Immunoglobulin G (IgG) antibodies have been used to treat cancer for many years (1). Another class of antibodies—immunoglobulin M (IgM)—has been overlooked in spite of offering unique advantages that make them highly desirable as cancer therapeutics. Serving a valuable function in our innate immune system, IgM antibodies are the first to be secreted when an abnormal cell is present (2). These antibodies play a critical role in recognition and elimination of infectious particles (3,4), in removal of intracellular components, and in immunosurveillance mechanisms against malignant cells (5,6). IgMs also can bind to multiple copies of a target on a cancer cell surface. Such high avidity leads to cross-linking and more effective cell killing (7).

PRODUCT FOCUS: ANTIBODIES (IGM)

PROCESS FOCUS: MANUFACTURING

WHO SHOULD READ: PROCESS ENGINEERS, PRODUCT DEVELOPMENT, AND MANUFACTURING

KEYWORDS: PER.C6 CELLS, SCALE-UP, OPTIMIZATION, MONOLITHS, FED BATCH

LEVEL: INTERMEDIATE

In spite of all those desirable biological activities, IgM antibodies have not been widely developed as biopharmaceuticals. A number of misconceptions about IgMs have prevented their more widespread adoption. For example, some people believe that these antibodies are too large to be effective, that their expression levels in bioproduction systems are too low to be economically feasible, and that they are unstable and too difficult to purify (8,9).

IgMs’ large size and complexity have slowed the creation of high-expressing cell lines. These antibodies contain five or six units, each of those containing an IgG-like structure of two heavy chains and two light chains, which are all assembled into a pentamer or hexamer. A J-chain also might be present, binding to the tail piece at the heavy-chain C terminus. Each IgM subunit can have five or six potential sites for N-linked glycosylation (10). Nevertheless, several nonlymphoid cell lines—such as C6 glioma, Chinese hamster ovary (CHO), and HeLa cells—have been used to successfully produce polymeric IgMs, albeit with low reported yields (10 ng/mL for CHO cells) (11). Using rat hybridoma cell lines has improved productivity, with final yields reported at ∼100–200 mg/L in batch cultures and 700 mg/L in a medium-exchange process (12).

On the downstream side, IgMs’ large size (>900 kDa), narrower solubility range of conditions than IgGs have, and susceptibility to denaturation have presented difficulties in purification. Those challenges have been overcome with available technologies to develop effective capture and purification procedures (9). Ion-exchange, hydrophobic-interaction, and hydroxyapatite chromatography can bind IgMs under proper conditions. And technologies such as membranes and monoliths that rely on convection rather than diffusion are especially well suited for IgM purification (13,14).

Figure 1:

Alternative expression systems have become available to express protein therapeutics. For example, the PER. C6 human cell line—developed by Percivia LLC (a joint venture between Crucell NV and DSM Biologics) as a generic modified human primary embryonic retinoblast line—has a well-documented history with evidence of safety testing. It has been extensively developed for producing recombinant proteins. Using that cell line, we have successfully expressed

IgMs at higher titers that are functional and stable. We used a straightforward and rapid stable cell-line generation method (10).

Here we describe the technology transfer and scale-up of a process for a PER.C6 line expressing the PAT-SM6 (SM6) IgM antibody. The SM6 protein is proprietary to Patrys Ltd. and is currently under evaluation in a phase 1 melanoma study. We outline the production, scalability, and purification processes for that antibody.

Bench-Scale Cell-Culture Studies: Following procedures recommended by Percivia for PER.C6 cell lines, we ran batch and fed-batch studies with shake flasks using HyClone CDM4PERMAb media from Thermo Scientific and a Percivia-developed custom feed. Then we transferred our shake-flask process to 12-L bioreactors and evaluated suitable Wave Bioreactor operating parameters for scale-up of inocula.

Process Evaluation in Shake Flasks: To initiate these studies, a vial from the SM6 master cell bank (MCB) was recovered using CDM4PERMAb media supplemented with l-glutamine and Pluronic F-68 solution. After serial passaging, we set up batch cultures in duplicate using EM 250-mL shake flasks at a seeding density of 5 × 10⁵ vc/mL. We set up fed-batch cultures in duplicate using EM 250-mL shake flasks comparing seeding densities of 5 × 10⁵ and 7.5 × 10⁵ vc/mL. SM6-expressing cells in batch cultures attained maximum viable densities of 8.7 and 9.7 × 10⁶ vc/mL in shaker flasks A and B, respectively (Figure 1). Fed-batch cultures with 5 × 10⁵ vc/mL seeding density reached densities of 35 and 36 × 10⁶ vc/mL in shaker flasks A and B, respectively. Fed-batch culture at the higher seeding density (7.5 ×10⁵ vc/mL) gave similar results, reaching 37.2 × 10⁶ vc/mL (Figure 2).

Figure 1: (Click to enlarge)

Figure 2: (Click to enlarge)

The viable cell density (VCD) for SM6-expressing cells in both fed-batch cultures was triple that attained with batch cultures. The productivity in batch cultures was negligible (0.002 g/L), whereas fed-batch IgM expression titers reached 0.849 g/L (Figure 3). That increased growth rate and productivity may be attributed to the concentration of glucose in fed-batch cell culture. When glucose levels were not replenished (in batch cultures), SM6-expressing cells entered the stationary phase of their growth cycle more rapidly, reducing productivity. However, daily addition of feed medium (in fed-batch culture) containing 50 g/L of glucose prolonged the exponential phase of the growth curve and enabled cells to reach high cell densities. Nutrient components in feed are key factors to triggering the production of our SM6 monoclonal antibodies.

