TECHNICAL FIELD OF THE INVENTION

The invention is generally directed to systems and methods for monitoring and controlling one or more critical quality attributes (CQAs) or parameters in downstream protein purification processes.

BACKGROUND OF THE INVENTION

The number of monoclonal antibodies (mAbs) that have been approved for therapeutic use has increased significantly over the past decade. This is due in part to improvements in large scale manufacturing processes which facilitate the production of large quantities of mAbs. In addition, initiatives such as The Process Analytical Technology (PAT) framework of the Food and Drug Administration (FDA) have led to innovative solutions to process development, process analysis, and process control to better understand processes and control the quality of products. Efficient recovery and purification of mAbs from cell culture media is a critical part of the production process. The purification process should reliably produce mAbs that are safe for use in humans. This includes monitoring critical quality attributes (CQAs) that include protein attributes and impurities such as host cell proteins, DNA, viruses, endotoxins, aggregates, concentrations, excipients, and other species that have the potential to impact patient safety, efficacy, or potency. Protein concentration is also often a CQA in purified material, and appropriate protein concentration in process intermediates can be a critical process parameter for unit operation performance. These CQAs need to be monitored throughout production as well as throughout the program lifecycle.

To ensure that the final formulations of mAbs do not contain impurities above determined levels, the mAb products are tested at various stages of the downstream processing. Quality control in the manufacturing of bioproducts such as mAbs is generally accomplished by analyzing purification intermediates and formulated drug substance samples with offline methods for each lot production. The samples are removed from the processing equipment, for example a UF/DF skid, and subjected to offline tests to measure product CQAs such as protein concentration (g/L), buffer excipients, and size variants. Real time monitoring and analysis during manufacturing is not available resulting in increases in processing time and a higher risk of batch failure due to not meeting CQAs. Accordingly, there is a need for rapid, in-line methods of real-time quality control monitoring of mAbs.

Therefore, it is an object of the invention to provide systems and methods for real-time monitoring of critical quality attributes during the downstream purification process.

SUMMARY OF THE INVENTION

In situ Raman spectroscopy methods and systems for characterizing or quantifying a protein purification intermediate during production or manufacture are provided. In one embodiment, in situ Raman spectroscopy is used to characterize or quantify critical quality attributes of a protein drug during downstream processing (i.e., after harvest of the protein purification intermediate from cell culture fluid). For example, the disclosed in situ Raman spectroscopy methods and systems can be used to characterize and quantify protein purification intermediate critical quality attributes as the protein purification intermediates are purified, prior to formulation into the final drug product to be sold or administered. Critical quality attributes include but are not limited to protein concentration, excipients, high molecular weight (HMW) species, antibody titer, and drug-antibody ratio.

One embodiment provides a method of producing a concentrated protein purification intermediate by determining concentrations of a protein purification intermediate in-real time using in situ Raman spectroscopy while concentrating/diafiltering the protein purification intermediate and adjusting parameters of the concentrating step in-real time to obtain the pre-determined concentration targets and excipient levels necessary for formulation of drug substance. The protein purification intermediate product can have a concentration of 5 mg/mL to 300 mg/mL, preferably 50 mg/mL to 300 mg/mL for subsequent formulation steps. In one embodiment, the protein purification intermediate is concentrated to a desired concentration target using ultrafiltration during primary or final concentration. Diafiltration is used during processing following primary concentration as a means for buffer exchange to achieve the desired final formulation components. The protein purification intermediate can be harvested from a bioreactor, a fed-batch culture, or a continuous culture. In another embodiment, determining the concentration of the protein purification intermediate can occur continuously or intermittently in real-time. Quantifying of protein concentration can be performed in intervals from approximately 5 seconds to 10 minutes, hourly, or daily. The protein purification intermediate can be an antibody or antigen binding fragment thereof, a fusion protein, or a recombinant protein. Spectral data can be collected at one or more wavenumber ranges selected from the group consisting of 977-1027 cm−1, 1408-1485 cm−1, 1621-1711 cm−1, 2823-3046 cm−1, and combinations thereof.

Another embodiment provides a method of producing a protein purification intermediate by independently performing Raman Spectroscopy analysis on a plurality of protein purification intermediates to produce a universal model capable of quantifying any one of the plurality of protein purification intermediates. The concentration of a protein purification intermediate can be determined using in situ Raman spectroscopy with the universal model during concentration of the protein purification intermediate from beginning to end of concentrating the protein purification intermediate. Another embodiment provides a method of producing a protein purification intermediate to produce a protein specific model capable of quantifying protein concentrations that would be used for commercial enabling productions of the protein.

The model can be produced using Partial Least Squares Regression Analysis of raw spectral data and using an orthogonal method for offline protein concentration data. Pre-processing techniques such as Standard Normal Variant (SNV) and/or point-smoothing technique can be 1stderivative with 21 cm−1smoothing can be performed on the Raman spectroscopy data to dampen model variability and prediction error. Further model refinement can be performed to isolate spectral regions that correlate to CQA predictions such as protein concentration. In one embodiment, the model has ≤5% error margin, preferably ≤3% error margin.

Still another embodiment provides a method for monitoring and controlling the levels of excipients in harvested cell culture fluid and/or protein purification intermediate during downstream purification by determining concentrations of the excipients in-real time using in situ Raman spectroscopy while purifying the cell culture fluid or protein purification intermediate, and adjusting parameters of the purification step in-real time to obtain or maintain predetermined amounts of the excipients in the harvested cell culture fluid and/or protein purification intermediate. The excipient can be acetate, citrate, histidine, succinate, phosphate, hydroxymethylaminomethane (Tris), proline, arginine, sucrose, or combinations thereof. The excipient can be a surface excipient such as polysorbate 80, polysorbate 20, and poloxamer 188.

