Article 
Recognizing the Variability and Complexity of Antibodies
While the steps needed to produce mAbs are well-defined, the variability and complexity of these proteins, and the possible presence of numerous impurities, require an optimized purification process. Relative to small molecule drugs, mAbs are much less stable, have the potential to aggregate, and have a range of charges which can evolve during the production process.
These factors influence the number and types of chromatography steps needed to achieve sufficient purity. Each chromatography resin works within a defined set of technical parameters. Multiple consecutive steps using two or more resins are typically employed to purify mAbs. Given the differences in mAbs, the ideal purification strategy must be tailored for each protein with an understanding of how different buffers affect stability, which conditions promote aggregate formation, and how to maximize yield and purity — all while considering costs.
Separating Charge Variants
Ensuring homogeneity of the final target protein is essential to maximize stability and activity while minimizing the risk of immunogenicity. Homogeneity is also critical for biosimilars which must match the characteristics of the originator product. On the other hand, some sources of heterogeneity can pollute a mAb product. Charge variation is one type of heterogeneity caused by posttranslational modification (PTM) of the target protein. Some PTMs directly modify the net charge of proteins, while others induce conformational changes and variation of local charge distribution (Leblanc et al. 2017).
Variants of a mAb resulting from protein deamidation can pose a challenge for downstream purification. Deamidation modifies asparagine residues to aspartic acid and isoaspartic residues, resulting in the appearance of a negative charge. Similarly, glutamines can deamidate to form glutamate residues. Using high-resolution ion exchange (IEX) chromatography to separate closely related charge variants can help achieve a homogeneous product (Bio-Rad bulletin 6888).
Isolating Specific Antibody Forms
Bispecific monoclonal antibodies (bsAbs) simultaneously bind two distinct targets or epitopes to achieve novel mechanisms of action and efficacy. As with mAbs, product- and process-specific impurities must be removed during downstream processing of bsAbs. Protein A chromatography has been used as both a capture and polishing modality (Tustian et al. 2016), and ceramic hydroxyapatite chromatography can separate byproducts (Gagnon et al. 2006). Indeed, with its unique separation properties and unmatched selectivity and resolution, ceramic hydroxyapatite is ideal for the purification of bsAbs as well as other antibody classes, antibody fragments, and enzymes (Bio-Rad bulletin 7080).
Minimizing On-Column Aggregation
mAb aggregates can form during purification and alter the pharmacodynamics and pharmacokinetics of the target protein. This in turn can increase the potential for immunogenicity, reduce product yield, and increase costs. Aggregates can also interfere with subsequent processing steps, such as filtration and formulation. The proportion of on-column aggregate formation depends partly on the surface extenders, resin pore sizes, and resin pore size distributions, and partly on the interactions between the mAbs and the resin structure. (Guo and Carter 2015; Guo et al. 2016; Reck et al. 2017)
The percentage of on-column aggregation that occurs with different resins can vary significantly; in one study of several cation exchange resins, aggregation ranged from 10–87% (Bio-Rad Bulletin 7000). This disparity underscores the importance and need for screening multiple approaches during process development and controlling process conditions such as pH, ionic strength, and protein concentration. Without diligent screening, it is difficult to predict the interaction and elution behavior of different mAbs on a particular resin.
Ensuring Product Purity
Consistent product purity and quality can be challenging to achieve due to the complexity of mAb molecules and the presence of impurities. Common impurities include host cell proteins (HCPs) and DNA derived from the expression systems used for production, endogenous and adventitious viruses, mycoplasma, endotoxin, and aggregates of the mAb. Additional impurities may be introduced during production and include Protein A leached off the chromatography column used to capture the target protein, extractables, buffers, and detergents used for virus reduction.
The template for mAb purification typically includes Protein A affinity chromatography followed by two additional polishing steps such as anion exchange (AEX), cation exchange (CEX), hydrophobic interaction (HIC), and/or mixed-mode chromatography (MMC). When optimized for the specific mAb and impurities that may be present in the process stream, these steps should provide orthogonal modes of purification, enabling the effective removal of contaminants.
Maximizing Yield
Several factors can affect the final yield of the production process, including the number of purification steps, as well as the resin and buffer conditions employed. Removal of product- and process-related impurities typically requires multiple separation steps. In some cases, yield can be increased by reducing the number of steps, such as changing a three-step process (capture, intermediate polishing, and final polishing) to a two-step process (capture and polishing).
