade® (infliximab), were shown to undergo rapid aggregation upon dilution into 5% dextrose when mixed with human plasma in vitro; however, the biochemical pathways leading to the aggregation were not clearly defined. Here, we show that dextrose-mediated aggregation of Avastin or Herceptin in plasma involves isoelectric precipitation of complement proteins. Using mass spectrometry, we found that dextrose-induced insoluble aggregates were composed of mAb itself and multiple abundant plasma proteins, namely complement proteins C3, C4, factor H, fibronectin, and apolipoprotein. These plasma proteins, which are characterized by an isoelectronic point of 5.5–6.7, lost solubility at the resulting pH in the mixture with formulated Avastin (pH 6.2) and Herceptin (pH 6.0). Notably, switching formulation buffers for Avastin (pH 6.2) and Remicade (pH 7.2) reversed their aggregation profiles. Avastin formed little, if any, insoluble aggregates in dextrose-plasma upon raising the buffer pH to 7.2 or above. Furthermore, dextrose induced pH-dependent precipitation of plasma proteins, with massive insoluble aggregates being detected at pH 6.5–6.8. These data show that isoelectric precipitation of complement proteins is a prerequisite of dextrose-induced aggregation of mAb in human plasma. This finding highlights the importance of assessing the compatibility of a therapeutic mAb with diluent and human plasma during product development.     

Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use

Ch-mAb7F9, a human-mouse chimeric monoclonal antibody (mAb) designed to bind (+)-methamphetamine (METH) with high affinity and specificity, was produced as a treatment medication for METH abuse. In these studies, we present the preclinical characterization that provided predictive evidence that ch-mAb7F9 may be safe and effective in humans. In vitro ligand binding studies showed that ch-mAb7F9 is specific for and only binds its target ligands (METH, (+)-amphetamine, and 3,4-methylenedioxy-N-methylamphetamine) with high affinity. It did not bind endogenous neurotransmitters or other medications and was not bound by protein C1q, thus it is unlikely to stimulate in vivo complement-dependent cytotoxicity. Isothermal titration calorimetry potency studies showed that METH binding by ch-mAb7F9 is efficient. Pharmacokinetic studies of METH given after ch-mAb7F9 doses in rats demonstrated the in vivo application of these in vitro METH-binding characteristics. While METH had little effect on ch-mAb7F9 disposition, ch-mAb7F9 substantially altered METH disposition, dramatically reducing the volume of distribution and clearance of METH. The elimination half-life of METH was increased by ch-mAb7F9, but it was still very fast compared with the elimination of ch-mAb7F9. Importantly, the rapid elimination of unbound METH combined with previous knowledge of mAb:target ligand binding dynamics suggested that ch-mAb7F9 binding capacity regenerates over time. This finding has substantial therapeutic implications regarding the METH doses against which ch-mAb7F9 will be effective, on the duration of ch-mAb7F9 effects, and on the safety of ch-mAb7F9 in METH users who use METH while taking ch-mAb7F9. These results helped to support initiation of a Phase 1a study of ch-mAb7F9.     

Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use

Ch-mAb7F9, a human-mouse chimeric monoclonal antibody (mAb) designed to bind (+)-methamphetamine (METH) with high affinity and specificity, was produced as a treatment medication for METH abuse. In these studies, we present the preclinical characterization that provided predictive evidence that ch-mAb7F9 may be safe and effective in humans. In vitro ligand binding studies showed that ch-mAb7F9 is specific for and only binds its target ligands (METH, (+)-amphetamine, and 3,4-methylenedioxy-N-methylamphetamine) with high affinity. It did not bind endogenous neurotransmitters or other medications and was not bound by protein C1q, thus it is unlikely to stimulate in vivo complement-dependent cytotoxicity. Isothermal titration calorimetry potency studies showed that METH binding by ch-mAb7F9 is efficient. Pharmacokinetic studies of METH given after ch-mAb7F9 doses in rats demonstrated the in vivo application of these in vitro METH-binding characteristics. While METH had little effect on ch-mAb7F9 disposition, ch-mAb7F9 substantially altered METH disposition, dramatically reducing the volume of distribution and clearance of METH. The elimination half-life of METH was increased by ch-mAb7F9, but it was still very fast compared with the elimination of ch-mAb7F9. Importantly, the rapid elimination of unbound METH combined with previous knowledge of mAb:target ligand binding dynamics suggested that ch-mAb7F9 binding capacity regenerates over time. This finding has substantial therapeutic implications regarding the METH doses against which ch-mAb7F9 will be effective, on the duration of ch-mAb7F9 effects, and on the safety of ch-mAb7F9 in METH users who use METH while taking ch-mAb7F9. These results helped to support initiation of a Phase 1a study of ch-mAb7F9.     

