Figure 3: Damage to the harvest pump mating surfaces.
During early development, fatty acid characterization was part of the product impurity profile. Purified GCMP prior to conjugation
contained <0.5% (w/w) fatty acids (palmitic, oleic, and 3-OH myristic). After the initial characterization, fatty acids were
not routinely analyzed, although residual protein and nucleic acid levels were. These data confirmed that most fatty acids
present were palmitic acid, the most prevalent fatty acid in Neisseria. Since the precipitate appeared in the 50K and 30K retentates, it was initially thought that there were flaws in the saponification
and diafiltration steps. A significant portion of the root cause investigation examined the purification process, but nothing
unusual about the deacetylation or saponification was uncovered. The in-process testing for residual protein and nucleic acid
gave acceptable and typical results (see Table II). Since no protein or nucleic acids could be detected, and purification
of GCMP was achieved, abnormalities in GCMP purification were ruled out as a root cause and the focus of the investigation
shifted to the fermentation and harvest steps.
Table II: In-Process testing of 30K retentate for purity of the precipitation lots: residual protein and nucleic acid
Many of the fermentation inputs can affect cell metabolism and as a consequence, GCMP yield. Prior to the appearance of precipitation,
the authors had seen increasing GCMP yields for several months. It was hypothesized that fermentation medium or operational
parameters had changed, increasing GCMP yield in the form of lipidated-GCMP and therefore overwhelming the purification system.
Several test runs were made to alter the fermentation process and reduce the GCMP yield, but had no apparent effect on the
precipitation problem.
Figure 4: Fatty acid/GCMP % (w/w) in fermentation lots.
The GCMP harvest was more closely examined. This process involved circulating the contents of the fermentation vessel through
0.2 µm hollow fiber cartridges using a circumferential piston pump. This pump is designed with moving part tolerances tighter
than most similarly-sized rotary lobe pumps. Since the product is in the filter permeate, the fermentation medium is continuously
circulated until the retained volume was low. We estimate that each cell passes through the harvest pump ~300 times. Upon
examination, the harvest pump showed damage on mating surfaces in the lobes and rotor housing (see Figure 3). This damage
had occurred when a catastrophic event scored these surfaces, rather than being the result of normal wear and tear. The pump
continued to deliver expected volumes and pressures but the authors decided to replace it with an identical pump. The abnormal
precipitation in the downstream process immediately ceased.
Table III: Concentration of palmitic acid entering the saponification step.
The authors concluded that the harvest pump, while operational, was damaged in such a way that it was also acting as a cell
disruptor. Not only was more lipidated-GCMP sheared from the cell capsule, but the cell membranes were disrupted, forming
fragments small enough to pass though the 0.2 µm harvest filters (see Figure 2, steps 2 and 3), but large enough to be retained
in the initial GCMP capture step (see Figure 2, step 4). Fatty acids in this material would be saponified in the deacetylation
step (see Figure 2, step 5). If the sodium salts were soluble, they would be removed from the system during diafiltration.
If they were not soluble, such as the sodium salt of palmitic acid, they would precipitate during this process. The ability
of the damaged pump to mechanically extract fatty acids from the cells must have been increasing for several months before
a critical point was reached where the amount or type entering the downstream process overwhelmed its ability to remove it.