Resins and buffer conditions must be selected to either capture a protein in adsorption mode or allow the desired target to flow through for collection. For example, whether a resin is in a high salt or low pH buffer can dictate whether a mAb will bind strongly to the resin or elute from it. Buffer extremes, however, can also lead to protein denaturation and aggregation, which can reduce yield. More mild buffer conditions may result in less protein in the elution, but the protein may be more stable, and the eluate may contain fewer contaminants.
Finding the optimal balance between yield and quality can be a challenge. It requires careful optimization of purification conditions, including buffer composition, pH, salt concentration, and column material, among others. It may be necessary to use different purification strategies or techniques for different types of proteins, as each protein may have unique requirements for yield and quality.
Facilitating Scale-up
Scale-up of chromatography operations is usually achieved by increasing the column diameter while maintaining the resin bed height and linear flow rate. These changes ensure that the mAb's residence time on the column is the same at all scales of operation. Among the other factors that should be consistent when scaling are the buffer setpoints and tolerances in terms of pH and conductivity ranges, as well as quality inputs and outputs.
Among the most fundamental factors to consider when scaling chromatography steps is whether the resin used at laboratory scale is applicable for clinical- and commercial-scale operations. For example, size exclusion chromatography (SEC) is a common way to separate by size at laboratory scale but is not scalable due to the time required for elution, buffer consumption, and limited pressure and flow rate tolerance.
Resin cost can also become problematic: at a much larger scale, some resins may become prohibitively expensive. Even if a particular chromatography media is available in large quantities and reasonably priced, process developers should remain mindful of their technical and physical limitations in terms of their scalability. If the chromatography process does not scale linearly, an investment in optimization will be essential. It is also important to note that smaller-diameter chromatography columns have wall effects that are not present in larger-diameter columns. Literature suggests that the wall effect is a support to the media given by the column walls, resulting in better flow properties in smaller columns. This effect decreases with an increasing diameter of the column. Another known feature of the wall effect in small columns is the distribution of the sample, which tends to flow near the column wall when the wall effect is large.
Gaining Efficiencies with Prepacked Columns
Ensuring the highest quality chromatographic separation depends on many factors, including how well the column is packed. Columns must have homogeneous, stable, and continuous media beds from top to bottom. Successful column packing ensures proper mobile-phase distribution and resin contact, which helps to ensure desired yield, separation, and product quality. Poor-quality packing can lead to solute band broadening and compromise quality parameters. Lack of consistency and performance of chromatography columns at the pilot and production scale can prove extremely costly resulting in disruptions and delays in the manufacturing workflow and mAb batches that may have to be scrapped.
On the other hand, using prepacked pilot- and production-scale columns can shorten workflows, decrease variability, and eliminate the need for validation protocols and asymmetry testing to physically characterize the column. Prepacked columns also support process intensification efforts and facilitate tech transfers around the world by reducing the time needed to get a column in place and inconsistencies that can result from different packing techniques.
Optimizing Process Economics
Balancing process efficiency and cost-effectiveness of the chromatography workflow can be challenging. Exploration of different resins can help identify those that offer improved process economics.
For example, while Protein A is a highly effective capture step and widely used across the biopharmaceutical industry, it is expensive and can cost nearly 50% more than chromatographic media with nonproteinaceous ligands. Moreover, Protein A resin has many challenges such as ligand leaching, caustic instability, and limited resin lifetime. As such, it may not be cost-effective, particularly for the manufacturing of biosimilars. CEX resins offer an excellent alternative to Protein A for affinity capture of mAbs. CEX provides scalability, high binding capacity with lower material costs, and the option to be used at high flow rates to facilitate antibody capture (Bio-Rad bulletin 7134). In a workflow involving three purification steps, a comparison study of CEX and Protein A for the capture of an adalimumab (HUMIRA®) biosimilar showed a significant improvement in process economics with comparable product purity and activity (Bio-Rad bulletin 7130).
Conclusion
In 2021, the US Food and Drug Administration approved the 100th mAb product, thirty-five years after the first approval (Mullard 2021). These therapeutics continue to fill drug development pipelines and account for nearly a fifth of the agency’s new drug approvals each year.
The processes for manufacturing mAbs have evolved since their introduction, and they continue to improve, becoming more streamlined and efficient. However, the diversity and changeable nature of mAbs require that each production process be optimized to deliver the desired levels of yield and purity, including fine-tuning chromatographic purification steps. Consideration of a range of factors that influence the success of the chromatography workflow, such as those described above, will help minimize production difficulties.
References
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