Development of process analytical technologies for chromatography based protein purification

While the integration of PAT is already well-advanced in upstream processing of therapeutic proteins, there is still a lack of suitable technologies for real-time process monitoring and control in downstream processing. The development of suitable PAT for chromatography based protein purification was hence the main objective of this thesis

Spectral deconvolution of chromatograms without offline analytics

Continuous bioprocessing has shifted into the focus of the pharmaceutical industry in recent past. An important driving force is the expected higher productivity compared to operations in batch mode. To ensure constant quality attributes over a complete production cycle, reliable process analytical technologies (PAT) are central. The deconvolution of spectral measurements in downstream processing has been introduced previously as a powerful PAT tool[1]. For spectral deconvolution, published research focused on partial least square (PLS) regression models. PLS models are able to deconvolute spectra even if the spectral differences of proteins are minute. They are widely used in chemometrics. However, PLS models depend on offline analytical calibration. An accurate calibration is difficult and the obtained model may be spectrometer dependent. Furthermore, the black box approach of PLS models make an interpretation and statistical evaluation challenging. To address these drawbacks, an alternative method is proposed making use of Lambert-Beer’s law. With a custom made algorithm, chromatograms were retrieved for multiple co-eluting species[2].The algorithm does not require offline analytics but deconvolutes absorbance data autonomously into component concentrations and the respective spectra. Chromatograms for two, three and four component mixtures were deconvoluted and analyzed. The deconvoluted chromatograms were compared to offline fraction analytics (Figure 1). A direct comparison was made between results obtained by a PLS model and the described method. Main advantages of the alternative method are easier statistical analysis as well as its semi-mechanistic background. Furthermore, during process development, the described method may allow to reduce dependencies on offline analytics and – to a certain degree – help to circumvent analytical bottlenecks. In summary, the alternative method is complementary to PLS regression as it provides a more flexible approach to spectral deconvolution but may not reach the same accuracy

Process analytical technologies for a continuous capture and connected downstream process

As we move towards the implementation of continuous and connected processes, it becomes apparent that we can no longer avail ourselves of traditional sampling points (pools). Therefore, the implementation of Process Analytical Technologies (PAT) to demonstrate process robustness or to monitor process consistency, becomes necessary. Methodologies to monitor mass and yield are still necessary and the need to have at- or on-line analytics becomes more real. In-between, it may be necessary to bridge between the traditional analysis of unit pools to a more dynamic process monitoring. Recovered product mass and step yields per unit operation are commonly used to monitor process robustness. This is however more challenging in a connected/continuous mode, where there are no longer pools. The presented approach will demonstrate how UV absorption measurements can be utilized to track yield and to show process consistency. To tackle the issue of signal saturation, variable pathlength measurements as well as UV measurements at 300 nm were used. While univariate UV measurements were used for post-Protein A steps, multivariate measurements in conjunction with Partial Least Squares Regression modelling were evaluated for mass tracking in harvest. The monitoring of quality attributes within continuous processes also requires an adapted approach. The most straightforward approach in this context is the automation of already existing assays. The poster will present how the turnover time of an offline SEC assay was reduced to 1 min and how the method was applied for automated aggregate analysis. Furthermore, it will be shown how an Online-UPLC assay was used to control the aggregate level in chromatography runs with variable column load densities and aggregate levels in the load. Until at- or on-line analytics can be developed for all critical quality attributes, a bridging method is required. Here, a testing strategy based on pseudo-product pools will be presented, which allows bridging of quality data from a continuous process stream to a traditional standard batch pool concept. Slip stream pumps were used to generate pseudo pools, whenever product pools were not available. It was demonstrated, that the pseudo pools are representative and that the data is applicable for statistical comparison with historical batch data.

