Efficient MILP formulations for the optimal synthesis of chromatographic protein purification processes

The use of recombinant proteins has increased greatly in recent years, as well as the techniques used for their purification. The selection of an efficient process to purify proteins is a major bottleneck found when trying to scale up results obtained in the laboratory to a large-scale industrial process. One of the main challenges in the synthesis of downstream purification stages in biotechnological processes is the appropriate selection and sequencing of chromatographic steps. The objective of this work is to develop mixed integer linear programming models for the synthesis of protein purification processes. Models for each chromatographic technique rely on physicochemical data of a protein mixture, which contains the desired product and provide information on its potential purification. Formulations that are based on convex hull representations are proposed to calculate the minimum number of steps from a set of chromatographic techniques that must achieve a specified purity level and alternatively to maximize purity for a given number of steps. The proposed models are tested in several examples with experimental data and present time reductions of up to three orders of magnitude when compared to big-M formulations.     

MINLP models for the synthesis of optimal peptide tags and downstream protein processing

The development of systematic methods for the synthesis of downstream protein processing operations has seen growing interest in recent years, as purification is often the most complex and costly stage in biochemical production plants. The objective of the work presented here is to develop mathematical models based on mixed integer optimization techniques, which integrate the selection of optimal peptide purification tags into an established framework for the synthesis of protein purification processes. Peptide tags are comparatively short sequences of amino acids fused onto the protein product, capable of reducing the required purification steps. The methodology is illustrated through its application on two example protein mixtures involving up to 13 contaminants and a set of 11 candidate chromatographic steps. The results are indicative of the benefits resulting by the appropriate use of peptide tags in purification processes and provide a guideline for both optimal tag design and downstream process synthesis.     

Optimal synthesis of chromatographic trains for downstream protein processing

Downstream bioprocessing and especially chromatographic steps, commonly used for the purification of multicomponent systems, are significant cost drivers in the production of therapeutic proteins. There has been an increased interest in the development of systematic methods for the design of such processes, and the appropriate selection of a series of chromatographic steps is still a major challenge to be addressed. Several models have been developed previously but have assumed that 100% recovery of the desired product is obtained at each chromatographic step. In this work, a mathematical framework is proposed, based on mixed integer optimisation techniques, that removes this assumption and allows full flexibility on the position of retention time cut-points, between which the desired product fraction is collected. The proposed model is demonstrated on three example protein mixtures, each containing up to 13 contaminants and selecting from a set of up to 21 candidate steps. The proposed model results in a reduction of one to three chromatographic steps over solutions that no losses are allowed.     

Is there a rational method to purify proteins? From expert systems to proteomics

The purification of recombinant proteins for therapeutic or analytical applications requires the use of several chromatographic steps in order to achieve a high level of purity. A range of techniques is available such as anion and cation exchange chromatography, which can be carried out at different pHs, and hence used at different steps, hydrophobic interaction chromatography, gel filtration and affinity chromatography. Evidently when confronted with a complex mixture of partially unknown proteins or a clarified cell extract there are many different routes one can take in order to choose the minimum and most efficient number of purification steps to achieve a desired level of purity (e.g. 98, 99.5 or 99.9%). In this review we will show how an initial "proteomic" characterization of the complex initial mixture of target protein and protein contaminants can be used to select the most efficient chromatographic separation steps in order to achieve a maximum level of purity with a minimum number of steps. The chosen methodology was implemented in a computer based expert system. The first algorithm developed was used to select the most efficient purification method to separate a protein from its contaminants based on the physicochemical properties of the protein product and the protein contaminants. The second algorithm developed was used to predict the number and concentration of contaminants after each separation as well as protein product purity. The successful application of the expert system approach, based on an initial proteomic characterization, to the practical cases of protein mixtures and clarified fermentation supernatant is presented and discussed. The purification strategy proposed was experimentally tested and validated with a mixture of four proteins and the experimental validation was also carried out with an "unknown" supernatant of Bacillus subtilis producing a recombinant beta-1,3-glucanase. The system was robust to errors <10% which is the range that can be found in the experimental determination of the properties in the database of product and contaminants. On the other hand, the system was sensitive both to larger variations (>20%) in the properties of the contaminant database and the protein product and to variations in one protein property (e.g. hydrophobicity).     