Molecular basis for the binding polyspecificity of an anti-cholera toxin peptide 3 monoclonal antibody

The onset of autoimmune diseases is proposed to involve binding promiscuity of antibodies (Abs) and T‐cells, an often reported yet poorly understood phenomenon. Here, we attempt to approach two questions: first, is binding promiscuity a general feature of monoclonal antibodies (mAbs) and second, what is the molecular basis for polyspecificity? To this end, the anti‐cholera toxin peptide 3 (CTP3) mAb TE33 was investigated for polyspecific binding properties. Screening of phage display libraries identified two epitope‐unrelated peptides that specifically bound TE33 with affinities similar to or 100‐fold higher than the wild‐type epitope. Substitutional analyses revealed distinct key residue patterns recognized by the antibody suggesting a unique binding mode for each peptide. A database query with one of the consensus motifs and a subsequent binding study uncovered 45 peptides (derived from heterologous proteins) that bound TE33. To better understand the structural basis of the observed polyspecificity we modeled the new cyclic epitope in complex with TE33. The interactions between this peptide and TE33 suggested by our model are substantially different from the interactions observed in the X‐ray structure of the wild‐type epitope complex. However, the overall binding conformation of the peptides is similar. Together, our results support the theory of a general polyspecific potential of mAbs. Copyright © 2005 John Wiley & Sons, Ltd.     

A monoclonal antibody driven biodiagnostic system for the quantitative screening of breast cancer

A prospective clinical parametric study comprising women afflicted by breast cancer and otherwise healthy participants was undertaken. The mean plasmatic concentration of putative leucine amino peptidase and nucleoside diphosphate phosphotransferase enzymatic complex in breast cancer cases was significantly elevated [43.9 +/- 2.8 microg ml(-1) (n = 9)] when compared to those found in otherwise healthy women [8.07 +/- 0.14 microg ml(-1) (n = 8)]. Women without images compatible with any tumours (n = 13) had a mean concentration of 10.77 +/- 1.49 microg ml(-1). The mean value obtained in women with fibroadenomas was 10.15 +/- 0.81 microg ml(-1) (n = 6) and with cystic fibrosis mastopathy 8.75 +/- 0.28 microg ml(-1) (n = 7). The efficacy of a tandem quantitative biodiagnostic system as a parametric screening tool for the early detection of breast cancer is underlined, raising the possibility of increasing the cost effectiveness of current imaging non-parametric technologies.     

Exploring the fate of inhaled monoclonal antibody in the lung parenchyma by microdialysis

Therapeutic antibodies (Abs) are emerging as major drugs to treat respiratory diseases, and inhalation may provide substantial benefits for their delivery. Understanding the behavior of Abs after pulmonary deposition is critical for their development. We investigated the pharmacokinetics of a nebulized Ab by continuous sampling in lung parenchyma using microdialysis in non-human primates. We defined the optimal conditions for microdialysis of Ab and demonstrated that lung microdialysis of Ab is feasible over a period of several days. The concentration-profile indicated a two-phase non-linear elimination and/or distribution of inhaled mAbX. Lung exposition was higher than the systemic one over a period of 33 hours and above MabX affinity for its target. The microdialysis results were supported by an excellent relationship with dosages from lung extracts.     

