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Downstream process development and manufacturing play a crucial role to ensure safety, quality, identity, purity, and efficacy.
A biotech company’s journey to develop a protein-based therapeutic drug from discovery, process development, manufacturing, and clinical trials to commercialization is long and complex. Multiple activities are involved, and many challenges are encountered when complying with chemistry, manufacturing, and control (CMC) requirements. Downstream process development and manufacturing play a crucial role to ensure the protein drug’s safety, quality, identity, purity, and efficacy (SQIPE) meet regulatory requirements for clinical trials and commercialization.
In this article, the authors analyze the challenges and seek effective solutions for CMC compliance (Figure 1), specifically focusing on protein complexity, raw material quality, upstream process (e.g., media source and titer), downstream process development (e.g., process capacity, efficiency, scalability, and control), manufacturing complexity, and product SQIPE.
Antibodies are complicated biologic molecules in their nature, including size (e.g., 150 kDa IgG–900 kDa IgM), heterogeneity (e.g., glycosylation, deamidation, oxidation), structure (e.g., monomer, dimer, trimer, hexamer), charge (e.g., pI of 5.0–9.0), hydrophobicity, and genetic source (e.g., rodent, human). Due to their intrinsic complexity, smart design for protein purification is necessary.
Raw material screening and selection is the first thing to be considered for purification. It is best practice to select a high grade of raw materials (e.g., chemicals) when process development is initiated. For chemicals, multi-compendial grade is the first option. Single compendial grade-such as United States Pharmacopeia–National Formulary (USP–NF), European Pharmacopoeia (Ph.Eur.), or Japanese Pharmacopeia (JP)-is acceptable if multi-compendial grade is not available. Finally, some chemicals may not be available in compendial grade. These need to be considered on a case-by-case basis regarding the purity, application, and option for substitution by a compendial-grade chemical.
Using a high grade of raw materials can minimize negative impact to product SQIPE and avoids potential timeline delays and reduces cost from process development, technology transfer, and manufacturing, to regulatory filing. The raw materials used are for upstream process (e.g., cell lines, media components, additives), downstream process development (e.g., chromatography resins, chemicals, buffers, membranes, filters, column housings, tubing, bags, tanks, water), formulation (e.g., excipients, buffers), and fill/finish (e.g., vial, prefilled syringe). It is important to monitor and control raw material critical attributes, grades, impurities (e.g., trace elements, leachables, extractables), lot-to-lot variations, safety (e.g., viruses, bacteria, fungi, mycoplasma, and endotoxin), and storage during manufacturing. Due diligence of vendors for critical raw material supply chain from primary and secondary vendors for late-stage and commercial production is highly recommended.
Harvest via clarification connects upstream and downstream processing. It is often conducted by centrifugation, depth filtration, microfiltration, or alternative tangential flow.
Depth filtration, a disposable technology, is routinely used for cell-culture harvest (bioreactor ≤ 2000 L). It removes cells, cell debris, and other impurities by size and/or charge. A number of depth filters are available on market (e.g., Millipore, 3M, Pall, and Sartorius). Although depth filters share similar mechanisms for particle and impurity removal, each filter has its unique properties in grade (e.g., low extractable and endotoxin), media feature (e.g., activated carbon, cellulose, glass fiber), charged or non-charged (e.g., positively charged ligand), capacity, and pore size (e.g., 2–20 µm). A properly designed depth filtration step could result in high viral clearance (>4 log10 reduction) (1) and impurity removal (e.g., host cell proteins [HCP]).
Disk-stacked centrifugation is designed for a large bioreactor harvest (e.g., ≥10,000L). A reliable scale-up and/or scale-down model between scales from bench, pilot, to manufacturing is essential for large-scale harvest and troubleshooting by small scale. A mature model can be established based on cell-culture conditions (e.g., cell line, cell viable density, cell viability, titer, and media osmolality), centrifugation system design, feed flowrate, bowl speed, sigma factor, discharge rate, and centrate quality (e.g., turbidity and/or lactate dehydrogenase activity). An automated centrifuge with a disposable flow-path marketed for cell therapy (Sartorius) eliminates cross-contamination, minimizes cell lysis, and enhances process efficiency, quality, and control.
The purification process for a monoclonal antibody (mAb) typically contains clarification, purification of capture, intermediate and polishing chromatography, viral clearance, final formulation, and bulk drug substance (BDS) filtration steps. The best downstream process should possess high capacity, efficiency, controllability, scalability, flexibility, cost-effectiveness, and biosafety (Figure 2).
A platform protein approach for mAb purification offers advantages (Figure 3) and can save time and resources, as well as expenses related to raw material screening, process parameter selection, process development, documentation preparation (e.g., using template strategy), technology transfer, scale-up, product characterization, formulation development, and regulatory filing. A platform approach also can anticipate process yield, product purity, and quality.