Protein purification using chromatography: selection of type, modelling and optimization of operating conditions

To achieve a high level of purity in the purification of recombinant proteins for therapeutic or analytical application, it is necessary to use several chromatographic steps. There is a range of techniques available including anion and cation exchange, which can be carried out at different pHs, hydrophobic interaction chromatography, gel filtration and affinity chromatography. In the case of a complex mixture of partially unknown proteins or a clarified cell extract, there are many different routes one can take in order to choose the minimum and most efficient number of purification steps to achieve a desired level of purity (e.g. 98%, 99.5% or 99.9%). This review shows how an initial 'proteomic' characterization of the complex mixture of target protein and protein contaminants can be used to select the most efficient chromatographic separation steps in order to achieve a specific level of purity with a minimum number of steps. The chosen methodology was implemented in a computer- based Expert System. Two algorithms were developed, the first algorithm was used to select the most efficient purification method to separate a protein from its contaminants based on the physicochemical properties of the protein product and the protein contaminants and the second algorithm was used to predict the number and concentration of contaminants after each separation as well as protein product purity. The application of the Expert System approach was experimentally tested and validated with a mixture of four proteins and the experimental validation was also carried out with a supernatant of Bacillus subtilis producing a recombinant beta-1,3-glucanase. Once the type of chromatography is chosen, optimization of the operating conditions is essential. Chromatographic elution curves for a three-protein mixture (alpha-lactoalbumin, ovalbumin and beta-lactoglobulin), carried out under different flow rates and ionic strength conditions, were simulated using two different mathematical models. These models were the Plate Model and the more fundamentally based Rate Model. Simulated elution curves were compared with experimental data not used for parameter identification. Deviation between experimental data and the simulated curves using the Plate Model was less than 0.0189 (absorbance units); a slightly higher deviation [0.0252 (absorbance units)] was obtained when the Rate Model was used. In order to optimize operating conditions, a cost function was built that included the effect of the different production stages, namely fermentation, purification and concentration. This cost function was also successfully used for the determination of the fraction of product to be collected (peak cutting) in chromatography. It can be used for protein products with different characteristics and qualities, such as purity and yield, by choosing the appropriate parameters.     

Experimental approaches to guarantee minimal risk of potential virus in purified monoclonal antibodies

Regarding biological products, increasing awareness of potential side effects have placed great importance not only at protein purity regarding other proteins but on the removal of biologicals such as DNA and especially virus the importance of which may not be known. Monoclonal antibodies (Mab) have come to be an important class of molecules obtained from hybridoma cells, i.e., nonrecombinant cells in culture. It has been noted during the last years, that with rare exceptions hybridoma cell lines contain retrovirus like particles. The infectious nature of the EM-visible particles has been tested for, however, in most cases not been substantiated. In order to bring these valuable biological reagents, Mab's, to good use in man for imaging or therapy, the remaining concern about a potential retroviral infection has to be reduced to an acceptable minimum. We describe experimental approaches for the validation of chromatographic and ultrafiltration steps used in the production of monoclonal antibodies to remove and inactivate murine retrovirus. Present day biotechnological manufacturing processes have been devised incorporating a number of strategic preventive measures that have found wide spread acceptance. They permit to answer the question: how can a potentially harmful infection by an unknown virus be excluded. Knowledge of the efficacy of purification steps to clear infectious model virus is fundamental to devise biotechnological manufacturing processes yielding a purified antibody for use in man.     

