Decisional tool to assess current and future process robustness in an antibody purification facility

Increases in cell culture titers in existing facilities have prompted efforts to identify strategies that alleviate purification bottlenecks while controlling costs. This article describes the application of a database‐driven dynamic simulation tool to identify optimal purification sizing strategies and visualize their robustness to future titer increases. The tool harnessed the benefits of MySQL to capture the process, business, and risk features of multiple purification options and better manage the large datasets required for uncertainty analysis and optimization. The database was linked to a discrete‐event simulation engine so as to model the dynamic features of biopharmaceutical manufacture and impact of resource constraints. For a given titer, the tool performed brute force optimization so as to identify optimal purification sizing strategies that minimized the batch material cost while maintaining the schedule. The tool was applied to industrial case studies based on a platform monoclonal antibody purification process in a multisuite clinical scale manufacturing facility. The case studies assessed the robustness of optimal strategies to batch‐to‐batch titer variability and extended this to assess the long‐term fit of the platform process as titers increase from 1 to 10 g/L, given a range of equipment sizes available to enable scale intensification efforts. Novel visualization plots consisting of multiple Pareto frontiers with tie‐lines connecting the position of optimal configurations over a given titer range were constructed. These enabled rapid identification of robust purification configurations given titer fluctuations and the facility limit that the purification suites could handle in terms of the maximum titer and hence harvest load. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1019–1028, 2012     

Pool‐less processing to streamline downstream purification of monoclonal antibodies

With cell culture titers and productivity increasing in the last few years, pressure has been placed on downstream purification to look at alternative strategies to meet the demand of biotech products with high dose requirements. Even when the upstream process is not continuous (perfusion based), adopting a more productive and/or continuous downstream process can be of significant advantage. Due to the recent trend in exploring continuous processing options for biomolecules, several enabling technologies have been assessed at Biogen. In this paper, we evaluate the capability of one of these technologies to streamline and improve our downstream mAb purification platform. Current conventional downstream polishing steps at Biogen are operated in flow‐through mode to achieve higher loadings while maintaining good selectivity. As titers increase, this would result in larger columns and larger intermediate product pool holding tanks. A semicontinuous downstream process linking the second and third chromatography steps in tandem can reduce/eliminate intermediate holding tanks, reduce overall processing time, and combine unit operations to reduce validation burdens. A pool‐less processing technology utilizing inline adjustment functionality was evaluated to address facility fit challenges for three high titer mAbs. Two different configurations of polishing steps were examined: (i) anion exchange and hydrophobic interaction and (ii) anion exchange and mixed mode chromatography. Initial laboratory scale proof of concept studies showed comparable performance between the batch purification process and the pool‐less process configuration.     

Comparison of batch and continuous multi‐column protein A capture processes by optimal design

Multi‐column capture processes show several advantages compared to batch capture. It is however not evident how many columns one should use exactly. To investigate this issue, twin‐column CaptureSMB, 3‐ and 4‐column periodic counter‐current chromatography (PCC) and single column batch capture are numerically optimized and compared in terms of process performance for capturing a monoclonal antibody using protein A chromatography. Optimization is carried out with respect to productivity and capacity utilization (amount of product loaded per cycle compared to the maximum amount possible), while keeping yield and purity constant. For a wide range of process parameters, all three multi‐column processes show similar maximum capacity utilization and performed significantly better than batch. When maximizing productivity, the CaptureSMB process shows optimal performance, except at high feed titers, where batch chromatography can reach higher productivity values than the multi‐column processes due to the complete decoupling of the loading and elution steps, albeit at a large cost in terms of capacity utilization. In terms of trade‐off, i.e. how much the capacity utilization decreases with increasing productivity, CaptureSMB is optimal for low and high feed titers, whereas the 3‐column process is optimal in an intermediate region. Using these findings, the most suitable process can be chosen for different production scenarios.     