A mAb-purification capture step often uses Protein A resin, a 42-kDa surface protein originally identified from Staphylococcus aureus (2). Protein A resins are made of either native or engineered protein cross-linked to different matrix (e.g., Eshmuno A from Millipore, PraestoAP from Purolite, and MabSelect SuRe from GE Healthcare). This protein has a high binding affinity to mAb or Fc-fusion protein by hydrophobic interaction with Fc domain. To select a Protein A resin, one needs to consider the resin-binding capacity, specificity, stability, cleanability, sensitivity to caustic (e.g., sodium hydroxide) solutions, ligand detecting kit availability, flow capability, good manufacturing practice (GMP) supply, and resin cost. For a thorough review of the progress seen in Protein A chromatographic media, see Bolton and Mehta (3). The current major drawback from Protein A resin is its high cost (approximately $14,000/L for MabSelect SuRe).
Low pH retrovirus inactivation is usually conducted at low pH (3.3–3.6) for ≥60 minutes hold time if the mAb is stable at the test pH. Low pH retrovirus inactivation is a robust viral clearance step, which can reach >4 Log10 reduction value (LRV). It was reported >5 LRV under pH 3.9-4.1 (4). A previous study showed >5 LRV at pH 3.8 held for 60 min (unpublished data). It is known that pH, hold time, buffer matrix, acid (e.g., acetate, citrate, glycine), and temperature (15–30 °C) have impact on retrovirus inactivation kinetics (4). The lower pH, higher temperature, and longer hold time maximize the retrovirus inactivation, but this must be tempered by the sensitivity of the antibody. Current industrial practice is toward a modular approach based on the understanding and historical data from mAb, buffer type, salt concentration, acid type, and operating range for pH, temperature, and hold time. If mAb is unstable for low pH retrovirus inactivation treatment, detergent inactivation (e.g., polysorbate 80) or UV-C inactivation system (UVivatec, Sartorius) can be an alternative.
This step can result in a significant impurity removal (>150-fold HCP reduction) due to impurity precipitation and/or adsorption by a charged depth filter. This may allow a two-column process. The intermediate depth filtration process design parameters include pH range, hold time, mixing, temperature, HCP testing method, and depth filter type.
In most cases, CEX step is conducted after the intermediate pH hold and depth filtration step, which provide for the needed pH and conductivity adjustment prior CEX loading. CEX is performed in a bind-and-elute mode, which removes both process- and product-related impurities (e.g., HCP, rProtein A, aggregates, and acidic species). The drawbacks from this step are from its efficiency (e.g., resin diffusion) and viral clearance capability (<3 LRV for parvovirus reduction). However, it is still possible to have >4 LRV retrovirus reduction when proper resin and operating condition are designed and developed.
In either resin or membrane format, AEX is often carried out after the CEX step to further remove process-related impurities (e.g., HCP, DNA, endotoxin) and viruses. The AEX step is an effective step to remove both retro- and parvoviruses. The CEX and AEX column order can be switched to overcome issues related to equipment, process, or facility. For example, if the CEX eluate salt concentration were too high, it would need a significant dilution to decrease conductivity prior AEX load and this could encounter limitation of equipment (e.g., tank size) or facility fit (e.g., suite size). Under these circumstances, AEX could be considered as second column step (unpublished data).
AEX-membrane chromatography (e.g., Sartobind Q) has unique advantages in process efficiency, buffer consumption, disposability, and is ready-to-use. Its shortcomings are mainly from its low-binding capacity and cost (e.g., at large scale-up). Therefore, it is ideal to put it after CEX column, which will minimize impurity breakthrough and enhance viral clearance capacity. Natrix Separations’ 3-D hydrogel membrane columns (e.g., NatriFlo HD-Q) overcome the limitations from conventional membrane chromatography (5).
Small viral retentive filtration is a robust viral clearance step (>6 LRV for parvovirus). A number of commercial viral filters are available (e.g., Sartorius, Merck Millipore, Pall, Asahi-Kasei). The viral filter selection depends on filter pore size (e.g., 15–35 nm), filter design (e.g., dead-ended, hollow-fiber), capacity (e.g., L/m2, g/m2), and cost. Process conditions (e.g., product purity, pH, conductivity, buffer matrix) and recovery must also be considered. Different viral filters have different load capacity (e.g., >10,000 g/m2 from Planova 20N BioEX) without significant flux decay (unpublished data). High capacity can reduce raw material cost significantly. To improve both viral clearance capability and process-related impurity removal (e.g., HCP, DNA), the authors expect vendors to develop innovative viral filters with dual functions of size exclusion and charge in the future.
Ultrafiltration and diafiltration is employed for mAb formulation by protein concentration and buffer exchange into final formulation buffer. Different types (e.g., A, C, D) of membranes (e.g., regenerated cellulose or polyethersulfone) are available for screening and selection based on recovery, product quality, and process efficiency. For bulk drug substance with high concentration (>100 g/L) and viscosity (>10 centipoise), C or D type of membrane with special system set-up and design (e.g., retentate solution level higher than inlet of membrane) is recommended.
Bioburden and endotoxin are strictly controlled to the lowest level during DSP and manufacturing to minimize safety risks to the patient. Bioburden and endotoxin excursions occasionally have been reported from poor environmental monitoring and control, insufficient equipment cleaning and maintenance (e.g., suite, biosafety cabinet), and lack of standardized in-process 0.22-μm filtration per unit operation. Personal hygiene and gowning are other possible causes.