正相和反相柱层析组合分离纯化紫杉醇

采用正相氧化铝柱层析和反相C₁₈柱层析从东北红豆杉培养细胞浸提物中分离纯化了紫杉醇。优化了氧化铝柱层析和反相柱层析的操作条件。实验发现,经过氧化铝柱层析后,测得的紫杉醇量大大增加。经两步层析,使紫杉醇的含量从小于1.0%提高到95%,样品中微量杂质继以重结晶步骤除去,即可获得纯度超过98%的紫杉醇晶体。采用13-CNMR对晶体分析,所得产物结构与文献上紫杉醇的结构一致。     

Quantifying process tradeoffs in the operation of chromatographic sequences

A method for the rapid representation of key process tradeoffs that need to be made during the analysis of chromatographic sequences has been proposed. It involves the construction of fractionation and maximum purification factor versus yield diagrams, which can be completed easily on the basis of chromatographic data. The output of the framework developed reflects the degree of tradeoff between levels of yield and purity and provides a fast and precise prediction of the sample fraction collection strategy needed to meet a desired process specification. The usefulness of this approach for the purposes of product purification and contaminant removal in a single chromatographic step has been successfully demonstrated in an earlier paper and it is now extended by application to a chromatographic sequence: the separation of a hypothetical three-component protein system by hydrophobic interaction chromatography (HIC) followed by size exclusion chromatography (SEC). The HIC operation has a strong impact upon the subsequent SEC step. The studies show how the analysis of performance in such a chromatographic sequence can be carried out easily and in a straightforward fashion using the fractionation diagram approach. The methodology proposed serves as a useful tool for identifying the process tradeoffs that must be made during operation of a sequence of chromatographic steps and indicates the impact on further processing of the cut-point decisions that are made.     

Strategy for a protein purification design using C-phycocyanin extract

A variety of techniques have been developed for the separation and recovery of proteins. The cost of purifying the product is frequently determined by the desired quality of the final product, which is evaluated by measuring the purity. In this work the design of a protein purification process for C-phycocyanin, a phycobiliprotein that can be used in the food and medical industries, was established. The study evaluated the use of ammonium sulfate precipitation, ion exchange chromatography and gel filtration to purify C-phycocyanin in a variety of sequences. The final design included the C-phycocyanin extraction step, precipitation with ammonium sulfate and ion exchange chromatography. When the elution step was studied, the kind of elution and pH were considered in order to obtain a product with a final purity of 4.0 with a purification factor of 6.35, so that, at the end of the strategy, C-phycocyanin of analytical grade would be obtained.     

Extension of the selection of protein chromatography and the rate model to affinity chromatography

The rational selection of optimal protein purification sequences, as well as mathematical models that simulate and allow optimization of chromatographic protein purification processes have been developed for purification procedures such as ion-exchange, hydrophobic interaction and gel filtration chromatography. This paper investigates the extension of such analysis to affinity chromatography both in the selection of chromatographic processes and in the use of the rate model for mathematical modelling and simulation. Two affinity systems were used: Blue Sepharose and Protein A. The extension of the theory developed previously for ion-exchange and HIC chromatography to affinity separations is analyzed in this paper. For the selection of operations two algorithms are used. In the first, the value of η, which corresponds to the efficiency (resolution) of the actual chromatography and, Σ, which determines the amount of a particular contaminant eliminated after each separation step, which determines the purity, have to be determined. It was found that the value of both these parameters is not generic for affinity separations but will depend on the type of affinity system used and will have to be determined on a case by case basis. With Blue Sepharose a salt gradient was used and with Protein A, a pH gradient. Parameters were determined with individual proteins and simulations of the protein mixtures were done. This approach allows investigation of chromatographic protein purification in a holistic manner that includes ion-exchange, HIC, gel filtration and affinity separations for the first time.     

Continuous preparative liquid chromatography in the downstrem processing of biotechnological products

Continuous counter‐current chromatographic processes have been successfully used in the petrochemical and sugar industry over the last 30 years. Only recently has simulated moving bed (SMB)‐technology attracted widespread interest in the pharmaceutical industry, mainly as a very efficient system for chromatographic enantioseparation. The application of this technique to the downstream processing of biotechnological products requires some specific changes to meet the special demands of bioproduct isolation. Production processes are set up on an multi‐ton scale, for example, for the purification of fructose with both yield and purity higher than 90%. Examples for other mono‐ and oligosaccharides are reported. In the purification of fatty acids or fat soluble vitamins, SMB technology under supercritical fluid conditions gives additional benefits and increases the productivity by a factor of four when a pressure gradient is applied. Another field of operation is the isolation of drug compounds from natural sources where different batch‐ and SMB‐chromatographic steps could be successfully combined. First examples are reported for cyclosporine A and paclitaxel isolation. Finally, step‐gradient elution modes can be used continuously, as demonstrated for the isolation of monoclonal antibodies.     

