Pre-analytical stability of ocrelizumab in serum after delayed centrifugation of whole blood

Ocrelizumab, an anti-CD20 antibody that depletes B cells (Hauser et al. 2017; Montalban et al. 2017; Carlson et al. 2024), is approved for treating various forms of multiple sclerosis (MS) in adults, including relapsing multiple sclerosis (RMS), active secondary progressive disease, and primary progressive multiple sclerosis (PPMS). Since its approval in 2017, this groundbreaking treatment has continued to expand its reach with ongoing clinical studies such as investigating its efficacy in pediatric MS (Bibinoglu Amirov et al. 2023) and testing subcutaneous injections as a way to enhance and expedite drug delivery in patients (Xu et al. 2023). This exploration into the subcutaneous route of administration highlights the continued efforts to maximize patient-centered therapy.

Accurate and precise pharmacokinetic (PK) measurements are crucial in the clinical development of therapeutic drugs, including ocrelizumab (Gibiansky et al. 2021) in order to evaluate drug exposure and the PK/PD (pharmacodynamics) relationship (Danhof et al. 2008), to inform the optimal dose for patients, the frequency of administration, and the duration of the therapy (Tang and Cao 2021).

During clinical trials, samples from patients are collected for drug measurements and PK characterization. This necessitates for patients to travel to clinical sites to have a phlebotomist collect blood samples which are processed within 30 min -1 h to generate serum/plasma used for drug measurements (Tuck et al. 2009). For some individuals living with debilitating diseases like multiple sclerosis (MS) making routine clinic visits can be challenging. A patient-centric approach would be to collect blood samples at patient’s home, so patients can undergo necessary tests without the burden of traveling to medical facilities. In addition to convenience, this approach promotes patient adherence to treatment and monitoring regimens and reducing patients’ risk of exposure to infections. However, prior to enabling in-home sample collection by nurses, the impact of collection logistics and processing must be evaluated. Therefore, for ocrelizumab, this necessitated a study to evaluate the impact of whole blood delayed centrifugation on serum drug concentration measurements.

Numerous investigations have assessed the influence of whole blood delayed centrifugation on the stability of endogenous proteins such as metabolites in serum or plasma (Debik et al. 2022; Qundos et al. 2013; Zhang et al. 1998; Salvagno et al. 2009). Amongst those studies, stability was tested and demonstrated for total protein (immunoglobulins represent ~ 40%) for up to 1 week at room temperature (Hedayati et al. 2020), for IgA, IgG and IgM for up to 10 h (Henriksen et al. 2014) and for IgE for up to 48 h (Ostergaard and Sandfeld-Paulsen 2023). However, to the authors knowledge, there is a lack of information on delayed centrifugation impact on the stability of antibody therapeutics.

Here, we demonstrated that ocrelizumab concentrations were not impacted by delayed centrifugation of whole blood under conditions that mimicked the logistics for in-home sample collection and processing. Therefore, this work bridges the knowledge gap by investigating whether delayed centrifugation affects PK measurement for ocrelizumab.

Biosample collection

Blood samples from 9 patients with MS treated with ocrelizumab were collected at the patients’ homes and processed to serum by Sanguine Biosciences (Woburn, MA). Collections were scheduled after ocrelizumab treatment as follows: anytime between day 1 and week 20 after a 600-mg infusion; at the initiation of treatment after the first 300-mg infusion; at the initiation of treatment between the first and second 300-mg infusion; at the initiation of treatment after the second 300-mg infusion.

Sample collection procedure

Four blood samples were collected from each patient in clot-activated serum separator tubes (SST, 1 mL) and allowed to clot at ambient temperature and processed to serum by centrifugation at four different timepoints: after collection time of 30–60 min (reference), as well as after 3 h, 6 h, and 24 h (Fig. 1). The reference blood sample was kept at ambient temperature for 30 to 60 min prior to centrifugation. The serum was then separated and frozen using dry ice. The remaining three tubes were placed upright into individual tube absorbent pouches for safe transport and wrapped in an ambient gel wrap. The tubes were then wrapped into a secondary absorbent material in a transport container and transported to a facility for processing. At each centrifugation time point, the tubes were centrifuged for 15 min at 2500 rpm. The serum (approximately 0.5 mL) was pipetted from the SST tube into cryovials and the cryovials were placed in a box layered with dry ice for shipping to the testing laboratory.

Fig. 1

Sample collection & analysis workflow

Full size image

PK assay procedure

A sandwich ELISA was used for quantification of ocrelizumab as described previously (Gibiansky et al. 2021; Fischer et al. 2012). Briefly, microtiter plates were coated with an anti-ocrelizumab goat polyclonal antibody. Diluted samples, standards, and controls were added to the plate and incubated. Subsequently, goat F(ab’)2 anti-human immunoglobulin G (IgG) (Fc gamma fragment specific) horseradish peroxidase conjugate was added for detection and incubated. A tetramethylbenzidine peroxidase (TMB) substrate was added to develop color, and the reaction was stopped with 1 M phosphoric acid. The plates were read at 450 nm using 630 nm for reference absorbance. The lower limit of quantitation in neat serum was 156 ng/mL. The samples were tested at PPD Laboratories (Richmond, VA).