Multiple major challenges for biologic drug development and manufacturing have been reported from early- to late-stage of clinical trials. Manufacturing changes that are implemented in late-stage development include upstream processes (e.g., cell line and media change to improve titer, implementation of centrifugation for late-stage harvest), downstream processes (e.g., resin and/or column order change to improve capacity, efficiency, and viral clearance capacity), technology transfer, scale-up (e.g., equipment), facility fit (e.g., disposable, stainless steel), formulation change at late stage, and DS/DP specification setting.
When harvest material with very high titer (>10g/L) is generated from the upstream process, the downstream processing can become the bottleneck due to its intrinsic limitation in process capacity, efficiency, and cost from resin-based purification technology.
Large-scale manufacturing (>10,000L stainless steel bioreactor) becomes less flexible due to its larger footprint of equipment, tank size, filter housing, cleaning-in-place, steaming-in-place, change-over, system validation, and maintenance.
In-line and on-line analytical methods are still not fully available to monitor product quality (e.g., charge variance, glycan), stability (e.g., aggregates, fragments), purity (e.g., monomer %), activity (e.g., enzyme-linked immunosorbent assay binding), and impurity (e.g., rHCP, rDNA, rProtein A).
Manufacturing site and scale changes cause unforeseen scale-up issues including cell culture grown profile, harvest quality, centrifugation efficiency, elution volume increase by additional buffer chase through pipe, filter clogging due to slightly material differences between lab and manufacturing.
Documentation is one of the crucial activities for the drug manufacturing lifecycle. It is important to capture correct information from process parameters, parts, in-process sampling, storage condition, retains, testing methods, and drug release specifications.
For higher titer harvest, continuous Protein A capture is the most promising option using new resins with high binding capacity, affinity, stable ligand, and low cost (Figure 4). This technology has been successfully conducted at lab, pilot, and manufacturing scale (e.g., Pall’s Cadence BioSMB, GEH’s periodic countercurrent chromatography, LEWA EcoPrime, Novasep BioSC, Semba Octave). It significantly reduces process time, resin cost, column size, and buffer amount. A nanofiber-based Protein A resin from GEH could make a revolutionary change to enhance process efficiency, flexibility, and robustness (6).
A non-Protein A process is an alternative to reduce resin cost and potential Protein A immunogenicity in some patients. A process that included CEX, multimodal, and AEX (e.g. Sartobind STIC) has demonstrated desirable recovery, purity, and impurity from mAb purification (7). Other technologies such as crystallization may be another alternative to replace Protein A (8, 9). Crystallization has been used for enzyme and insulin purification for decades (10).
The low pH retrovirus inactivation step can be integrated into the Protein A step by controlling elution buffer pH and volume without pH titration. As described previously, it is feasible to have >4 LRV for retroviral inactivation when pH is controlled between 3.4–3.7 and hold time for 60–90 minutes. Alternatively, a single-use virus inactivation system (e.g., Cadence VI system) can be adopted as a robust tool for the continuous intermediate pH hold (e.g., pH 5.3 for further HCP removal) and subsequent step load conditioning.
To connect the second column (e.g., CEX) with the third one (e.g., AEX), in-line buffer dilution and pH titration system can achieve the desire load pH and conductivity for the third column load. A flow-through (overloaded) mode CEX showed higher capability and impurity removal (11). This allows both second and third columns in flow-through mode (e.g., AEX). Biogen has demonstrated a flow-through mode platform for both AEX and HIC (12). The platform eliminates intermediates pH/conductivity adjustment between two columns and reduces process time, cost, and hardware (e.g., tank).
In conclusion, mAb purification from process development to large-scale manufacturing is evolving rapidly. To overcome bottlenecks from the upstream process, lower efficiency, capacity, and discontinuous processing nature from downstream processing, the continuous downstream process strategy is currently one of the most promising options. This approach relies on new type of Protein A resin, which should have higher binding capacity and resistance to CIP. The intermediate polishing steps should possess high capacity and throughput. To transform the laboratory-scale continuous process into large-scale manufacturing, a biopharmaceutical company has to smartly design a “new era” mAb purification process based on molecule complexity, manufacturing complexity, online product purity and quality monitoring, control, biosafety assurance, CMC compliance, and regulatory requirements.
It is exciting for downstream scientists to explore novel disruptive technologies such as crystallization to replace Protein A step by direct capture mAb from high titer harvest to make a revolutionary move for future mAb purification process development and manufacturing.
The authors sincerely thank Dr. Michael Ultee for his review and invaluable inputs.
Yanhuai (Richard) Ding, PhD, is director, downstream process development and manufacturing; Peggy Marino, PhD, is vice-president, program management; and Hemant Kumar, PhD, is senior vice-president, head of process development and manufacturing, all at Anaptysbio.
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Vol. 32, No. 12
When referring to this article, please cite it as Y. Ding, M. Marino, H. Kumar, "Antibody Purification Process Development and Manufacturing," BioPharm International 32 (12) 2019.