Introduction of a process mass intensity metric for biologics.

Biopharmaceuticals (or biologics), large molecule therapeutics typically produced using biotechnology, are a rapidly growing segment of the pharmaceutical market. As such, the environmental footprint of the production of these molecules is coming under scrutiny from various stakeholders such as healthcare providers, investors, and even employees. Process mass intensity (PMI), originally adopted for small molecules by the Green Chemistry Institute Pharmaceutical Roundtable, is a simple metric that can also be applied to evaluate the process efficiency of biopharmaceutical production. PMI for biologics is defined as the total mass input in kg of water, raw materials and consumables, required to make 1 kg of active pharmaceutical ingredient. Six large pharmaceutical companies participated in a benchmarking exercise to calculate the PMI for monoclonal antibody (mAb) production. On average, 7700 kg of input is required to produce 1 kg of mAb. Over 90% of the mass is due to water use, highlighting the water-intensive nature of biologics production.

Integrated continuous bioprocessing: Economic, operational, and environmental feasibility for clinical and commercial antibody manufacture.

This paper presents a systems approach to evaluating the potential of integrated continuous bioprocessing for monoclonal antibody (mAb) manufacture across a product's lifecycle from preclinical to commercial manufacture. The economic, operational, and environmental feasibility of alternative continuous manufacturing strategies were evaluated holistically using a prototype UCL decisional tool that integrated process economics, discrete-event simulation, environmental impact analysis, operational risk analysis, and multiattribute decision-making. The case study focused on comparing whole bioprocesses that used either batch, continuous or a hybrid combination of batch and continuous technologies for cell culture, capture chromatography, and polishing chromatography steps. The cost of goods per gram (COG/g), E-factor, and operational risk scores of each strategy were established across a matrix of scenarios with differing combinations of clinical development phase and company portfolio size. The tool outputs predict that the optimal strategy for early phase production and small/medium-sized companies is the integrated continuous strategy (alternating tangential flow filtration (ATF) perfusion, continuous capture, continuous polishing). However, the top ranking strategy changes for commercial production and companies with large portfolios to the hybrid strategy with fed-batch culture, continuous capture and batch polishing from a COG/g perspective. The multiattribute decision-making analysis highlighted that if the operational feasibility was considered more important than the economic benefits, the hybrid strategy would be preferred for all company scales. Further considerations outside the scope of this work include the process development costs required to adopt continuous processing. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 33:854-866, 2017.

Systematic Investigation of EDC/sNHS-Mediated Bioconjugation Reactions for Carboxylated Peptide Substrates.

1-Ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) bioconjugations have been utilized in preparing variants for medical research. While there have been advances in optimizing the reaction for aqueous applications, there has been limited focus toward identifying conditions and side reactions that interfere with product formation. We present a systematic investigation of EDC/N-hydroxysulfosuccinimide (sNHS)-mediated bioconjugations on carboxylated peptides and small proteins. We identified yet-to-be-reported side products arising from both the reagents and substrates. Model peptides used in this study illustrate particular substrates are more susceptible to side reactions than others. From our studies, we found that bioconjugations are more efficient with high concentrations of amine nucleophile but not sNHS. Performing bioconjugations on a model affibody protein show that the trends established with model peptides hold for more complex systems.

N-terminal mono-PEGylation of growth hormone antagonist: correlation of PEG size and pharmacodynamic behavior.

Growth hormone antagonist (GHA), an analog of growth hormone (GH), can inhibit GH action and treat acromegaly. However, GHA suffers from a short plasma half-life of 15-20 min that has limited its clinical application. PEGylation, conjugation with polyethylene glycol (PEG), can increase the plasma half-life of GHA. Single PEG attachment (mono-PEGylation) at N-terminus of GHA has the advantages of product homogeneity and minimization of the bioactivity loss. Conjugation of large PEG molecule may increase the plasma half-life but could potentially decrease the bioactivity of GHA, due to the steric shielding effect of PEG. Thus, N-terminal mono-PEGylation of GHA with 20 kDa and 40 kDa PEG were used to look for a balance of the two competing factors. Sedimentation velocity analysis suggested that 40 kDa PEG was more efficient than 20 kDa PEG to elongate the molecular shape of the conjugate. As reflected by marginal suppression of insulin-like growth factor I (IGF-I), GHA conjugated with 40 kDa PEG was statistically indistinguishable from the saline solution that could not inhibit GH action. In contrast, GHA conjugated with 20kDa PEG can apparently inhibit GH action, as reflected by IGF-I suppression of 30-43%. Thus, our work demonstrated the effective therapeutic potency of N-terminally mono-PEGylated GHA.