Caprylic acid‐induced impurity precipitation from protein A capture column elution pool to enable a two‐chromatography‐step process for monoclonal antibody purification

This article presents the use of caprylic acid (CA) to precipitate impurities from the protein A capture column elution pool for the purification of monoclonal antibodies (mAbs) with the objective of developing a two chromatography step antibody purification process. A CA‐induced impurity precipitation in the protein A column elution pool was evaluated as an alternative method to polishing chromatography techniques for use in the purification of mAbs. Parameters including pH, CA concentrations, mixing time, mAb concentrations, buffer systems, and incubation temperatures were evaluated on their impacts on the impurity removal, high‐molecular weight (HMW) formation and precipitation step yield. Both pH and CA concentration, but not mAb concentrations and buffer systems, are key parameters that can affect host–cell proteins (HCPs) clearance, HMW species, and yield. CA precipitation removes HCPs and some HMW species to the acceptable levels under the optimal conditions. The CA precipitation process is robust at 15–25°C. For all five mAbs tested in this study, the optimal CA concentration range is 0.5–1.0%, while the pH range is from 5.0 to 6.0. A purification process using two chromatography steps (protein A capture column and ion exchange polishing column) in combination with CA‐based impurity precipitation step can be used as a robust downstream process for mAb molecules with a broad range of isoelectric points. Residual CA can be effectively removed by the subsequent polishing cation exchange chromatography. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1515–1525, 2015     

Antigen itself antibody: an alternative one‐step immunoassay for measuring the anti‐idiotypic antibody titer

Immunoassay designs rely on the great specificity of antibodies and a suitable marker that facilitates generation of a quantitative signal. Currently, there is no reliable method for measuring the titers of an anti‐idiotypic antibody. Our initial attempt to measure titers of mouse anti‐idiotypic antibody after idiotypic vaccination with HM‐1 killer toxin neutralizing monoclonal antibody (nmAb‐KT) failed. Because the injected antigen, nmAb‐KT, is a mouse IgG, using a commercial antibody to measure the antibody titer always gave a false positive signal against control mouse serum antibody in parallel with the antigen‐treated immunized serum antibodies. To get a reliable and clearly differentiable signal by ELISA, idiotypic antigen was labeled with HRP and HRP‐conjugated‐nmAb‐KT used to measure the antibody titers in the antigen‐treated mice. Compared with control mice, signals were found in high anti‐nmAb‐KT IgG responses in test mice; however, untreated control mice had a significant amount of purified non‐specific IgG. This method is amenable to long read lengths and will likely enable anti‐idiotypic antibody titer measurement in a more specific and cost effective way without requiring commercial antibody.     

Single‐step affinity purification of enzyme biotherapeutics: A platform methodology for accelerated process development

Downstream sample purification for quality attribute analysis is a significant bottleneck in process development for non‐antibody biologics. Multi‐step chromatography process train purifications are typically required prior to many critical analytical tests. This prerequisite leads to limited throughput, long lead times to obtain purified product, and significant resource requirements. In this work, immunoaffinity purification technology has been leveraged to achieve single‐step affinity purification of two different enzyme biotherapeutics (Fabrazyme® [agalsidase beta] and Enzyme 2) with polyclonal and monoclonal antibodies, respectively, as ligands. Target molecules were rapidly isolated from cell culture harvest in sufficient purity to enable analysis of critical quality attributes (CQAs). Most importantly, this is the first study that demonstrates the application of predictive analytics techniques to predict critical quality attributes of a commercial biologic. The data obtained using the affinity columns were used to generate appropriate models to predict quality attributes that would be obtained after traditional multi‐step purification trains. These models empower process development decision‐making with drug substance‐equivalent product quality information without generation of actual drug substance. Optimization was performed to ensure maximum target recovery and minimal target protein degradation. The methodologies developed for Fabrazyme were successfully reapplied for Enzyme 2, indicating platform opportunities. The impact of the technology is significant, including reductions in time and personnel requirements, rapid product purification, and substantially increased throughput. Applications are discussed, including upstream and downstream process development support to achieve the principles of Quality by Design (QbD) as well as integration with bioprocesses as a process analytical technology (PAT). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:708–717, 2014     

Single pass tangential flow filtration to debottleneck downstream processing for therapeutic antibody production

