Discussed is our strategy for assessing and developing a downstream process for complex IgGs. With a focus on product-related variants, quantitating and eliminating chain-mispaired species such as homodimers is a priority. Our approach leverages high-throughput screening and orthogonal analytics, and examples are presented from a few case studies.

A novel diabody-cytokine fusion protein presented unique impurity and stability challenges. To address these issues, purification process was developed via a tiered approach which consists of initial device/condition screening, end-to-end process confirmation, and key step robustness evaluation. The final process achieved 20-fold reduction in aggregate and nearly 6-log reduction in host cell protein (HCP). This exploration enabled high-quality purification of the novel diabody and expanded the purification development toolbox with new technologies.

In this study, we explored how a new downstream AEX single-use technology adds flexibility to a traditional downstream unit operation. This novel technology combines a guanidinium-functionalized membrane with Q-functionalized fibers to enhance process flexibility. Four feed streams were evaluated for HCP clearance, DNA reduction, and viral clearance at different buffer and salt conditions. These results conclude that it can be applied across multiple modalities, simplifying the downstream development and manufacturing process.

It has been suggested that "new therapeutic modalities will require new modes of separation." This laboratory has developed a novel family of polymer fiber stationary phases to affect the isolation of exosomes and viruses using a hydrophobic interaction chromatography elution scheme. In the case of isolation from cell culture media, samples are injected on-column under high salt conditions, the ionic strength reduced to elution host cell proteins, and finally, the salt eliminated with high purity vesicles readily collected. The platform technology is operable across many scales, from clinical to research to date. Challenges, accomplishments, and paths forward will be presented.

Gene therapy attempts to modify or manipulate genes to treat or stop diseases. Applications of viral vector gene delivery systems have shown some encouraging results in treatments of inherited retinal diseases and spinal muscular atrophy (SMA). Viral vectors packaged with genes through a transfection process are frequently produced from cells. Vectors are released from production cells at the end of the cell culture through surfactant-based cell lysis. Triton X-100 (TX-100) is one of the most popular lysis reagents used in gene therapy industry. However, Triton X-100 has endocrine disrupting properties which elicit environmental concerns. Therefore, this chemical is listed as an emerging compound of concern by many European countries. This study evaluated a few environmentally friendly surfactants as lysis reagents for vector release. The lysis efficiency and filterability of resulted lysate of each surfactant were first studied through bench-scale experiments.

Despite the rapidly growing commercial interest in extracellular vesicles, manufacturing processes for EV production are still in the development phase. The use of the glycosaminoglycan, heparin, to capture extracellular vesicles is a relatively novel and unexplored method of purification. In this study, we demonstrated the use of heparin affinity chromatography to both purify and fractionate extracellular vesicles into subpopulations.

Successful gene therapy using AAV vectors requires high titers of functional virus. A challenge for AAV production is enriching full capsids by anion-exchange chromatography. Each capsid and transgene modification requires specific chromatography buffers with discreet, critical parameters for optimized production. Teknova’s ability to manufacture high-quality custom buffer formulations, combined with our in-house purification platform, allows us to identify the ideal buffer candidate quickly and effectively for use in scale-up and GMP production.

The advantages of processing platforms include shorter development times and reduced risks in terms of developability and manufacturability. Additionally, extensive knowledge gained from the broad and frequent application generates high problem-solving competence along the whole process chain. To leverage this huge potential for non-mAb proteins, we developed the CASPase based fusiON (CASPON) platform consisting of an immobilized metal affinity chromatography step, followed by enzymatic tag removal via a modified human caspase-2 enzyme incorporating a poly-histidine-tag. The platform not only allows for generic DSP but introducing additional functionalities into the tag sequence makes the technology largely universal for the USP process.

Polysorbate 80 is commonly used as a surfactant in the therapeutic protein formulation. Lipase-mediated enzymatic polysorbate degradation is one of the challenges in the manufacturing and storage of the biologics. Typically, a trace amount of residual host cell proteins (HCPs) with lipase enzymatic activity could result in the polysorbate degradation in drug substance during storage. In this study, a series of the chromatographic approaches were evaluated to minimize the lipase risk in a bispecific antibody downstream process which has successfully been scaled up to the manufacturing scale.

For cell-derived products, it is critical to demonstrate removal of viral contaminants, host cell DNA, and protein impurities. This presentation will discuss use of novel additives to drive downstream process efficiency. Case studies will show various processes from mAb to viral vector. Examples from a biodegradable detergent and a novel Protein A resin will be explored.

