Cost Effective Protein A Media for MAb, BsMAb, and Tribody Purification and SMB Process Platform Consideration

The use of protein A media in the mAb purification platform is a well understood and characterized method used in the biopharmaceutical industry. Protein A was first discovered in 1958 by Klaus Jensen. Klaus discovered that the antigen from S. aurens precipitated ~500 human serum samples. In 1965 it was found to bind to the FC region of IgG's. Furthermore, peer reviewed consensus states that the affinity interaction with IgG is within its VH3 region. The binding can be achieved at neutral pH and dissociated via decreasing pH. The first immobilization occurred in 1972 to a polymeric backbone (not agarose) and commercialized in 1978. A patent was filed in 1973 and granted in 1986.

The first significant protein A sales started in about 2000. The cost of protein A is very expensive (up to 16K/L) which makes it hard for smaller companies to produce MAb's of interest. If the company cannot achieve funding, it must license the MAb, sell the company, or simply dissolve. Alternative and cost effective technologies will take time before they replace the existing preferred (FDA accepted processes that use these resins) vendors and platforms. One of the bottle necks seen in the COVID crisis was that few pharmaceutical companies have qualified secondary or tertiary sources for protein A (or other processing media) and the supply chain consequently was at risk.

Protein A is a relatively simple technology that binds an antigen to a support. The support itself must be biocompatible with a MAb to prevent non-specific interaction. Such interaction can affect MAb recovery, increase HCP carry-over, and also lead to higher EU residuals. The protein A resin itself must not leach protein A and have GRAS raw materials used in its manufacture. All of this information is provided in a regulatory support file (RSF) giving the manufacturer assurance that the processing material complies to their cGMP standards.

Sepax Technologies has invested significant resources in making a unique cGMP compliant protein A media that not only has high MAb capacity (DBC) vs. the competition but has characteristics that give it a competitive advantage in the BsMAb and Tribody purification process.

Figure 1: Sepax Protein A Characteristics:

The figure above shows a 50-70mg/mL capacity with 4-5 minutes residence times with 0.5M caustic soda stability. All back pressures are stable up to 10 bar making the line attractive to higher velocity systems like SMB and processes that benefit from higher aspect ratio columns. Higher aspect ratio columns stretch the adsorption band allowing for delayed breakthrough and more use of the resin bed.

Another aspect that makes the Sepax Protein A (proA) resin suite interesting is the mono dispersed particle size distribution and residual HCP vs. the competition. A monosphere bead distribution allows for the most efficient elution kinetics (normalized), lower pressure drop, and easy packing qualification. Figure 2 below plots HCP/DBC level vs the competitors as well as illustration of particle size distribution.

Figure 2: DBC and Particle Attributes-P45

A critical issue for new process or design is the use of equipment that is cost effective and reliable. The current platform on the market uses low aspect ratio "short and fat" columns that are expensive and have high maintenance costs. These systems were designed for agarose based resin since they are soft and compressible. The drawbacks of these systems are significant downtime in bed stratification, clogging/fouling, and counter-current cleaning (for fouling). Flow velocities have to remain low to prevent irreversable compression of the bed. Such compression could be very expensive...ie.. 300L x ~12-13K/L Resin to replace.

Figure 3 looks at the hydraulic characteristics of the Sepax ProA product line.

Figure 3: DBC Vs. Residence Time and Pressure Plots A45/P45

Flow rates for P45 (polymeric) are linear up to 500cm/hr under 2.5 bar which make it attractive to systems that is easier to manufacture and cost significantly less. Also SMB systems require higher aspect ratios to utilize the entire resin bed (unlike agarose based resin traditional platforms). It is important to note that SMB systems are expected to replace existing affinity platforms since they use 80% less resin and have higher throughput. Such systems will require more mechanically stable resins with smaller particle size to support the demands of continuous chromatography.

Link 1: Describes SMB and Its Uses

Another important aspect of protein A is its stability in sodium hydroxide. Many vendors claim stability in 1M caustic soda at the expense of resin life time. The figure below shows Sepax protein A resins vs. the competition in 0.5M NaOH.

Figure 4: Caustic Stability Data Vs. Competitors-P45

Figure 4 compares our media against MabSelect Sure. The P45 shows ~87% recovery after 200 cycles with 15 minutes of contact time per cycle. The stability is not only due to the resin base matrix but also the genetically engineered ligand as well.

A critical attribute of protein A resins is their relative life cycle and HCP carry over. Figure 5 looks at both protein A leaching and HCP residual analysis.

Figure 5: Lifetime Studies and HCP Analysis

BsMAb purification with Sepax proA. Bispecific MAb's for therapeutics is a growing trend in the industry due to its affinity for more than one antigen site. This MAb type aids the immune system or ADC to target multiple sites increasing the effectiveness of treatment. The data below summarizes a BsMAb purification using our media. The type of MAb used is this study is unique because it has no FC region just a Kappa light chain type. The type of MAb also has a unique hydrophobicity giving it a different selectivity for processing crude feedstock.

Protein L is typically used to purify these MAb's because of the affinity difference compared to standard protein A. Since the lead times of protein L long and the cost was triple then our cost, it was an attractive option for them to evaluate our P45. After an extensive study showing the purification run and the analysis confirmed with SEC, the customer reported less LMW fragments (4% vs. 6-7%), and 2X less residual HCP vs. the competition. The cost was also significantly less as well.

Figure 5: Purification of BsMAb (No FC region Kappa Light Chain Type)-P45

Another attractive therapeutic is tribody MAb's for multiple point attachment in various therapeutic targets. A customer was interested in evaluating our P45 for a tribody purification to see if it can be used in this application. The crude feedstock via SEC analysis had ~ 57% MAb. The results of the DBC curve in Figure 6 revealed a capacity of 46g/L.

Figure 6: Tribody DBC-P45

Figure 7 looks at the 3 individual elution conditions with acetate, citrate, and glycine and compares the purity of all three via purification fraction analysis with SEC. All three conditions revealed ~96% purity proving that our P45 protein A is suitable for this tribody purification.

Figure 7: Tribody Purification and Elution Buffer Robustness-P45

Conclusions:

The above data demonstrates a unique protein A that can be used as a candidate across standard MAb, BsMAb, and Tribody MAb. The hydraulic characteristics of this particle (rigidity, hydrophilicity, and uniform particle) make it suitable for more advanced platforms like SMB, CCTC (continous Counter-Current Tangential Chromatography), and other higher pressure/flow systems to reduce processing costs and increasing throughput. There are many process alternatives to consider that can decrease costs and increase throughput with the equipment that has been proven safe and reliable throughout the years.