Optimising the design and operation of semi-continuous affinity chromatography for clinical and commercial manufacture.

This paper presents an integrated experimental and modelling approach to evaluate the potential of semi-continuous chromatography for the capture of monoclonal antibodies (mAb) in clinical and commercial manufacture. Small-scale single-column experimental breakthrough studies were used to derive design equations for the semi-continuous affinity chromatography system. Verification runs with the semi-continuous 3-column and 4-column periodic counter current (PCC) chromatography system indicated the robustness of the design approach. The product quality profiles and step yields (after wash step optimisation) achieved were comparable to the standard batch process. The experimentally-derived design equations were incorporated into a decisional tool comprising dynamic simulation, process economics and sizing optimisation. The decisional tool was used to evaluate the economic and operational feasibility of whole mAb bioprocesses employing PCC affinity capture chromatography versus standard batch chromatography across a product's lifecycle from clinical to commercial manufacture. The tool predicted that PCC capture chromatography would offer more significant savings in direct costs for early-stage clinical manufacture (proof-of-concept) (∼30%) than for late-stage clinical (∼10-15%) or commercial (∼5%) manufacture. The evaluation also highlighted the potential facility fit issues that could arise with a capture resin (MabSelect) that experiences losses in binding capacity when operated in continuous mode over lengthy commercial campaigns. Consequently, the analysis explored the scenario of adopting the PCC system for clinical manufacture and switching to the standard batch process following product launch. The tool determined the PCC system design required to operate at commercial scale without facility fit issues and with similar costs to the standard batch process whilst pursuing a process change application. A retrofitting analysis established that the direct cost savings obtained by 8 proof-of-concept batches would be sufficient to pay back the investment cost of the pilot-scale semi-continuous chromatography system.

Fed-batch and perfusion culture processes: economic, environmental, and operational feasibility under uncertainty.

This article evaluates the current and future potential of batch and continuous cell culture technologies via a case study based on the commercial manufacture of monoclonal antibodies. The case study compares fed-batch culture to two perfusion technologies: spin-filter perfusion and an emerging perfusion technology utilizing alternating tangential flow (ATF) perfusion. The operational, economic, and environmental feasibility of whole bioprocesses based on these systems was evaluated using a prototype dynamic decision-support tool built at UCL encompassing process economics, discrete-event simulation and uncertainty analysis, and combined with a multi-attribute decision-making technique so as to enable a holistic assessment. The strategies were compared across a range of scales and titres so as to visualize how their ranking changes in different industry scenarios. The deterministic analysis indicated that the ATF perfusion strategy has the potential to offer cost of goods savings of 20% when compared to conventional fed-batch manufacturing processes when a fivefold increase in maximum viable cell densities was assumed. Savings were also seen when the ATF cell density dropped to a threefold increase over the fed-batch strategy for most combinations of titres and production scales. In contrast, the fed-batch strategy performed better in terms of environmental sustainability with a lower water and consumable usage profile. The impact of uncertainty and failure rates on the feasibility of the strategies was explored using Monte Carlo simulation. The risk analysis results demonstrated the enhanced robustness of the fed-batch process but also highlighted that the ATF process was still the most cost-effective option even under uncertainty. The multi-attribute decision-making analysis provided insight into the limited use of spin-filter perfusion strategies in industry. The resulting sensitivity spider plots enabled identification of the critical ratio of weightings of economic and operational benefits that affect the choice between ATF perfusion and fed-batch strategies.

Microcosmic mechanisms for protein incomplete release and stability of various amphiphilic mPEG-PLA microspheres.