As the therapeutic monoclonal antibody (mAb) market continues to grow, optimizing production processes is becoming more critical in improving efficiencies and reducing cost-of-goods in large-scale production. With the recent trends of increasing cell culture titers from upstream process improvements, downstream capacity has become the bottleneck in many existing manufacturing facilities. Single Pass Tangential Flow Filtration (SPTFF) is an emerging technology, which is potentially useful in debottlenecking downstream capacity, especially when the pool tank size is a limiting factor. It can be integrated as part of an existing purification process, after a column chromatography step or a filtration step, without introducing a new unit operation. In this study, SPTFF technology was systematically evaluated for reducing process intermediate volumes from 2× to 10× with multiple mAbs and the impact of SPTFF on product quality, and process yield was analyzed. Finally, the potential fit into the typical 3-column industry platform antibody purification process and its implementation in a commercial scale manufacturing facility were also evaluated. Our data indicate that using SPTFF to concentrate protein pools is a simple, flexible, and robust operation, which can be implemented at various scales to improve antibody purification process capacity.     

Virus elimination during the purification of monoclonal antibodies by column chromatography and additional steps

The theoretical potential for virus transmission by monoclonal antibody based therapeutic products has led to the inclusion of appropriate virus reduction steps. In this study, virus elimination by the chromatographic steps used during the purification process for two (IgG‐1 & ?3) monoclonal antibodies (MAbs) have been investigated. Both the Protein G (>7log) and ion‐exchange (5 log) chromatography steps were very effective for eliminating both enveloped and non‐enveloped viruses over the life‐time of the chromatographic gel. However, the contribution made by the final gel filtration step was more limited, i.e., 3 log. Because these chromatographic columns were recycled between uses, the effectiveness of the column sanitization procedures (guanidinium chloride for protein G or NaOH for ion‐exchange) were tested. By evaluating standard column runs immediately after each virus spiked run, it was possible to directly confirm that there was no cross contamination with virus between column runs (guanidinium chloride or NaOH). To further ensure the virus safety of the product, two specific virus elimination steps have also been included in the process. A solvent/detergent step based on 1% triton X‐100 rapidly inactivating a range of enveloped viruses by >6 log inactivation within 1 min of a 60 min treatment time. Virus removal by virus filtration step was also confirmed to be effective for those viruses of about 50 nm or greater. In conclusion, the combination of these multiple steps ensures a high margin of virus safety for this purification process. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1341–1347, 2014     

Exploring the linkage between cell culture process parameters and downstream processing utilizing a plackett‐burman design for a model monoclonal antibody

Linkage of upstream cell culture with downstream processing and purification is an aspect of Quality by Design crucial for efficient and consistent production of high quality biopharmaceutical proteins. In a previous Plackett‐Burman screening study of parallel bioreactor cultures we evaluated main effects of 11 process variables, such as agitation, sparge rate, feeding regimens, dissolved oxygen set point, inoculation density, supplement addition, temperature, and pH shifts. In this follow‐up study, we observed linkages between cell culture process parameters and downstream capture chromatography performance and subsequent antibody attributes. In depth analysis of the capture chromatography purification of harvested cell culture fluid yielded significant effects of upstream process parameters on host cell protein abundance and behavior. A variety of methods were used to characterize the antibody both after purification and buffer formulation. This analysis provided insight in to the significant impacts of upstream process parameters on aggregate formation, impurities, and protein structure. This report highlights the utility of linkage studies in identifying how changes in upstream parameters can impact downstream critical quality attributes. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:163–170, 2017     

Purification of recombinant protein by cold‐coacervation of fusion constructs incorporating resilin‐inspired polypeptides

Polypeptides containing between 4 and 32 repeats of a resilin‐inspired sequence AQTPSSYGAP, derived from the mosquito Anopheles gambiae, have been used as tags on recombinant fusion proteins. These repeating polypeptides were inspired by the repeating structures that are found in resilins and sequence‐related proteins from various insects. Unexpectedly, an aqueous solution of a recombinant resilin protein displays an upper critical solution temperature (cold‐coacervation) when held on ice, leading to a separation into a protein rich phase, typically exceeding 200 mg/mL, and a protein‐poor phase. We show that purification of recombinant proteins by cold‐coacervation can be performed when engineered as a fusion partner to a resilin‐inspired repeat sequence. In this study, we demonstrate the process by the recombinant expression and purification of enhanced Green fluorescent protein (EGFP) in E. coli. This facile purification system can produce high purity, concentrated protein solutions without the need for affinity chromatography or other time‐consuming or expensive purification steps, and that it can be used with other bulk purification steps such as low concentration ammonium sulfate precipitation. Protein purification by cold‐coacervation also minimizes the exposure of the target protein to enhanced proteolysis at higher temperature. Biotechnol. Bioeng. 2012; 109: 2947–2954. © 2012 Wiley Periodicals, Inc.     