Impact of media and antifoam selection on monoclonal antibody production and quality using a high throughput micro‐bioreactor system

Monoclonal antibody production in commercial scale cell culture bioprocessing requires a thorough understanding of the engineering process and components used throughout manufacturing. It is important to identify high impact components early on during the lifecycle of a biotechnology‐derived product. While cell culture media selection is of obvious importance to the health and productivity of mammalian bioreactor operations, other components such as antifoam selection can also play an important role in bioreactor cell culture. Silicone polymer‐based antifoams were known to have negative impacts on cell health, production, and downstream filtration and purification operations. High throughput screening in micro‐scale bioreactors provides an efficient strategy to identify initial operating parameters. Here, we utilized a micro‐scale parallel bioreactor system to study an IgG1 producing CHO cell line, to screen Dynamis, ProCHO5, PowerCHO2, EX‐Cell Advanced, and OptiCHO media, and 204, C, EX‐Cell, SE‐15, and Y‐30 antifoams and their impacts on IgG1 production, cell growth, aggregation, and process control. This study found ProCHO5, EX‐Cell Advanced, and PowerCHO2 media supported strong cellular growth profiles, with an IVCD of 25‐35 × 10⁶ cells‐d/mL, while maintaining specific antibody production (Q_p > 2 pg/cell‐d) for our model cell line and a monomer percentage above 94%. Antifoams C, EX‐Cell, and SE‐15 were capable of providing adequate control of foaming while antifoam 204 and Y‐30 noticeably stunted cellular growth. This work highlights the utility of high throughput micro bioreactors and the importance of identifying both positive and negative impacts of media and antifoam selection on a model IgG1 producing CHO cell line. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:262–270, 2018     

High-throughput methods for miniaturization and automation of monoclonal antibody purification processes

In the last decade, high-throughput downstream process development techniques have entered the biopharmaceutical industry. As chromatography is the standard downstream purification method, several high-throughput chromatographic methods have been developed and applied including miniaturized chromatographic columns for utilization on liquid handling stations. These columns were used to setup a complete downstream process on a liquid handling station for the first time. In this article, a monoclonal antibody process was established in lab-scale and miniaturized afterwards. The scale-down methodology is presented and discussed. Liquid handling in miniaturized single and multicolumn processes was improved and applicability was demonstrated by volume balances. The challenges of absorption measurement are discussed and strategies were shown to improve volume balances and mass balances in 96-well microtiter plates. The feasibility of miniaturizing a complete downstream process was shown. In the future, analytical bottlenecks should be addressed to gain the full benefit from miniaturized complete process development.     

Effect of antigen-dependent clearance on pharmacokinetics of anti-heparin-binding EGF-like growth factor (HB-EGF) monoclonal antibody

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family and is an important therapeutic target in some types of human cancers. KM3566 is a mouse anti-HB-EGF monoclonal antibody that neutralizes HB-EGF activity by inhibiting the binding of HB-EGF to its receptors. Based on the results of our pharmacokinetics study, a humanized derivative antibody, KHK2866, is rapidly cleared from serum and shows nonlinear pharmacokinetics in cynomolgus monkeys. In this study, we examined the antigen-dependent clearance of an anti-HB-EGF monoclonal antibody in vivo and in vitro in order to pharmacokinetically explain the rapid elimination of KHK2866. We revealed tumor size-dependent clearance of KM3566 in in vivo studies and obtained good fits between the observed and simulated concentrations of KM3566 based on the two-compartment with a saturable route of clearance model. Furthermore, in vivo imaging analyses demonstrated tumor-specific distribution of KM3566. We then confirmed rapid internalization and distribution to lysosome of KM3566 at a cellular level. Moreover, we revealed that the amounts of HB-EGF on cell surface membrane were maintained even while HB-EGF was internalized with KM3566. Recycled or newly synthesized HB-EGF, therefore, may contribute to a consecutive clearance of KM3566, which could explain a rapid clearance from serum. These data suggested that the rapid elimination in pharmacokinetics of KM3566 is due to antigen-dependent clearance. Given that its antigen is expressed in a wide range of normal tissue, it is estimated that the rapid elimination of KHK2866 from cynomolgus monkey serum is caused by antigen-dependent clearance.