Two imaging techniques were used to visualize and characterize sterilizing grade filtration of liposomes, examining a dual-layer polyethersulfone system. X-ray computed tomography resolved internal nanoporous structure that enabled pore size distributions to be calculated for both layers. Confocal microscopy identified the location where liposomes were retained within membranes after filtration, with three differential pressures compared for processing performance and retention profiles as a function of distance through each layer.

The current study explores the use of DSPX, a mechanistic modeling software, to simulate an HIC process used in the downstream purification of a therapeutic protein expressed from E. coli. A mechanistic model is the basis for digital twin, allowing the simulation and prediction of chromatography performance, which may be applied to design space characterization, process monitoring, and control. In this study, following calibration of the initial model, its application for process optimization was explored.  

An FTIR model calibration for the quantitative determination of tier in harvested broth to facilitate the downstream purification. We applied design of experiments and data pretreatment methods to generate the model based on a relatively small sample set.

We have developed a powerful purification platform based on a self-removing affinity tag that can serve both basic research and clinical manufacturing applications. In practice, a simple shift in buffer pH releases the purified tagless target from the affinity column, which can then be stripped and reused. The power and versatility of this system is demonstrated here using several case studies on proteins expressed in bacterial and mammalian host cells.

Lack of consistent, scalable Protein A membranes has limited membrane chromatography’s potential in antibody-based therapies. Gore’s high-throughput affinity capture devices surpass the limits of existing bead and membrane options. GORE® Protein Capture Devices achieve rapid cycling, high binding capacity at short residence times and low, consistent column pressure-drop. Our data demonstrate increased productivity and scalable performance across commercial sizes up to 232 mL and larger next-generation devices. 

A novel single-use based clarification platform was developed combining fluidized bed centrifugation with integrated filtration. The feasibility of this streamlined setup was investigated up to 200-liter bioreactor scale, where low harvest turbidity (2 NTU) and superior antibody recovery (95%) were achieved. A cost-of-goods (CoG) analysis, comparing conventional depth filtration with the developed unit operation, showed enormous potential to save more than half of the clarification costs in industrial biopharmaceutical manufacturing.

Adeno-associated viruses (AAV) have allowed for rapid advancements in the biotherapeutic landscape. Although AAVs can deliver genes in a targeted and efficient manner, the presence of empty capsids – a by-product of AAV production – raises great safety concerns. Anion exchange chromatography (AEX), often used for full-capsid enrichment, commonly requires extensive development work. Hence, we established a systematic workflow for the AEX optimization of different AAV serotypes. This platform utilizes high-throughput technology for stability studies and resin-binding optimization, along with a methodical screening of elution conditions. Our strategy has demonstrated to be to an efficient route for AEX development.

Chromatography is becoming an increasingly popular tool for the purification of rAAV capsids. However, efforts toward the development of a platform purification process for rAAV vectors are still in their nascent stages, unlike those of protein therapeutics. The purification of rAAV capsid with these resins can present challenges. Product recovery due to nonspecific binding and diffusional limitation into the porous bead may play a role in resin selection. In this talk, we share our investigation of how capsid-resin interaction impacts purification performance, product recovery, and product stability.

AAV continues to provide clinical success, but still needs significant improvements in the processes used to purify it. Alongside this, there remains a lack of information on the purification of various serotypes and transgenes. This presentation will describe our efforts to report the purification of multiple serotype and transgene combinations across platform purification steps.

In recent years, significant resources have been invested into increasing the productivity of AAV manufacturing. Optimization of upstream processes has led to significant increases in AAV titer, and downstream purification strategies designed around lower-yielding production must be reevaluated to better accommodate the large increases in vector. This presentation highlights work done to modify downstream purification steps to handle a titer increase seen during the implementation of a next-generation upstream process.

Variable empty/full distribution coming out of the upstream unit ops coupled with the fact that empty particles co-purify with full particles remain a challenge in rAAV manufacturing. Modulating the mobile phase composition during the AEX load enables the flow-through or “partitioning” of empty capsids while selectively retaining full rAAV vectors. Minimizing empty capsid binding simplifies the elution strategy which can be a significant benefit during tech transfer and scale-up.

This talk will discuss transformative technologies in the production, filtration, and chromatography-based purification of AAV with real-life examples of the challenges that have been overcome. Some real-life examples will include the development of depth filtration for suspension AAV production, which increased capacity and recovery by 4x and 2x respectively, and a new strategy for ensuring viral clearance that resulted in >85% recovery.