Real-time monitoring and control of the load phase of a protein A capture step

The load phase in preparative Protein A capture steps is commonly not controlled in real‐time. The load volume is generally based on an offline quantification of the monoclonal antibody (mAb) prior to loading and on a conservative column capacity determined by resin‐life time studies. While this results in a reduced productivity in batch mode, the bottleneck of suitable real‐time analytics has to be overcome in order to enable continuous mAb purification. In this study, Partial Least Squares Regression (PLS) modeling on UV/Vis absorption spectra was applied to quantify mAb in the effluent of a Protein A capture step during the load phase. A PLS model based on several breakthrough curves with variable mAb titers in the HCCF was successfully calibrated. The PLS model predicted the mAb concentrations in the effluent of a validation experiment with a root mean square error (RMSE) of 0.06 mg/mL. The information was applied to automatically terminate the load phase, when a product breakthrough of 1.5 mg/mL was reached. In a second part of the study, the sensitivity of the method was further increased by only considering small mAb concentrations in the calibration and by subtracting an impurity background signal. The resulting PLS model exhibited a RMSE of prediction of 0.01 mg/mL and was successfully applied to terminate the load phase, when a product breakthrough of 0.15 mg/mL was achieved. The proposed method has hence potential for the real‐time monitoring and control of capture steps at large scale production. This might enhance the resin capacity utilization, eliminate time‐consuming offline analytics, and contribute to the realization of continuous processing. Biotechnol

PAT concepts for chromatography: Real-time monitoring, real-time control, and cause of error diagnostics

In the recent years, the growing competition in the biopharmaceutical industry due to biosimilars combined with market fluctuations and increasing product pipelines have given rise to the evaluation of continuous processing. Advantages include smaller equipment that may be disposable, an overall process intensification as well as steady-state operation. As variability should be managed by the continuous process itself to insure a consistent product quality, the development of advanced Process Analytical Technology (PAT) concepts is one crucial aspect for the implementation of continuous processing. In chromatographic protein purification, process variability can lead to variations in retention volumes or peak shapes of the eluting species. If the contaminants are co-eluting with the product, a peak deconvolution is required to enable real-time process monitoring and control. In order to overcome this analytical bottleneck, a tool will be presented, which allows for real-time peak deconvolution and pooling decisions in chromatography [1]. The peak deconvolution is obtained by Partial Least Squares Regression (PLS) modelling with spectroscopic data. The applicability of the peak deconvolution tool for real-life applications was successfully demonstrated in a case study with co-eluting monoclonal antibody, its aggregates, and fragments as well as in case study with co-eluting serum proteins [2]. Aside from the detection of variations in elution profiles, a fast diagnostics of their causes is crucial for a comprehensive process understanding and control. The identification of causes for variations in chromatography processes requires however a mechanistic understanding. In this presentation, a case study will be shown, where the deconvoluted peaks of three co-eluting model-proteins of several linear gradient elutions were used for the calibration of a mechanistic chromatography model. Variations in the elution profiles of all proteins were subsequently induced by deliberately generated errors in the process parameters flow rate, salt concentration in loading buffer, and salt concentration in elution buffer. The individual elution profiles of all proteins were determined by the peak deconvolution tool. The peak deconvolution in combination with the mechanistic model allowed for a fast estimation of the actual values of the three process parameters by curve fitting. In summary, peak deconvolution by PLS modelling with spectroscopic data might enable real-time monitoring and control in future chromatographic processes. A combination of peak deconvolution with mechanistic modelling allows furthermore for a fast cause of error-diagnostics, which might contribute to better process understanding and control and might minimize lot rejections

In-line Fourier-transform infrared spectroscopy as a versatile process analytical technology for preparative protein chromatography

Fourier-transform infrared spectroscopy (FTIR) is a well-established spectroscopic method in the analysis of small molecules and protein secondary structure. However, FTIR is not commonly applied for in-line monitoring of protein chromatography. Here, the potential of in-line FTIR as a process analytical technology (PAT) in downstream processing was investigated in three case studies addressing the limits of currently applied spectroscopic PAT methods. A first case study exploited the secondary structural differences of monoclonal antibodies (mAbs) and lysozyme to selectively quantify the two proteins with partial least squares regression (PLS) giving root mean square errors of cross validation (RMSECV) of 2.42 g/l and 1.67 g/l, respectively. The corresponding Q$^{2}$ values are 0.92 and, respectively, 0.99, indicating robust models in the calibration range. Second, a process separating lysozyme and PEGylated lysozyme species was monitored giving an estimate of the PEGylation degree of currently eluting species with RMSECV of 2.35 g/l for lysozyme and 1.24 g/l for PEG with Q$^{2}$ of 0.96 and 0.94, respectively. Finally, Triton X-100 was added to a feed of lysozyme as a typical process-related impurity. It was shown that the species could be selectively quantified from the FTIR 3D field without PLS calibration. In summary, the proposed PAT tool has the potential to be used as a versatile option for monitoring protein chromatography. It may help to achieve a more complete implementation of the PAT initiative by mitigating limitations of currently used techniques

