Purification of IgM Monoclonal Antibodies - Manufacturing challenges surround the use of IgM monoclonal - BioPharm International

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Purification of IgM Monoclonal Antibodies
Manufacturing challenges surround the use of IgM monoclonal


BioPharm International Supplements


Results and Discussion

Initial Screening


Figure 1. Hydroxyapatite screening profiles at different monitor settings. Conditions as in Table 2
A few milligrams of highly enriched product are a valuable asset in early method development because they allow product and contaminant behavior to be evaluated visually from chromatograms. This evaluation permits initial screening and process optimization to be done without secondary testing, and it accelerates development. Enriched product is easily obtained by Protein A affinity chromatography for human IgG monoclonals. Hydroxyapatite provides a useful alternative for IgM. Figure 1 illustrates screening results for an IgM on hydroxyapatite. The IgM peak is clearly identifiable, and the majority of contaminants flow through the column during sample application. Elution between 200 and 300 mM phosphate is the norm.7


Figure 2. Comparison of screening and optimized profiles on hydroxyapatite. Conditions as in Table 2
Collecting the IgM peak from an initial hydroxyapatite screening run typically provides antibody of 65–80% purity. This purity is sufficient to proceed with initial screening of anion and cation exchange, but only a few more experiments are required to produce a reference sample that better reflects antibody purity under process conditions, which is typically approximately 90%. Figure 2 compares the initial screening profile with the profile from an optimized gradient. Conditions were set so that most contaminants were either eliminated in the pre-elution wash, or were left on the column after the antibody had eluted. The 1-mL column was subsequently loaded with 50 mL of CCS containing approximately 12.5 mg of IgM, then eluted with the optimized gradient. IgM from this run was used to evaluate other methods.


Figure 2. Comparison of screening and optimized profiles on hydroxyapatite. Conditions as in Table 2
Figure 3 illustrates screening results for an IgM on cation exchange at pH 7.0. The profile is reminiscent of hydroxyapatite in the sense that the IgM binds much more strongly than is usual for IgG antibodies, and the majority of host cell proteins fail to bind. Figure 4 compares retention characteristics at pH 6.0, 7.0, and 8.0. Binding is weaker at pH 8.0, but purification is improved remarkably. These results suggest a process strategy where the IgM can be bound at pH 6.0 to maximize capacity, then washed and eluted at pH 8.0 to maximize contaminant removal.


Figure 3. Cation exchange screening profile at pH 7.0 with different monitor settings. Conditions as in Table 3
Figure 5 illustrates screening results for an IgM on anion exchange at pH 7.0. The upper trace was produced from hydroxyapatite-purified reference, the lower trace from CCS. Again, the IgM binds much more strongly than is typical for IgG monoclonals, and the degree of purification appears to be excellent; however, a substantial proportion of contaminants also bind. Strong binding by IgMs may support a process strategy such as that discussed above for cation exchange: Bind at the pH that supports the highest capacity, and wash and elute at the pH that supports the most effective contaminant removal.


Figure4. Cation exchange screening profiles at pH 6.0, 7.0, and 8.0. Conditions as in Table 3
IgM typically elutes from weakly hydrophobic columns in a single well-defined peak, as shown by the ETH profile in Figure 6, but this antibody was severely denatured by the more strongly hydrophobic PHE column. The small first peak was the only remnant of native IgM. The later eluting dominant peak and shoulders represent denatured forms. They were turbid on elution and precipitated overnight at 4 C. Although weakly hydrophobic columns avoid the denaturation problem, they leave another challenge in its place: The IgM elutes at very high salt concentrations, which can be difficult to accommodate in later process steps.