A simple method for large-scale purification of plasma-derived apo-transferrin

We investigated and optimized a purification process, suitable for industrial scale, to obtain pharmaceutical grade apo-Tf (apo-transferrin), preserving its physiological properties and functions. Apo-Tf was obtained from fraction IV subfraction 1 and IV subfraction 4 (fraction IV-1,4), a waste product of the Cohn fractionation process, performing a single chromatographic run and two viral inactivation/removal steps. The structural integrity and the biological activity of the final product were extensively tested. The yield of apo-Tf produced was 80% on laboratory scale and 90% in scale-up lots, and the purity was higher than 95%. The purified protein preserves iron- and receptor-binding activities and shows a normal glycosylation pattern. The single chromatographic step process presented here provides an efficient means to prepare commercial quantities of the protein. The final product is sterile and two viral inactivation/removal steps were introduced into the process.     

Purification of a synthetic oligonucleotide by anion exchange chromatography: method optimisation and scale-up

A single-step chromatographic method for purification of a synthetic 20-mer oligonucleotide is described. Method optimisation was conducted at laboratory scale where 30 mg crude sample was purified per run with a yield of 17 mg pure oligonucleotide. The protocol was scaled-up in steps to achieve 5-, 58- and a final 230-fold scale-up. At the final scale, 7.0 g of crude material was purified with a yield of 4.1 g product. The purity of the oligonucleotide was in all scales higher than 97%. The cycle time was 110 min, which corresponds to a purification capacity of about 90 g crude oligonucleotide material per 24 h.     

Automated, High Productivity Purification and Analysis of Synthetic Peptides

Methods for peptide assembly consist of techniques that allow for construction of complex sequences. The advantage of solid-phase methodologies is automation of the repetitive processes of deprotecting, washing, and coupling protected amino acids (acylation). However, for difficult sequences the crude product contains a variety of side products that must be removed to provide the desired target peptide in sufficient concentration and purity. This paper illustrates that high efficiency purification method-development can be achieved by combining purification and analysis on a single platform. Incorporation of fast LC-based assays using polystyrene-based POROS® Perfusion Chromatography media permitted rapid overall processing times from crude peptide purification through fraction pooling and product verification. Application of these technologies to the purification of peptides at scales of 100 mg is demonstrated.     

Automated,High Productivity Purification and Analysis of Synthetic Peptides

Methods for peptide assembly consist of techniques that allow for construction of complex sequences. The advantage of solid-phase methodologies is automation of the repetitive processes of deprotecting, washing, and coupling protected amino acids (acylation). However, for difficult sequences the crude product contains a variety of side products that must be removed to provide the desired target peptide in sufficient concentration and purity. This paper illustrates that high efficiency purification method-development can be achieved by combining purification and analysis on a single platform. Incorporation of fast LC-based assays using polystyrene-based POROS® Perfusion Chromatography media permitted rapid overall processing times from crude peptide purification through fraction pooling and product verification. Application of these technologies to the purification of peptides at scales of 100 mg is demonstrated.     

Expression, purification, and molecular analysis of the Necator americanus glutathione S-transferase 1 (Na-GST-1): a production process developed for a lead candidate recombinant hookworm vaccine antigen

The enzyme Necator americanus glutathione S-transferase 1 (Na-GST-1) belongs to a unique Nu class of GSTs and is a lead candidate antigen in a bivalent human hookworm vaccine. Here we describe the expression of Na-GST-1 in the yeast Pichia pastoris at the 20 L manufacturing scale and its purification process performed by three chromatographic steps, comprised of a Q Sepharose XL anion exchange column, followed by a Butyl Sepharose HP hydrophobic affinity column and a Superdex 75 size-exclusion column. Approximately 1.5 g of recombinant protein was recovered at an overall process yield of 51%, with a purity grade of 98% and the absence of detectable host cell protein. By mass spectrometry the recombinant protein exhibits a mass of 23,676Da, which closely matches the predicted molecular mass of the protein. The expression and purification methods described here are suitable for further scale-up product development and for its use to design formulation processes suitable to generate a vaccine for clinical testing.     

