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 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.
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.