Figure 5. Anion exchange screening profiles of CCS and hydroxyapatite-purified reference. Conditions as in Table 3
HIC media with intermediate hydrophobicity offer a practical solution. As shown in Figure 7, the IgM elutes from the PPG column as a well-defined peak near the end of the gradient. Conductivity of the eluted IgM pool is sufficiently low to support binding to an ion exchanger with moderate dilution. Another benefit of HIC on moderately hydrophobic supports is that the high salt required for binding can dissociate ionic complexes that may exist between IgM and contaminants of opposite charge.7 This fact can be important, because the same positive charges that cause IgM to bind strongly to cation exchangers can just as easily bind DNA fragments. This is highlighted by the use of immobilized DNA for affinity purification of IgM.28 As with hydroxyapatite and cation exchange, the majority of contaminants flow through HIC columns on sample application (Figure 7).7

Sample Application and Binding Capacity


Figure 6. HIC profiles on weak and strong hydrophobic media. Blue profile: RESOURCE PHE (GE Healthcare). Green profile: RESOURCE ETH, 1 mL, 2 mL/min (approximately 600 cm/hr). Equilibrate with 1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0. Inject 20 μL (200 μg) hydroxyapatite-purified mouse IgM. Wash 2.5 Cv with equilibration buffer. Elute with a 10 Cv linear gradient to 50 mM sodium phosphate, pH 7.0.The colored areas represent native fully active IgM.
Loading sufficient IgM to obtain antibody for characterization is simple on hydroxyapatite because it accommodates filtered CCS with only minor adjustments. If the CCS contains less than 5 mM phosphate, then phosphate should be added to that concentration to stabilize the hydroxyapatite during large-volume sample applications. Titration may also be required to bring the sample to operating pH. Dynamic binding capacity (5% breakthrough at 200 cm/hr with a 10 cm bed height) was approximately 23 mg/mL for one IgM, and approximately 19 mg/mL for another. Dilution to reduce conductivity increases binding capacity for purified IgM. For CCS, however, it may allow more contaminants to bind, and so it may potentially reduce net product binding capacity. Dilution also increases preparative sample application time, which already accounts for several hours at 200 cm/hr. IgM capacity increases at slower flow rates, or with longer residence time at the same flow rate on a taller bed.

Sample loading is more complicated for ion exchangers because many IgMs begin to develop turbidity soon after they are equilibrated to the low conductivity conditions required to support high binding capacities. Turbidity may be absent initially, but it tends to form progressively over time. This fact not only risks product integrity, it also creates a source of process variation, because sample composition varies over the duration of the load. Turbidity can be measured conveniently on a spectrophotometer at 600 nm. Solubility limitations generally disqualify bulk offline sample equilibration of IgMs for ion exchange. Sample application by inline dilution provides an alternative.7 Sample is titrated to target pH and loaded through one inlet line. Diluent buffer at target pH is loaded through another. Residence time of the IgM at dilution, defined as the elapsed time from the point of mixing to column contact, represents seconds or fractions of a second, depending on flow rate and configuration of the chromatography system. The short duration is generally insufficient time for turbidity to develop. An equally important benefit is that sample composition is uniform for the duration of the load, no matter how long that duration might be.7

Inline dilution factors vary according to the salt concentration of the sample, the charge characteristics of the individual antibody, and the desired binding capacity. A dilution ratio of 1 part sample to 2 parts diluent is a reasonable starting point with feed-streams at roughly physiological conductivity, although higher dilution is likely to support higher binding capacity. Samples with higher conductivities may require still higher dilution factors. Any dilution invites criticism for water or buffer consumption and increased column loading time, but it is important to consider that inline dilution eliminates the need for diafiltration with its attendant equipment, equilibration, and chase buffers; preparation, process, and maintenance time; cleaning validation; and inevitable product losses. Moreover, monoliths support such high volumetric flow rates that loading time ceases to be a consideration. Capacities for hydroxyapatite-purified reference IgM on monoliths ranged from 30 to 40 mg/mL for both anion and cation exchangers at flow rates of 12 Cv.

Inline dilution is necessary for HIC as well, because the concentration of salt required to support good binding capacity on moderately hydrophobic columns is typically sufficient to precipitate the antibody.7,29 Development of inline dilution conditions for HIC on such ligands is discussed in depth by Gagnon, Grund and Lindback.29


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