Polishing approach with fully connected flow‐through purification for therapeutic monoclonal antibody

The biopharmaceutical industry is evolving toward process intensification that can offer increased productivity and improved economics without sacrificing process robustness. A semi‐continuous downstream process linking purification/polishing unit operations in series can reduce or eliminate intermediate holding tanks and reduce overall processing time. Accordingly, we have developed a therapeutic monoclonal antibody polishing template comprised of a connected flow‐through polishing technologies that include activated carbon, cation exchange, and anion‐exchange chromatography. In this report, we evaluated fully‐connected pool‐less polishing with three flow‐through technologies, operating as a single skid to streamline and improve an mAb purification platform. Laboratory‐scale pool‐less processing was achieved without utilizing in‐line pH adjustment and conductivity dilution based on the previously optimized single process parameter. Two connected flow‐through configurations of polishing steps were evaluated: a two‐step process using anion exchange and cation exchange and a three step process using activated carbon, anion exchange and cation exchange chromatography. Laboratory‐scale proof of concept studies showed comparable performance between the batch purification process and the pool‐less process configuration. Three step polishing highly intensified the processes and provided higher process loading and achieved bulk drug specification with higher impurity clearance (>95%) and high overall mAb yield (>95%).     

Multipronged approach to managing beta‐glucan contaminants in the downstream process: Control of raw materials and filtration with charge‐modified nylon 6,6 membrane filters

(1→3)‐β‐d ‐Glucans (beta‐glucans) have been found in raw materials used in the manufacture of recombinant therapeutics. Because of their biological activity, beta‐glucans are considered process contaminants and consequently their level in the product needs to be controlled. Although beta‐glucans introduced into the cell culture process can readily be removed by bind‐and‐elute chromatography process steps, beta‐glucans can also be introduced into the purification process through raw materials containing beta‐glucans as well as leachables from filters made from cellulose. This article reports a multipronged approach to managing the beta‐glucan contamination in the downstream process. Raw material screening and selection can be used to effectively limit the level of beta‐glucan introduced into the downstream process. Placement of a cellulosic filter upstream of the last bind‐and‐elute column step or effective preuse flushing can also limit the level of contaminant introduced. More importantly, this article reports the active removal of beta‐glucan from the downstream process when necessary. It was discovered that the Posidyne^® filter, a charge‐modified nylon 6,6 membrane filter, was able to effectively remove beta‐glucans from buffers at relatively low pH and salt concentrations. An approach of using low beta‐glucan buffer components combined with filtration of the buffer with a Posidyne membrane has been successfully demonstrated at preparative scale. Additionally, the feasibility of active removal of beta‐glucan from in‐process product pools by Posidyne membrane filtration has also been demonstrated. Based on the data presented, a mechanism for binding is proposed, as well as a systematic approach for sizing of the Posidyne filter. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:672–680, 2013     

Clarification and capture of high‐concentration refold pools for E. coli‐based therapeutics using expanded bed adsorption chromatography

Expanded bed adsorption (EBA) chromatography was investigated for clarification and capture of high‐concentration refold pools of Escherichia coli‐based therapeutics. Refolding of denatured inclusion bodies (IBs) at high protein concentration significantly improved product throughput; however, direct filtration of the refold materials became very challenging because of high content of protein precipitates formed during refolding. In addition, irreversible protein precipitation caused by high local concentration was encountered in packed bed capture during cation exchange chromatography elution, which limited column loading capacity and capture step productivity. In this study, the two issues are addressed in one unit operation by using EBA. Specifically, EBA can handle feed streams with significant amount of particles and precipitates, which eliminated the need for refold pool clarification through filtration. The relatively broad EBA elution profile is particularly suitable for proteins of low solubility and can effectively avoid product loss previously associated with on‐column precipitation during capture. As the EBA resin (RHOBUST^® FastLine SP IEX) used here has unique properties, it can be operated at high linear velocity (800–1,600 cm/h), while achieving a selectivity and impurity clearance largely comparable to the packed bed resin of the same ligand chemistry (SP Sepharose FF). Furthermore, the filtration of the EBA elution pool is easily manageable within facility capability. Overall, this study demonstrates that the EBA process helps debottleneck the purification of high‐turbidity refold pools by removing precipitates and concurrently capturing the product, which can be applied to other E. coli‐based therapeutics that also requires refolding of IBs. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:113–123, 2014     