The microcosmic mechanisms of protein (recombinant human growth hormone, rhGH) incomplete release and stability from amphiphilic poly(monomethoxypolyethylene glycol-co-D,L-lactide) (mPEG-PLA, PELA) microspheres were investigated. PELA with different hydrophilicities (PELA-1, PELA-2, and PELA-3) based on various ratios of mPEG to PLA were employed to prepare microspheres exhibiting a narrow size distribution using a combined double emulsion and premix membrane emulsification method. The morphology, rhGH encapsulation efficiency, in vitro release profile, and rhGH stability of PELA microspheres during the release were characterized and compared in detail. It was found that increasing amounts of PLA enhanced the encapsulation efficiency of PELA microspheres but reduced both the release rate of rhGH and its stability. Contact angle, atomic force microscope (AFM), and quartz crystal microbalance with dissipation (QCM-D) techniques were first combined to elucidate the microcosmic mechanism of incomplete release by measuring the hydrophilicity of the PELA film and its interaction with rhGH. In addition, the pH change within the microsphere microenvironment was monitored by confocal laser scanning microscopy (CLSM) employing a pH-sensitive dye, which clarified the stability of rhGH during the release. These results suggested that PELA hydrophilicity played an important role in rhGH incomplete release and stability. Thus, the selection of suitable hydrophilic polymers with adequate PEG lengths is critical in the preparation of optimum protein drug sustained release systems. This present work is a first report elucidating the microcosmic mechanisms responsible for rhGH stability and its interaction with the microspheres. Importantly, this research demonstrated the application of promising new experimental methods in investigating the interaction between biomaterials and biomacromolecules, thus opening up a range of exciting potential applications in the biomedical field including drug delivery and tissue regeneration.

A novel sustained-release formulation of recombinant human growth hormone and its pharmacokinetic, pharmacodynamic and safety profiles.

An effective and safe formulation of sustained-release rhGH for two months using poly(monomethoxypolyethylene glycol-co-D,L-lactide) (mPEG-PLA, PELA) microspheres was developed to reduce the frequency of medication. The rhGH-loaded PELA microspheres with a narrow size distribution were successfully prepared by a double emulsion method combined with a premix membrane emulsification technique without any exogenous stabilizing excipients. The narrow size distribution of the microspheres would guarantee repeatable productivity and release behavior. Moreover, the amphiphilic PELA improved the bioactivity retention of protein drugs since it prevented protein contact with the oil/water interface and the hydrophobic network, and modulated diffusion of acidic degradation products from the carrier system. These PELA microspheres were compared in vivo with commercial rhGH solution, conventional poly(D,L-lactic acid) (PLA) and poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres. Administration of rhGH-PELA could extend the duration of rhGH release (for up to 56 days) and increase area under the curve (AUC) compared to rhGH solution, PLA or PLGA microspheres in Sprague-Dawley (SD) rats. In addition, rhGH-PELA microspheres induced a greater response in total insulin-like growth factor-1 (IGF-1) and insulin-like growth factor binding protein-3 (IGFBP-3) than other rhGH formulations. With a hypophysectomized SD rat model, the pharmacological efficacy of rhGH-PELA microspheres was shown to be better than that from daily administration of rhGH solutions over 6 days based on body weight gain and width of the tibial growth plate. Histological examination of the injection sites indicated a significantly milder inflammatory response than that observed after injection of PLA and PLGA microspheres. Neither anti-rhGH antibodies nor the toxic effects on heart, liver and kidney were detectable after administration of rhGH-PELA microspheres in SD rats. These results suggest that rhGH-PELA microspheres have the potential to be clinically effective and safe when administered only once every two months, a dose regimen for better patient acceptance and compliance.

Mass spectrometry to describe product and contaminant adsorption properties for bioprocess development.

Process development for biologics is expensive and lengthy, tools are needed to rapidly understand where the difficulties will lie, and, hence, rationally deploy resources. In this work we introduce and evaluate a methodology to determine the manufacturability of a protein candidate. The methodology determines protein impurities by mass spectrometry and separation difficulty from the product based on adsorption properties deduced from a single set of experiments. This information can aid early process strategy decisions to target hard to remove protein impurities (nearest neighbors) and allow the re-evaluation of conventional process synthesis. The methodology chosen gives consideration to the fact that at this point in early phase development, material, and established analytical methods are limiting. This study uses surface enhanced laser desorption ionization mass spectroscopy (SELDI-MS), for its rapid analysis and minimal sample requirement to measure product and contaminant adsorption properties. The technique is used to provide an array of hydrophobic and electrostatic conditions for protein adsorption. The adsorption pattern produced for each protein is analyzed and visualized via a star plot. Dendrograms then define nearest neighbor protein contaminants by quantifying differences in the adsorption pattern between the product and contaminants. By comparison to an existing process to manufacture a 28 kDa recombinant protein expressed in Escherichia coli, we confirm the method is capable of determining where the greatest separation difficulty lies and what separation methods should be considered. The technique identified that the nearest neighbor contaminants were product-related proteins (28.6 and 29.1 kDa/e). Thus demonstrating a capability to measure the relative difficulty of purifying early stage protein candidates where little is known about the separation properties of products and contaminants, or the process sequence for their production.

