Using NIR to Move Bioprocessing into a PAT Framework - Pharmaceutical Technology

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Using NIR to Move Bioprocessing into a PAT Framework
The author provides a review of PAT and tools such as near infrared analysis that may facilitate the use of PAT in the biopharmaceutical sector.


Pharmaceutical Technology
Volume 33, Issue 7

Bioprocessing

Biological materials are an increasingly important field in the pharmaceutical industry. There are currently more than 350 biologicals approved by FDA (5). Of these, 56 are vaccines, 130 are antibodies, and 30 are labeled as recombinant. Bioprocesses that produce pharmaceuticals rely on intricate biological systems to synthesize useful products, many of which are large complex proteins, hormones, or polysaccharides that are difficult, or even impossible, to manufacture in large quantities any other way. The complexity of these biological systems and the finished product makes variability among batches inevitable. In fact, it is estimated that 30% of production batches require reprocessing for quality reasons and, although some of these reprocessed batches continue through to production, this additional process, along with the discarding of poor quality product, can result in an approximately 10-fold loss in profit (2). Therefore, if there ever was an industry in need of PAT control, the biopharmaceutical industry is it.

Many types of cells, including genetically engineered bacterial and yeast cells are used to culture and produce biopharmaceutical products. The majority of products, however, are produced within mammalian systems such as Chinese hamster ovary (CHO), green monkey (VERO), or human embryonic kidney (HEK) cell lines. These mammalian-cell lines require more controlled feeding and care than other lines because they originate from multicellular organisms that have been adapted to survive as free-floating cells in a synthetic or complex artificial medium. The protein products are well-characterized biologics that require precise control of the culture parameters. Often, proper glycosylation of the target protein is important to its function, and post-translational modifications such as glycosylation are dependent on subtle glutamine feeding as well as the control and removal of ammonia waste products (2). Specifically, the ratio of N-acetyl-neuraminic acid (NeuAc) to N-glycolyl-neuraminic acid (NeuGc) of the target protein needs to be maximized to optimize the in vivo half-life of the biopharmaceutical. A major influence on this ratio is the composition of the cell culture medium (6). Controlling the cell culture medium through NIR and other PAT instruments helps ensure the correct NeuAc/NeuGc ratio.


Figure 1: Process schematic for manufacturing biopharmaceuticals. Raw materials (A) and inoculating cell cultures (B) are nurtured for several days in a process tank (C). The resulting target molecule is purified from the spent media by centrifugation (D) and filtration (E). The target molecule is further purified with an affinity column (F) or chromatography (G) and lyophilized. Near infrared spectroscopic analysis can be performed automatically in real-time at all stages of the process.
Figure 1 represents a typical industrial-scale bioprocess system producing and purifying a target complex molecule. Raw materials (see Figure 1A) used in media preparation and culture feeding are mixed and diluted to appropriate concentrations. Inoculums containing the carefully produced and stored cell lines are grown in small containers (see Figure 1B) prior to introduction into the large-scale process tank (see Figure 1C). Depending on the process, the cells are nurtured in the tank for several days where they grow and produce the target molecule. Typically, for mammalian cells, the target molecule is a protein that is secreted out into the cell medium where it accumulates, but other expression systems such as bacterial cells, may produce and store the target molecule within inclusion bodies and require eventual break-up of the cell for recovery. After a prescribed time, the spent medium and products are withdrawn from the process tank and purified, which may involve microfiltering or centrifugation (see Figure 1D). For mammalian cells, this step is a 'soft' method so as not to disrupt the cells, as cellular debris is more difficult to remove from the target protein. The resulting material is typically concentrated approximately 10-fold by tangential flow filtration (see Figure 1E). Often, diafiltration further purifies the filtrate and places it in a binding buffer that is suitable for the subsequent chromatography step. Large low-pressure chromatography columns (see Figure 1F) containing a binding resin (frequently, Protein A in the case of antibody purification) selectively bind the target molecule and bring it out of solution. Following wash steps that are optimized for impurity removal, the columns are flushed with an elution buffer allowing the product molecule to be released and collected. Finally, polishing steps (see Figure 1G) may occur where ion and buffer exchange is performed. Otherwise, chromatography is used to separate and collect the different possible glyocoforms. This step is followed by sterile filtration, liquid fill, and lyophilization.


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