Online integrity monitoring in the Protein A step of mAb Production Processes—increasing reliability and process robustness

The purification of recombinant proteins and antibodies using large packed‐bed columns is a key component in most biotechnology purification processes. Because of its efficiency and established practice in the industry, column chromatography is a state of the art technology with a proven capability for removal of impurities, viral clearance, and process efficiency. In general, the validation and monitoring of chromatographic operations—especially of critical process parameters—is required to ensure robust product quality and compliance with health authority expectations. One key aspect of chromatography that needs to be monitored is the integrity of the packed bed, since this is often critical to achieving sufficient separation of protein species. Identification of potential column integrity issues before they occur is important for both product quality and economic efficiency. In this article, we examine how transition analysis techniques can be utilized to monitor column integrity. A case study on the application of this method during a large scale Protein A capture step in an antibody purification process shows how it can assist with improving process knowledge and increasing the efficiency of manufacturing operations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:383–390, 2014     

Development of a polishing step using a hydrophobic interaction membrane adsorber with a PER.C6®‐derived recombinant antibody

Membrane chromatography has already proven to be a powerful alternative to polishing columns in flow‐through mode for contaminant removal. As flow‐through utilization has expanded, membrane chromatography applications have included the capturing of large molecules, including proteins such as IgGs. Such bind‐and‐elute applications imply the demand for high binding capacity and larger membrane surface areas as compared to flow‐through applications. Given these considerations, a new Sartobind Phenyl? membrane adsorber was developed for large‐scale purification of biomolecules based on hydrophobic interaction chromatography (HIC) principles. The new hydrophobic membrane adsorber combines the advantages of membrane chromatography—virtually no diffusion limitation and shorter processing time—with high binding capacity for proteins comparable to that of conventional HIC resins as well as excellent resolution. Results from these studies confirmed the capability of HIC membrane adsorber to purify therapeutic proteins with high dynamic binding capacities in the range of 20 mg‐MAb/cm³‐membrane and excellent impurity reduction. In addition the HIC phenyl membrane adsorber can operate at five‐ to ten‐fold lower residence time when compared to column chromatography. A bind/elute purification step using the HIC membrane adsorber was developed for a recombinant monoclonal antibody produced using the PER.C6® cell line. Loading and elution conditions were optimized using statistical design of experiments. Scale‐up is further discussed, and the performance of the membrane adsorber is compared to a traditional HIC resin used in column chromatography. Biotechnol. Bioeng. 2010; 105: 296–305. © 2009 Wiley Periodicals, Inc.     

Scale-up of monoclonal antibody purification processes

Mammalian cell culture technology has improved so rapidly over the last few years that it is now commonplace to produce multi-kilogram quantities of therapeutic monoclonal antibodies in a single batch. Purification processes need to be scaled-up to match the improved upstream productivity. In this chapter key practical issues and approaches to the scale-up of monoclonal antibody purification processes are discussed. Specific purification operations are addressed including buffer preparation, chromatography column sizing, aggregate removal, filtration and volume handling with examples given.     

Displacement to separate host‐cell proteins and aggregates in cation‐exchange chromatography of monoclonal antibodies

An efficient and consistent method of monoclonal antibody (mAb) purification can improve process productivity and product consistency. Although protein A chromatography removes most host‐cell proteins (HCPs), mAb aggregates and the remaining HCPs are challenging to remove in a typical bind‐and‐elute cation‐exchange chromatography (CEX) polishing step. A variant of the bind‐and‐elute mode is the displacement mode, which allows strongly binding impurities to be preferentially retained and significantly improves resin utilization. Improved resin utilization renders displacement chromatography particularly suitable in continuous chromatography operations. In this study we demonstrate and exploit sample displacement between a mAb and impurities present at low prevalence (0.002%–1.4%) using different multicolumn designs and recycling. Aggregate displacement depends on the residence time, sample concentration, and solution environment, the latter by enhancing the differences between the binding affinities of the product and the impurities. Displacement among the mAb and low‐prevalence HCPs resulted in an effectively bimodal‐like distribution of HCPs along the length of a multi‐column system, with the mAb separating the relatively more basic group of HCPs from those that are more acidic. Our findings demonstrate that displacement of low‐prevalence impurities along multiple CEX columns allows for selective separation of mAb aggregates and HCPs that persist through protein A chromatography.     

