Accelerated development of a SEC-HPLC procedure for purity analysis of monoclonal antibodies using design of experiments.

The complex structure of biopharmaceutical products poses an inherent need for their thorough characterization to ensure product quality, safety, and efficacy. Analytical size exclusion chromatography (SEC) is a widely used technique throughout the development and manufacturing of monoclonal antibodies (mAbs) which quantifies product size variants such as aggregates and fragments. Aggregate and fragment content are critical quality attributes (CQAs) in mAb products, as higher contents of such size heterogeneities impact product quality. Historically, SEC methods have achieved sufficient separation between the high molecular weight (HMW) species and the main product. In contrast, some low molecular weight (LMW) species are often not sufficiently different in molecular mass from the main product, making it difficult to achieve appropriate resolutions between the two species. This lack of resolution makes it difficult to consistently quantify the LMW species in mAb-based therapeutics. The following work uses a design of experiments (DoE) approach to establish a robust analytical SEC procedure by evaluating SEC column types and mobile phase compositions using two mAb products with different physiochemical properties. The resulting optimized procedure using a Waters™ BioResolve column exhibits an improved ability to resolve and quantify mAb size variants, highlighting improvement in the resolution of the LMW species. Additionally, the addition of L-arginine as a mobile phase additive showed to reduce secondary interactions and was beneficial in increasing the recoveries of the HMW species.

A case study application of AQbD to the re-development and validation of an affinity chromatography analytical procedure for mAb titer quantitation.

Protein A (ProA) high-performance liquid chromatography (HPLC) is a common analytical procedure for measuring monoclonal antibody (mAb) titers due to its high specificity and efficiency. Accurate and reliable results of this procedure are imperative, as the quantitation of the total mAb present for in-process samples directly impacts downstream purification steps related to the removal of process-related impurities. This study aimed to improve a platform ProA HPLC analytical procedure which was previously developed using traditional approaches and was not always reliable. By retrospectively applying Analytical Quality by Design (AQbD) principles and statistical assessments of performance, a bias in the calibration standard due to protein-adsorption to common sample vial materials was identified. The inclusion of Tween® 20 into the mobile phase used as sample diluent was optimized to ensure procedure performance and improve analytical range. The resulting procedure robustness was evaluated using Design of Experiment (DoE) approaches and performance was verified against Analytical Target Profile (ATP) criteria as recommended by regulatory agencies. The resulting linearity displayed R2 values of 1.00 with intercept biases of 1.2 % (analyst 1) and 0.8 % (analyst 2), accuracy across all levels was reported at 99.2 % recovery, and intermediate precision was reported as 3.0 % RSD. Application of this new platform procedure has since reduced development timelines for new mAb products by 50 % and allowed for accurate titer determination to support >5 early phase product-specific process decisions without requiring extensive analytical procedure development. This work demonstrates the utility and relative ease of adopting AQbD concepts, even for established procedures, and supporting them with a lifecycle approach to managing procedure performance.

Analytical Quality by Design as applied to the development of a SEC-HPLC platform procedure for the determination of monoclonal antibody purity without mobile phase additives.

This work presents the application of AQbD principles to the development of a size exclusion chromatography (SEC) HPLC procedure for the determination of monoclonal antibody (mAb) product purity using state-of-the-art column technology available via the Waters™ XBridge Premier Protein SEC column. Analytical Quality by Design (AQbD) emphasizes a systematic, risk-based lifecycle approach to analytical procedure development based on sound statistical methodologies. It has recently become increasingly recommended by regulatory agencies as a response to the need for greater efficiency, improved reliability, and increased robustness among modern analytical procedures in the pharmaceutical industry. Use of an Analytical Target Profile (ATP) and formal risk assessments informed the application of Design of Experiments (DoE) to optimize this analytical procedure, as well as assess its robustness and ruggedness. Importantly, our ruggedness results demonstrated the transferability of this procedure between two laboratories within the Catalent Biologics Global Network. Application of this analytical procedure as a platform approach for evaluating mAb purity is expected to support expedited, first-in-human timelines of mAb molecules by enabling great quantitative performance with simple mobile phase buffer compositions. Taken together, this case study demonstrates the utility of adopting AQbD principles in analytical procedure development.

Report on the international workshop on alternative methods for human and veterinary rabies vaccine testing: state of the science and planning the way forward.

Potency testing of most human and veterinary rabies vaccines requires vaccination of mice followed by a challenge test using an intracerebral injection of live rabies virus. NICEATM, ICCVAM, and their international partners organized a workshop to review the availability and validation status of alternative methods that might reduce, refine, or replace the use of animals for rabies vaccine potency testing, and to identify research and development efforts to further advance alternative methods. Workshop participants agreed that general anesthesia should be used for intracerebral virus injections and that humane endpoints should be used routinely as the basis for euthanizing animals when conducting the mouse rabies challenge test. Workshop participants recommended as a near-term priority replacement of the mouse challenge with a test validated to ensure potency, such as the mouse antibody serum neutralization test for adjuvanted veterinary rabies vaccines for which an international collaborative study was recently completed. The workshop recommended that an in vitro antigen quantification test should be a high priority for product-specific validation of human and non-adjuvanted veterinary rabies vaccines. Finally, workshop participants recommended greater international cooperation to expedite development, validation, regulatory acceptance, and implementation of alternative test methods for rabies vaccine potency testing.