Figure 3: (Click to enlarge)

Cell Culture Scale-Up in WAVE Bioreactor Systems: Scalability of cell culture expansion is very important for manufacturing process development. Our SM6 cell scale-up process began with recovery of a vial of SM6 MCB in CDM4PERMAb media supplemented by l-glutamine and Pluronic F-68 solution in a T25 flask at a seeding density of 5.0 × 10⁵ vc/mL. This initial culture was then subcultured sequentially in shake flasks and then Wave Bioreactor systems at the same seeding density every three to four days for seven passages. At passage 7, to generate enough cells for inoculating the bioreactor at a higher seeding density (6.5 ± 0.5 × 10⁵ vc/mL), we seeded the Wave bag at 6.5 ± 0.5 ×10⁵ vc/mL. After the second passage, growth rate and viability of SM6-expressing cells in both shaker-flask and Wave system remained comparable, with VCDs reaching 1.9 × 10⁶ vc/mL in shake flasks and 1.8 × 10⁶ vc/mL in the Wave bag after 72 hours.

Process Evaluation in 12-L Bioreactors: To evaluate the performance of our SM6-producing cell line in fed-batch culture at Laureate—and in an effort to achieve higher titers in a bioreactor—we inoculated a 12-L Applikon glass bioreactor based on previously developed 10-L bioreactor parameters with a modified pH control. Instead of using constant 5% CO₂ to the headspace, CO₂ was cascaded to pH control and sparged to the culture based on demand.

Our cell culture inoculum expansion process was the same as described above. Data were consistent with the previous Wave study, indicating that during scale-up, four-day splits (rather than three-day) should be performed when culturing in bags.

Before inoculation, the bioreactor was charged with 5.93 L of CDM4PERMAb medium supplemented by l-glutamine and Pluronic F-68 solution and held overnight at 36.0 °C. At the seventh passage (n – 1), we used a total of 1.67 L of cell suspension from the WAVE bag to inoculate the bioreactor, which gave a postinoculation reactor volume of 7.60 L. We ran this bioreactor for 14 days, during which the VCD reached a maximum of 29.7 ×10⁶ cells/mL, with the viability dropping to 70% at harvest (Figure 4).

Figure 4: (Click to enlarge)

We used a continuous-feed strategy for fed-batch production. The feed consisted of two feed streams: custom feed media and a high-pH amino-acid supplement. Those were initiated when glucose levels dropped <2.5 g/L or on day 4, whichever came first. We then adjusted feed rates based on daily glucose readings, targeting a glucose level of 2.0–3.5 g/L. An average population doubling time (PDT) of 37.8 hours for this bioreactor was calculated from days 1 through 7 (data not shown).

After day 10, when cell densities reached 25 × 10⁶ vc/mL, cell culture growth leveled off. Despite the addition of glucose-containing feed medium and amino-acid supplement at increasing rates, glucose levels could not be maintained at our targeted amount under the high-density conditions that these cultures reached. By day 11, daily glucose reading was 0 g/L before each daily feed, indicating that the cells were consuming all that was fed to them each day (Figure 5). However, even with the steady decline of glucose levels in the culture, cells continued to proliferate and maintain high viability, with lactate levels remaining ∼1 g/L during the final days.

Figure 5: (Click to enlarge)

We harvested this bioreactor on day 14 at a viability of 72% (Figure 6). The overall titer for this first 12-L bioreactor was 0.585 g/L (Figure 7), 30% higher than that of the transferred process (0.450 g/L) run at a comparable scale (10 L). The total amount of product recovered from the bioreactor after filtration through two BioCap Cuno depth filters and one 0.2-µm Meissner filter was 60%.

Figure 6: (Click to enlarge)

Figure 7: (Click to enlarge)

Bioreactor Process Modification: To further improve productivity and make the process more suitable for large-scale production, we implemented a second 12-L bioreactor with a few modifications to its operating parameters. We increased the feed rate, removed one of two scoping impellers to match the design of our production bioreactor, and extended the run time. Results indicate that those modifications retained cell growth and viability similar to the first 12-L run. Cells reached a maximum VCD of 22.9 ×10⁶ cells/mL with a slightly longer average population doubling time of 48.2 hours from days 1–10 (Figure 6). This culture extended one additional day, with an increase in titer from 0.741 mg/mL on day 14 to 0.904 mg/mL on day 15 (Figure 7).

Despite the increased feed rate, glucose levels could not be maintained at targeted levels from days 9–15; they fell to 1.0 –1.4 g/L from day 9 to day 13 and dropped to 0 g/L by day 15 (Figure 8). However, even with the steady decline in glucose levels, cells continued to proliferate and maintain relatively high viabilities (Figure 7), which dropped to 72% at harvest on day 15. To test scalability, we transferred the process from the second 12-L bioreactor to the 250-L scale.

Figure 8: (Click to enlarge)

250-L Single-Use Bioreactor (SUB) Production: Our modified fed-batch process for a 250-L Thermo Scientific HyClone SUB system was based on expansion and production procedures used for the second 12-L run. Figure 9 and 10 compare cell growth and productivity of the two runs. The 250-L run ended on day 14 when the cell viability target level of 70% was approached. In this modified process, both cell growth and productivity were better than those in original process. Modifications made to that original process showed no adverse effect on either growth rate or productivity. So this bioreactor process is scalable.

Figure 9: (Click to enlarge)

Figure 10: (Click to enlarge)