Fourier-transform infrared spectroscopy as a process analytical technology for near real time in-line estimation of the degree of PEGylation in chromatography.

PEGylation of biological macromolecules is a well-established strategy to increase circulation half-life, decrease renal clearance and improve biocompatibility. PEGylation is a process in which polyethylene glycol (PEG) is covalently attached to a target molecule. The production of PEGylated biopharmaceuticals is usually executed by first producing and purifying the base molecule followed by the PEGylation reaction and purification of the modified molecule. Most PEGylated pharmaceuticals are produced by random PEGylation in batch mode and need to be purified as mainly the mono-PEGylated form is the desired drug product. In this work we propose a method to estimate the degree of PEGylation (DOP) of modified protein eluting from a chromatography column in near real-time. extended multiplicative signal correction (EMSC) is used in conjunction with asymmetric least squares (aaLS) to alleviate the influence of a salt gradient during ion exchange chromatography (IEX) on the spectral data. To convert the raw data obtained from spectral data to the actual DOP additional information obtained from off-line measurements is utilized. Once the signal correction is applied to in-line spectral data the DOP can be estimated without further use of off-line analytics. As the prerequisites for the application of this method are relatively easy to obtain it may also find use to speed up process development.

Precipitation of complex antibody solutions: influence of contaminant composition and cell culture medium on the precipitation behavior.

Preparative protein precipitation is known as a cost-efficient and easy-to-use alternative to chromatographic purification steps. This said, at the moment, there is no process for monoclonal antibodies (mAb) on the market, although especially polyethylene glycol-induced precipitation has shown great potential. One reason might be the highly complex behavior of each component of a crude feedstock during the precipitation process. For different investigated mAbs, significant variations in the host cell protein (HCP) reduction are observed. In contrast to the precipitation behavior of single components, the interactions and interplay in a complex feedstock are not fully understood yet. This work discusses the influence of contaminants on the precipitation behavior of two different mAbs, an IgG1, and an IgG2. By spiking the mAbs with mock solution, a complex feedstock could successfully be mimicked. Spiking contaminants influenced the yield and purity of the mAbs after the precipitation step, compared to the precipitation behavior of the single components. The mixture showed a decrease in the contaminant and mAb solubility. By re-buffering the mock solution prior to spiking, special salts, small molecules like amino acids, vitamins, or sugars could be depleted while larger ones like HCP or DNA were still present. Therefore, it was possible to distinguish the influence of small molecules and larger ones. Hence, mAb-macromolecular interaction could be identified as a possible reason for the observed higher precipitation propensity, while small molecules of the cell culture medium were identified as solubilisation factors during the precipitation process.

Impact of additives on the formation of protein aggregates and viscosity in concentrated protein solutions.

In concentrated protein solutions attractive protein interactions may not only cause the formation of undesired aggregates but also of gel-like networks with elevated viscosity. To guarantee stable biopharmaceutical processes and safe formulations, both phenomenons have to be avoided as these may hinder regular processing steps. This work screens the impact of additives on both phase behavior and viscosity of concentrated protein solutions. For this purpose, additives known for stabilizing proteins in solution or modulating the dynamic viscosity were selected. These additives were PEG 300, PEG 1000, glycerol, glycine, NaCl and ArgHCl. Concentrated lysozyme and glucose oxidase solutions at pH 3 and 9 served as model systems. Fourier-transformed-infrared spectroscopy was chosen to determine the conformational stability of selected protein samples. Influencing protein interactions, the impact of additives was strongly dependent on pH. Of all additives investigated, glycine was the only one that maintained protein conformational and colloidal stability while decreasing the dynamic viscosity. Low concentrations of NaCl showed the same effect, but increasing concentrations resulted in visible protein aggregation.

Prediction of salt effects on protein phase behavior by HIC retention and thermal stability.

In the biopharmaceutical industry it is mandatory to know and ensure the correct protein phase state as a critical quality attribute in every process step. Unwanted protein precipitation or crystallization can lead to column, pipe or filter blocking. In formulation, the formation of aggregates can even be lethal when injected into the patient. The typical methodology to illustrate protein phase states is the generation of protein phase diagrams. Commonly, protein phase behavior is shown in dependence of protein and precipitant concentration. Despite using high-throughput methods for the generation of phase diagrams, the time necessary to reach equilibrium is the bottleneck. Faster methods to predict protein phase behavior are desirable. In this study, hydrophobic interaction chromatography retention times were correlated to crystal size and form. High-throughput thermal stability measurements (melting and aggregation temperatures), using an Optim(®)2 system, were successfully correlated to glucose isomerase stability. By using hydrophobic interaction chromatography and thermal stability determinations, glucose isomerase conformational and colloidal stability were successfully predicted for different salts in a specific pH range.

Determination of protein phase diagrams by microbatch experiments: exploring the influence of precipitants and pH.

Knowledge of protein phase behavior is essential for downstream process design in the biopharmaceutical industry. Proteins can either be soluble, crystalline or precipitated. Additionally liquid-liquid phase separation, gelation and skin formation can occur. A method to generate phase diagrams in high throughput on an automated liquid handling station in microbatch scale was developed. For lysozyme from chicken egg white, human lysozyme, glucose oxidase and glucose isomerase phase diagrams were generated at four different pH values ­ pH 3, 5, 7 and 9. Sodium chloride, ammonium sulfate, polyethylene glycol 300 and polyethylene glycol 1000 were used as precipitants. Crystallizing conditions could be found for lysozyme from chicken egg white using sodium chloride, for human lysozyme using sodium chloride or ammonium sulfate and glucose isomerase using ammonium sulfate. PEG caused destabilization of human lysozyme and glucose oxidase solutions or a balance of stabilizing and destabilizing effects for glucose isomerase near the isoelectric point. This work presents a systematic generation and extensive study of phase diagrams of proteins. Thus, it adds to the general understanding of protein behavior in liquid formulation and presents a convenient methodology applicable to any protein solution.