Arylsulfonyltetrazoles, new coupling reagents and further improvements in the triester method for the synthesis of deoxyribooligonucleotides.

The modified triester approach has been further improved and refined to the synthesis of defined sequences of deoxyribo-oligonucleotides. Improvements include arylsulfonyltetrazoles as faster and milder condensing agents, benzenesulfonic acid to avoid depurination during deblocking of trityl protecting groups and improved chromatographic procedures for purification of triester intermediates and purification of the final product containing 3'-5' phosphodiester linkages.     

A combined screening and in silico strategy for the rapid design of integrated downstream processes for process and product-related impurity removal

Integrated designs of chromatographic processes for purification of biopharmaceuticals provides potential gains in operational efficiency and reductions of costs and material requirements. We describe a combined method using screening and in silico algorithms for ranking chromatographic steps to rapidly design orthogonally selective integrated processes for purifying protein therapeutics from both process- and product-related impurities. IFN-α2b produced in Pichia pastoris containing a significant product variant challenge was used as a case study. The product and product-related variants were screened on a set of 14 multimodal, ion exchange, and hydrophobic charge induction chromatography resins under various pH and salt linear gradient conditions. Data generated from reversed-phase chromatography of the fractions collected were used to generate a retention database for IFN-α2b and its variants. These data, in combination with a previously constructed process-related impurity database for P. pastoris, were input into an in silico process development tool that generated and ranked all possible integrated chromatographic sequences for their ability to remove both process and product-related impurities. Top-ranking outputs guided the experimental refinement of two successful three step purification processes, one comprising all bind-elute steps and the other having two bind-elute steps and a flowthrough operation. This approach suggests a new platform-like approach for rapidly designing purification processes for a range of proteins where separations of both process- and product-related impurities are needed.     

Current perspectives on stability of protein drug products during formulation, fill and finish operations

Commercialization of protein-based therapeutics is a challenging task in part due to the difficulties in maintaining protein solutions safe and efficacious throughout the drug product development process, storage, transportation and patient administration. Bulk drug substance goes through a series of formulation, fill and finish operations to provide the final dosage form in the desired formulation and container or delivery device. Different process parameters during each of these operations can affect the purity, activity and efficacy of the final product. Common protein degradation pathways and the various physical and chemical factors that can induce such reactions have been extensively studied for years. This review presents an overview of the various formulation-fill-finish operations with a focus on processing steps and conditions that can impact product quality. Various manufacturing operations including bulk freeze-thaw, formulation, filtration, filling, lyophilization, inspection, labeling, packaging, storage, transport and delivery have been reviewed. The article highlights our present day understanding of protein instability issues during biopharmaceutical manufacturing and provides guidance on process considerations that can help alleviate these concerns.     

Hybrid optimization of preparative chromatography for a ternary monoclonal antibody mixture

In the purification of monoclonal antibodies, ion-exchange chromatography is typically used among the polishing steps to reduce the amount of product-related impurities such as aggregates and fragments, whilst simultaneously reducing HCP, residual Protein A and potential toxins and viruses. When the product-related impurities are difficult to separate from the products, the optimization of these chromatographic steps can be complex and laborious. In this paper, we optimize the polishing chromatography of a monoclonal antibody from a challenging ternary feed mixture by introducing a hybrid approach of the simplex method and a form of local optimization. To maximize the productivity of this preparative bind-and-elute cation-exchange chromatography, wide ranges of the three critical operational parameters—column loading, the initial salt concentration, and gradient slope—had to be considered. The hybrid optimization approach is shown to be extremely effective in dealing with this complex separation that was subject to multiple constraints based on yield, purity, and product breakthrough. Furthermore, it enabled the generation of a large knowledge space that was subsequently used to study the sensitivity of the objective function. Increased design space understanding was gained through the application of Monte Carlo simulations. Hence, this work proposes a powerful hybrid optimization method, applied to an industrially relevant process development challenge. The properties of this approach and the results and insights gained, make it perfectly suited for the rapid development of biotechnological unit operations during early-stage bioprocess development.