Statistical analysis

The paired t-test analysis was employed to assess whether there is a significant difference in drug concentration values of the 9 patients between two distinct timepoints, by examining the mean differences (within each patient) between the reference and each subsequent time point.

Table 1 presents an overview of the findings of ocrelizumab in samples obtained from nine patients. The concentrations measured in samples centrifuged at 3 h, 6 h and 24 h after collection were compared to the concentrations measured in the reference tube which was centrifuged at 30–60 min and the change calculated as %Difference. Negative %Difference values signify a decrease from the reference reading and positive values signify an increase. The data shows that all the samples (except for the 24-h timepoint sample from patient 2 showing an increase of 24.5%) exhibited changes within ± 12% of the reference sample which fall within the ± 20% pre-set acceptance criteria. In addition, the paired t-test indicates that there is no significant change between timepoints of each individual (Fig. 2). Therefore, delayed centrifugation of blood samples for up to 24 h at ambient temperature did not impact ocrelizumab concentrations in serum demonstrating pre-analytical stability of ocrelizumab under those conditions.

Table 1 Comparison of ocrelizumab concentrations at various delayed centrifugation time points with those at the reference time point (30–60 min)

Full size table

Fig. 2

The paired t-test over 4 timepoints of 9 individuals. The drug concentration values of 9 patients at each centrifugation time point were used for the paired t-test. ns indicates non-significance

Full size image

Recently, in-home blood collection by healthcare providers has become a feasible option for clinical study conduct, particularly for enrollment of patients with mobility disabilities like the elderly and patients with severe neurological diseases. However, despite these advantages health providers encounter challenges regarding sample stability due to sample handling and delayed centrifugation. While portable miniaturized centrifuges are accessible for home use, the timing of centrifugation after blood collection may still be susceptible to human error as well as logistical issues related to serum separation and storage. Therefore, pre-analytical drug stability studies that replicate and extend the in-home sample collection and processing conditions are warranted.

This study demonstrates pre-analytical stability of ocrelizumab with delayed centrifugation of blood samples of up to 24 h at ambient temperature, which supports in-home blood collection for ocrelizumab. Before initiating in-home sample collection, it is essential to conduct pre-analytical stability studies tailored to each analyte.

In this study, we demonstrate that PK measurement of ocrelizumab in serum is unaffected by delaying whole blood centrifugation for up to 24 h at ambient temperature. This finding facilitates sampling of patients at home, enhancing convenience for both patients and their caregivers in participating in clinical trials. We anticipate that this finding may be applicable to other monoclonal antibody therapeutics. However, further studies are required for verification.

Not applicable.

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The authors would like to thank Justin Low and Jihong Yang for providing the critical reagents for ocrelizumab PK assay, Anthony Cheung for coordinating with Sanguine Biosciences, and Reginald Delarosa and Bodrey Ro for coordinating sample testing with PPD.

All work described in this paper was funded by Genentech, Inc. The authors are or were all employees of Genentech, Inc., during the conduct of the study and stockholders of the Roche group.

Authors and Affiliations

1. BioAnalytical Sciences, Development Sciences, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080, USA

   Jeongsup Shim, Montserrat Carrasco-Triguero & Saloumeh K. Fischer

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Contributions

J.S.: project administration, formal analysis, and writing—original draft; M.C-T.: conceptualization, data curation, formal analysis, methodology, project administration, review and editing, and supervision; S.K.F.: conceptualization, formal analysis, project administration, writing—review and editing, and supervision.

Corresponding author

Correspondence to Saloumeh K. Fischer.

Ethics approval and consent to participate

Sample collection was conducted by Sanguine who obtained all approvals from the patients. The samples were purchased by Genentech. All results or intellectual property in or arising out of the use of the material is sole property of Genentech.

Competing interests

The authors are (or were) employees of Genentech, Inc., and own stock in F. Hoffmann-La Roche Ltd. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

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Shim, J., Carrasco-Triguero, M. & Fischer, S.K. Pre-analytical stability of ocrelizumab in serum after delayed centrifugation of whole blood. AAPS Open 10, 11 (2024). https://doi.org/10.1186/s41120-024-00098-9

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• Received: 08 June 2024

• Accepted: 28 July 2024

• Published: 16 September 2024

• DOI: https://doi.org/10.1186/s41120-024-00098-9

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Keywords

• Ocrelizumab
• Delayed whole blood centrifugation
• PK analysis