Effects of vaccine route and dosage on protection from rabies after intracerebral challenge in mice.

OBJECTIVE: To evaluate the effect of various routes of administration and number of doses of 3 commercially produced rabies vaccines on serum antibody responses and protection in mice challenged by intracerebral injection with fixed-strain rabies virus. ANIMALS: 2,213 mice. PROCEDURE: Inactivated, adjuvanted rabies vaccines were administered to mice in either 2, 1, or 0 (control) doses via IP, IM, and SC routes, and mice were challenged intracerebrally with fixed-strain rabies virus. RESULTS: Vaccination route and dose number significantly influenced serum antibody responses and protection from rabies virus challenge, independent of vaccine strain origin and mouse strain, although mouse age significantly affected results. Extended challenge studies revealed that IM vaccination of mice resulted in the highest serum neutralizing antibody responses and protection levels equivalent to IP vaccination. Even multiple doses administered SC (a vaccination route used in dogs) resulted in poor serum anti-rabies neutralizing antibody responses in mice and were far less protective than other routes. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggest possible improvements for the current rabies vaccine potency test in mice by using 1 dose, the IM route, and a delayed time of challenge. These modifications would more closely model vaccination practices in target species and yield more accurate information regarding primary immunogenicity of a vaccine.

Effect of heterogeneity of rabies virus strain and challenge route on efficacy of inactivated rabies vaccines in mice.

OBJECTIVE: To determine effect of route of challenge and strain of rabies virus on efficacy of inactivated rabies vaccines in mice. ANIMALS: 3,056 mice. PROCEDURE: Challenge was performed with fixed and street rabies virus strains by use of footpad and intracerebral routes as well as IM injection into the hip, shoulder, neck, and masseter muscles. Intraperitoneal and IM vaccination was performed with 1 or 2 doses of 1 of 3 vaccine-strain inactivated rabies vaccines. For 2 of the vaccine strains, the vaccines were adjuvanted and nonadjuvanted. RESULTS: Incubation periods were dependent on route, dose, and virus strain used for challenge. Use of an intramasseter challenge route with challenge virus-strain rabies virus, which more accurately models natural exposure to rabies virus, resulted in reproducible mortality rates in mice. Use of this route revealed that differences among vaccines and challenge virus strains affected mortality rate less than that observed in the National Institutes of Health potency test, even when street isolates of widely variant origin were used for challenge. CONCLUSIONS AND CLINICAL RELEVANCE: These results, combined with earlier data, support a proposal for a new rabies potency test that more closely models current vaccine administration practices and natural infection routes.

Phase 1 first-in-human studies of the reactogenicity and immunogenicity of a recombinant meningococcal NspA vaccine in healthy adults.

BACKGROUND: Neisserial surface protein A (NspA) is a highly conserved, surface-exposed outer membrane protein of Neisseria meningitidis that has been shown to induce a bactericidal immune response in animals against all pathogenic Neisserial serogroups. METHODS: Healthy 18-50-year-old adults were assigned to receive, in a dose escalating manner, 3 doses of 1 of 5 formulations of an experimental, unfolded, recombinant NspA (rNspA) vaccine or placebo, or 1 dose of commercially available quadravalent (A, C, Y, W-135) meningococcal polysaccharide vaccine (Menomune((R))). Adverse events were collected during the first week post-immunization, prior to the next dose and 1 month after the last dose. Serum for measurement of hematological and biochemical parameters and antibodies by enzyme immunoassay and bactericidal assay were measured before the first dose, prior to the second dose and 1 month after the last dose of vaccine. RESULTS: The rNspA vaccine was well tolerated by recipients. Injection-site pain was reported more frequently by recipients of the three highest doses of rNspA compared to placebo but at similar rates to the licensed meningococcal polysaccharide vaccine. Adverse events were reported less frequently after subsequent doses in the three-dose series. An antibody rise measured by enzyme immunoassay was elicited with a dose-related increase that reached a maximum with the 125mug dose. Prolongation of the dosing interval between the second and third dose appeared to be associated with increased antibody levels. No bactericidal antibodies were detected after any of the rNspA formulations. CONCLUSIONS: The unfolded rNspA meningococcal vaccine was well tolerated and immunogenic in healthy adult volunteers but did not elicit bactericidal antibodies.

The rabies peripheral challenge test: more accurate determination of vaccine potency.

The National Institutes of Health (NIH) rabies vaccine potency test is used internationally for evaluating the efficacy of inactivated rabies vaccines, despite concerns about its methods. An alternative test has been developed, using a simplified in vivo method for rabies vaccine testing which has several advantages over the currently recommended method of efficacy testing. The rabies peripheral challenge test more closely models practical vaccine application in target species; decreases the observed effect of vaccine virus strain in testing results and allows sensitive analysis of vaccine and production lot testing.