NS1 Specific CD8(+) T-Cells with Effector Function and TRBV11 Dominance in a Patient with Parvovirus B19 Associated Inflammatory Cardiomyopathy

Background: Parvovirus B19 (B19V) is the most commonly detected virus in endomyocardial biopsies (EMBs) from patients with inflammatory cardiomyopathy (DCMi). Despite the importance of T-cells in antiviral defense, little is known about the role of B19V specific T-cells in this entity. Methodology and Principal Findings: An exceptionally high B19V viral load in EMBs (115,091 viral copies/mg nucleic acids), peripheral blood mononuclear cells (PBMCs) and serum was measured in a DCMi patient at initial presentation, suggesting B19V viremia. The B19V viral load in EMBs had decreased substantially 6 and 12 months afterwards, and was not traceable in PBMCs and the serum at these times. Using pools of overlapping peptides spanning the whole B19V proteome, strong CD8(+) T-cell responses were elicited to the 10-amico-acid peptides SALKLAIYKA (19.7% of all CD8(+) cells) and QSALKLAIYK (10%) and additional weaker responses to GLCPHCINVG (0.71%) and LLHTDFEQVM (0.06%). Real-time RT-PCR of IFN gamma secretion-assay-enriched T-cells responding to the peptides, SALKLAIYKA and GLCPHCINVG, revealed a disproportionately high T-cell receptor Vbeta (TRBV) 11 expression in this population. Furthermore, dominant expression of type-1 (IFN gamma, IL2, IL27 and Tbet) and of cytotoxic T-cell markers (Perforin and Granzyme B) was found, whereas gene expression indicating type-2 (IL4, GATA3) and regulatory T-cells (FoxP3) was low. Conclusions: Our results indicate that B19V Ag-specific CD8(+) T-cells with effector function are involved in B19V associated DCMi. In particular, a dominant role of TRBV11 and type-1/CTL effector cells in the T-cell mediated antiviral immune response is suggested. The persistence of B19V in the endomyocardium is a likely antigen source for the maintenance of CD8(+) T-cell responses to the identified epitopes

Putting a price tag on novel autologous cellular therapies

This paper was accepted for publication in the journal Cytotherapy and the definitive published version is available at http://dx.doi.org/10.1016/j.jcyt.2016.05.005Cell therapies, especially autologous therapies, pose significant challenges to researchers who wish to move from small, probably academic, methods of manufacture to full commercial scale. There is a dearth of reliable information about the costs of operation, and this makes it difficult to predict with confidence the investment needed to translate the innovations to the clinic, other than as small-scale, clinician-led prescriptions. Here, we provide an example of the results of a cost model that takes into account the fixed and variable costs of manufacture of one such therapy. We also highlight the different factors that influence the product final pricing strategy. Our findings illustrate the need for cooperative and collective action by the research community in pre-competitive research to generate the operational models that are much needed to increase confidence in process development for these advanced products

Generation of cytomegalovirus specific T-cell lines from healthy and immunocompromised donors for adoptive therapy