Integrated solution to purification challenges in the manufacture of a soluble recombinant protein in E. coli.

Apolipoprotein A 1 Milano (ApoA-1M), the protein component of a high-density lipoprotein (HDL) mimic with promising potential for reduction of atherosclerotic plaque, is produced at large scale by expression in E. coli. Significant difficulty with clearance of host cell proteins (HCPs) was experienced in the original manufacturing process despite a lengthy downstream purification train. Analysis of purified protein solutions and intermediate process samples led to identification of several major HCPs co-purifying with the product and a bacterial protease potentially causing a specific truncation of ApoA-1M found in the final product. Deletion of these genes from the original host strain succeeded in substantially reducing the levels of HCPs and the truncated species without adversely affecting the overall fermentation productivity, contributing to a much more efficient and robust new manufacturing process.

Product and contaminant measurement in bioprocess development by SELDI-MS.

Bioprocesses for therapeutic protein production typically require significant resources to be invested in their development. Underlying these efforts are analytical methods, which must be fit for the purpose of monitoring product and contaminants in the process. It is highly desirable, especially in early-phase development when material and established analytical methods are limiting, to be able to determine what happens to the product and impurities at each process step with small sample volumes in a rapid and readily performed manner. This study evaluates the utility of surface-enhanced laser desorption ionization mass spectroscopy (SELDI-MS), known for its rapid analysis and minimal sample volumes, as an analytical process development tool. In-process samples from an E. coli process for apolipoprotein A-IM (ApoA-IM) manufacture were used along with traditional analytical methods such as HPLC to check the SELDI-MS results. ApoA-IM is a naturally occurring variant of ApoA-I that appears to confer protection against cardiovascular disease to those that carry the mutated gene. The results show that, unlike many other analytical methods, SELDI-MS can handle early process samples that contain complex mixtures of biological molecules with limited sample pretreatment and thereby provide meaningful process-relevant information. At present, this technique seems most suited to early-phase development particularly when methods for traditional analytical approaches are still being established.

Ultra scale-down approaches for clarification of mammalian cell culture broths in disc-stack centrifuges.

Ultra-scale down (USD) methodology developed by University College London for cell broth clarification with industrial centrifuges was applied to two common cell lines (NS0 and GS-CHO) expressing various therapeutic monoclonal antibodies. A number of centrifuges at various scales were used with shear devices operating either by high speed rotation or flow-through narrow channels. The USD methodology was found effective in accounting for both gravitational and shear effects on clarification performance with three continuous centrifuges at pilot and manufacturing scales. Different shear responses were observed with the two different cell lines and even with the same cell line expressing different products. Separate particle size analysis of the treated broths seems consistent with the shear results. Filterability of the centrifuged solutions was also evaluated to assess the utility of the USD approach for this part of the clarification operation.

Separation of product associating E. coli host cell proteins OppA and DppA from recombinant apolipoprotein A-I(Milano) in an industrial HIC unit operation.

We have shown how product associating E. coli host cell proteins (HCPs) OppA and DppA can be substantially separated from apolipoprotein A-I(Milano) (apo A-I(M)) using Butyl Sepharose hydrophobic interaction chromatography (HIC). This work illustrates the complex problems that frequently arise during development and scale-up of biopharmaceutical manufacturing processes. Product association of the HCPs is confirmed using co-immunoprecipitation and Western blotting techniques. Two-dimensional gel electrophoresis and mass spectrometry techniques are used to confirm the identity of OppA and DppA. In this example, clearance of these difficult to separate HCPs decreased significantly when the process was scaled to a 1.4 m diameter column. Laboratory-scale experimentation and trouble shooting identified several key parameters that could be further optimized to improve HCP clearance. The key parameters included resin loading, peak cut point on the ascending side, wash volume, and wash salt concentration. By implementing all of the process improvements that were identified, it was possible to obtain adequate HCP clearance so as to meet the final specification. Although it remains speculative, it is believed that viscosity effects may have contributed to the lower HCP clearance observed early in the manufacturing campaign.