Development of at‐line assay to monitor charge variants of MAbs during production

One major challenge currently facing the biopharmaceutical industry is to understand how MAb microheterogeneity affects therapeutic efficacy, potency, immunogenicity, and clearance. MAb micro‐heterogeneity can result from post‐translational modifications such as sialylation, galactosylation, C‐terminal lysine cleavage, glycine amidation, and tryptophan oxidation, each of which can generate MAb charge variants; such heterogeneity can affect pharmacokinetics (PK) considerably. Implementation of appropriate on‐line quality control strategies may help to regulate bioprocesses, thus enabling more homogenous material with desired post‐translational modifications and PK behavior. However, one major restriction to implementation of quality control strategies is the availability of techniques for obtaining on‐line or at‐line measurements of these attributes. In this work, we describe the development of an at‐line assay to separate MAb charge variants in near real‐time, which could ultimately be used to implement on‐line quality control strategies for MAb production. The assay consists of a 2D‐HPLC method with sequential in‐line Protein A and WCX‐10 HPLC column steps. To perform the 2D‐HPLC assay at‐line, the two columns steps were integrated into a single method using a novel system configuration that allowed parallel flow over column 1 or column 2 or sequential flow from column 1 to column 2. A bioreactor system was also developed such that media samples could be removed automatically from bioreactor vessels during production and delivered to the 2D‐HPLC for analysis. With this at‐line HPLC assay, we have demonstrated that MAb microheterogeneity occurs throughout the cell cycle whether the host cell line is grown under different or the same nominal culture conditions. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:249–255, 2014     

Ultra scale‐down approach to correct dispersive and retentive effects in small‐scale columns when predicting larger scale elution profiles

Ultra scale‐down approaches represent valuable methods for chromatography development work in the biopharmaceutical sector, but for them to be of value, scale‐down mimics must predict large‐scale process performance accurately. For example, one application of a scale‐down model involves using it to predict large‐scale elution profiles correctly with respect to the size of a product peak and its position in a chromatogram relative to contaminants. Predicting large‐scale profiles from data generated by small laboratory columns is complicated, however, by differences in dispersion and retention volumes between the two scales of operation. Correcting for these effects would improve the accuracy of the scale‐down models when predicting outputs such as eluate volumes at larger scale and thus enable the efficient design and operation of subsequent steps. This paper describes a novel ultra scale‐down approach which uses empirical correlations derived from conductivity changes during operation of laboratory and pilot columns to correct chromatographic profiles for the differences in dispersion and retention. The methodology was tested by using 1 mL column data to predict elution profiles of a chimeric monoclonal antibody obtained from Protein A chromatography columns at 3 mL laboratory‐ and 18.3 L pilot‐scale. The predictions were then verified experimentally. Results showed that the empirical corrections enabled accurate estimations of the characteristics of larger‐scale elution profiles. These data then provide the justification to adjust small‐scale conditions to achieve an eluate volume and product concentration which is consistent with that obtained at large‐scale and which can then be used for subsequent ultra scale‐down operations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Isolation and characterization of a fraction from brain that inhibits 1,4-[3H]dihydropyridine binding and L-type calcium channel current

Bovine brain was subjected to acid extraction and several purification steps. A fraction from brain that eluted from C18 reverse-phase columns at 30-35% acetonitrile inhibited [3H]nitrendipine binding to cardiac membranes. Further purification of this fraction on a sizing column in the presence of 40% acetonitrile yielded a low molecular mass fraction (less than 1 kDa) that produced a time- and voltage-dependent inhibition of L-type (but not T-type) Ca2+-channel current in GH3 cells. The results suggest that this fraction contains an endogenous substance that binds directly to slowly-inactivating Ca2+ channels and thereby inhibits current flow.