Titelblatt und Verzeichnisse Einleitung Ziele der Arbeit Methoden Ergebnisse Diskussion Literaturverzeichnis Abkürzungen Eidesstattliche ErklärungIn der Transplantation stellen Zytomegalovirus- (CMV-) Infektionen ein ernstzunehmendes Problem dar und können sowohl das Überleben des Patienten als auch das des Transplantats negativ beeinflussen. Zur Behandlung von CMV- Komplikationen ist die medikamentöse antivirale Therapie die erste therapeutische Option. Eine langfristige Behandlung durch antivirale Substanzen ist aber aufgrund toxischer Nebenwirkungen sowie viraler Resistenzbildungen nur bedingt möglich. Die einzige Möglichkeit einen langfristigen Schutz vor CMV-Komplikationen zu gewährleisten, ist die Etablierung einer effektiven CMV-spezifischen Immunantwort. Bei transplantierten Patienten ist die natürliche Entwicklung einer protektiven Antwort durch die medikamentöse Immunsuppression, welche die Abstoßung des transplantierten Organs verhindern soll, stark erschwert. Darum setzen neuere Therapieansätze auf den adoptiven Transfer in vitro-manipulierter CMV- spezifischer T-Zelllinien zur Überwindung dieses iatrogenen Effektes. Obwohl klinische Studien die Sicherheit und Effektivität adoptiv transferierter T-Zelllinien in der Bekämpfung von CMV-Komplikationen belegten, kommen diese bis heute lediglich bei wenigen Patienten zur Anwendung. Dies ist vor allem durch den technisch aufwendigen Herstellungsprozess, sowie die mangelnde Übertragbarkeit vieler Generierungsmethoden in die klinische Praxis bedingt. Kürzlich wurde in unserer Arbeitsgruppe ein Protokoll entwickelt, das die Herstellung CMV-spezifischer T-Zelllinien aus gesunden Probanden ermöglicht. Dieses Protokoll basiert auf der Stimulation mit überlappenden Peptidpools, der anschließenden magnetischen Isolierung der Interferon-γ\- sezernierenden Zellen, sowie deren in vitro-Expansion. Dieses Protokoll überwindet viele Limitierungen bisheriger Herstellungsmethoden, da es für jeden HLA-Typ einsetzbar ist und keine Kenntnis von immundominanten Epitopen voraussetzt. Die vorliegende Arbeit beschäftigte sich mit der Weiterentwicklung und Verbesserung dieses Herstellungsprotokolls. Es konnte gezeigt werden, dass die beschriebene Methode für die Herstellung von CMV-spezifischen T-Zelllinien aus immunsupprimierten Patienten genutzt werden kann. Dadurch können Patienten nach solider Organstransplantation behandelt und somit die Anwendung der Methode um ein Vielfaches ausgeweitet werden. Des Weiteren wurden T-Zelllinien gegen mehrere virale Proteine aus demselben Spender generiert. Die dadurch erreichte breitere Antigenreaktivität sollte die Effizienz der adoptiven Immuntherapie weiter erhöhen. Abschließend wurde in dieser Arbeit die Übertragbarkeit der Generierungsmethode in die klinische Praxis demonstriert, und es konnte ein Patient mit CMV-spezifischen T-Zelllinien behandelt werden. Somit stellt das entwickelte Herstellungsprotokoll einen wichtigen Fortschritt in der adoptiven Immuntherapie dar und erlaubt eine bessere Versorgung von Patienten mit schweren CMV-Komplikationen. Des Weiteren ist zukünftig eine Übertragung dieser Methode auf die Behandlung anderer viraler Erkrankungen oder Tumoren denkbar.Transplant recipients are at increased risk for severe human cytomegalovirus (HCMV) infection, which impairs graft survival and increases patient mortality. Prolonged virostatic therapy is limited by its side effects and the development of viral resistance. Reconstitution of cellular immunity by adoptive transfer of HCMV-specific T cells is a safe and effective treatment option but is limited to haematopoietic stem cell transplantation. A novel protocol based on stimulation with overlapping peptide libraries followed by isolation of the IFN- γ secreting cells was developed in our group to generate HCMV specific T-cell lines from healthy volunteers. The aim of this study was to determine whether this protocol is applicable to generate functional HCMV- specific T cells from chronically immunosuppressed solid-organ-transplant (SOT) patients. Autologous CD4+/8+ T-cell lines directed against two immunodominant HCMV proteins were generated from the peripheral blood of SOT patients. Despite chronic immunosuppression, T-cell lines with HCMV-specific killing behavior were generated from 8/10 SOT patients. Cell lines recognized multiple epitope targets of the viral proteins and were oligoclonal at the T-cell receptor V-beta level. Additionally, the feasibility of the protocol to generate T-cell lines under clinical conditions was demonstrated and one patient was treated with HCMV specific T cells. These data indicate the potential of this procedure for application in clinical relevant situations, like treatment of SOT patients with HCMV infection resistant to virostatic therapy