Separation of recombinant apolipoprotein A-I(Milano) modified forms and aggregates in an industrial ion-exchange chromatography unit operation.

We have shown how protein self-association impacts the ion-exchange separation of modified forms and aggregates for apolipoprotein A-I(Milano). It is well known that reversible self-association of a protein can lead to chromatographic band broadening, peak splitting, merging, fronting, and tailing. To mitigate these effects, urea or an organic modifier can be added to the chromatography buffers to shift the equilibrium distribution of the target molecule to the dissociated form. A first generation process that did not utilize urea resulted in low yield and low purity as it was not possible to separate protein aggregates. A second generation process run in the presence of 6M urea resulted in high purity and high yield, but throughput was limited due to low resin binding capacity when the protein was completely denatured. A third generation process achieved high purity, high yield, and high throughput by shifting the urea concentration during the process to continually operate in the optimal window for maximum loading and selectivity. Key to these systematic process improvements was the rational understanding of the interplay of urea concentration and ion-exchange chromatographic behavior. Results from pilot and industrial scale operations are presented, demonstrating the suitability of the techniques described in this work for the large scale manufacture of recombinant therapeutic proteins.

Use of cyclohexanedimethanol as a nonflammable organic solvent for industrial scale reversed phase chromatography.

This work demonstrates that cyclohexanedimethanol is an effective nonflammable organic solvent for the pilot scale reversed phase chromatography of recombinant apolipoprotein A-I(Milano). Cyclohexanedimethanol has low viscosity in water, enabling the use of conventional low pressure process scale chromatography columns and hardware. Results from pilot scale manufacturing using a 30 cm diameter CG71M packed column indicate that a 5.7 log reduction in E. coli host cell protein with >80% yield can be obtained for the purification of apolipoprotein A-I(Milano).

Ultra scale-down to define and improve the relationship between flocculation and disc-stack centrifugation.

The recovery of intracellular recombinant proteins produced in microbial systems typically requires physical, chemical or thermal treatment of the cells post-harvest to release the product into the broth, followed by removal of the cell debris using centrifugation or tangential flow filtration. Often a precipitation or flocculation step is introduced to facilitate the liquid-solid separation. Due to the complex nature of the cell materials and the unit operations, it is difficult to obtain data at laboratory scale that closely reflect the performance of these operations on larger scales (pilot or manufacturing). This study uses a predictive scale-down model that enables rapid optimization of the operating conditions for a flocculation followed with a centrifugation step using only small volumes (20 mL) of a high solids ( approximately 20% w/w) E. coli heat extract. Results obtained show that, with proper theoretical and experimental consideration to account for high cell density, conditions could be found that improve the beneficial interaction between flocculation and centrifugation. These experiments suggested that adding a higher level of a cationic polymer could substantially increase the strength of the flocculated particles produced, thereby enhancing overall clarification performance in a large scale centrifuge. This was subsequently validated at pilot scale.

Impact of denaturation with urea on recombinant apolipoprotein A-IMilano ion-exchange adsorption: equilibrium uptake behavior and protein mass transfer kinetics.

We have studied the equilibrium uptake behavior and mass transfer rate of recombinant apolipoprotein A-I(Milano) (apo A-I(M)) on Q Sepharose HP under non-denaturing, partially denaturing, and fully denaturing conditions. The protein of interest in this study is composed of amphipathic alpha helices that serve to solubilize and transport lipids. The dual nature of this molecule leads to the formation of micellar-like structures and self association in solution. Under non-denaturing conditions equilibrium uptake is 134 mg/mL media and the isotherm is essentially rectangular. When fully denatured with 6 M urea, the equilibrium binding capacity decreases to 25 mg/mL media and the isotherm becomes less favorable. The decrease in both binding affinity and media capacity when the protein is completely denatured with 6 M urea can be explained by the loss of all alpha helical structure. The rate of apo A-I(M) mass transfer on Q Sepharose HP was characterized using a macropore diffusion model. Results of modeling studies indicate that effective pore diffusivity increases from 4.5 x 10(-9) cm2/s in the absence of urea to 6.0 x 10(-8) cm2/s when apo A-I(M) is fully denatured with 6 M urea. Based on light-scattering data reported for apo A-I, protein self association appears to be the dominant cause of slow protein mass transfer observed under non-denaturing conditions.