Defining the right diluent for intravenous infusion of therapeutic antibodies

Figures & data

Figure 1. Routes of administration for therapeutic antibodies

(a) Routes of administration for 97 therapeutic antibodies that were approved by the U.S. Food and Drug Administration (as of December 2018): intravenous infusion (63%, n = 61), subcutaneous (SC) injection (34%, n = 33), intramuscular (IM) and intravitreal injection (3%, n = 3). (b) Among the 57 mAbs that require further dilution for IV infusion, saline solution is acceptable for use with 98% (n = 56) products, while 5% dextrose solution and other diluents (0.45% sodium chloride or lactated ringer’s solution) are only recommended for 30% (n = 17) and 11% (n = 6) therapeutic antibodies, respectively. Remarkably, dextrose solution is prohibited for use with 33% (n = 19) antibody products. Note that four of the 61 antibodies that are administered through IV infusion require no dilution. Data were obtained from the Drugs@FDA website (https://www.accessdata.fda.gov/scripts/cder/daf/).

Table 1. Diluents approved for intravenous infusion of therapeutic antibodies

				Number of products with specific diluents^c
Formulation pH^a	Number of products	Formulation NaCl concentration	Number of products	Saline	Dextrose	Others^d	No dextrose^e
5.0–6.5	38	0	25	24	3	4	12
		40 − 154 mM	13	13	5	0	5
6.6–8.0	14	0	6	6	2	1	2
		10 − 154 mM	8	8	4	1	0
Total	52^b		52	51	14	6	19

^aTherapeutic antibodies (n = 52) are divided into two groups based on formulation pH in the range of pH 5.0–6.5 (n = 38) and pH 6.6–8.0 (n = 14) which are further grouped for salt concentrations in the formulations. Information were obtained from FDA approved product labels that are publicly available.

^bFifty-seven mAbs require a diluent for intravenous infusion, five are excluded due to the lack of public information on their formulation pH values.

^cSome products are compatible with more than one diluent.

^d0.45% sodium chloride or Lactated Ringer’s Solution

^eDextrose is prohibited for use with those products.

Table 2. Therapeutic antibodies tested for compatibility with dextrose and human serum in vitro

Formulation pH*	Number of products	Formulation NaCl concentration*	Number of products	Number of products tested in this study	Number of products aggregated when mixed with dextrose and serum
5.0–6.5	38	0	25	5	5
		40 − 154 mM	13	4	1
6.6–8.0	14	0	6	1	0
		10 − 154 mM	8	1	0
Total	52**		52	11	6

*Therapeutic antibodies that require a diluent for IV infusion are divided into two groups based on formulation pH in the range of pH 5.0–6.5 and pH 6.6–8.0, which are further grouped for salt concentrations in the formulations. Information were obtained from FDA approved product labels that are publicly available.

**Only included the mAbs (n = 52) with formulation information (pH and salt concentrations) that are publicly available, out of 57 mAbs that require a diluent for IV infusion (Figure 1b).

Figure 2. Aggregation of therapeutic mAbs after mixing with dextrose and human serum in vitro

A panel of 11 therapeutic mAbs, named mAb-1 to mAb-11, were tested for their compatibility with diluents and human serum under in vitro conditions (see Materials and Methods). Aliquots of therapeutic mAbs, which were prepared from their original formulations, were diluted into (a) 5% dextrose in water (D5W), (b) saline (0.9% sodium chloride), and (c) lactated Ringer’s solution (containing 0.6% sodium chloride), followed by mixing with human serum. After incubation at 25°C for 30 minutes, the resultant insoluble pellets, if any, were collected by centrifugation and analyzed by SDS-PAGE electrophoresis. When diluted with 5% dextrose (a), insoluble protein aggregates were detected for therapeutic mAbs (mAb-1, mAb-3, mAb-5, mAb-7, and mAb-8) which were present in acidic pH (5.2 to 6.5) and nonionic formulation buffers. A lower amount of aggregates was also detected for mAb-2, which was formulated in pH 5.5 buffer containing 50 mM sodium chloride. In contrast, little or no aggregates were detected for mAb-10 and mAb-11 that were both formulated in pH 7.2 to 7.5 or other mAbs (mAb-4, mAb-6, and mAb-9) whose formulations were acidic (pH 5.8–6.5) and contained sodium chloride. When diluted with ionic diluent (saline or lactated Ringer’s solution) (b and c), no protein aggregates were detected across the 11 mAbs when mixed with human serum. * Shown are the pH values and NaCl concentrations of individual mAb formulation buffers and the corresponding IgG isotypes. ** The faint bands indicate basal protein adsorption to the microtube inner surface (Supplement I). Shown are representative images of duplicate experiments.

Figure 3. pH-dependence of protein aggregation of therapeutic mAbs in mixtures with dextrose and human serum

The indicated D5W-mAb mixtures were adjusted to (a) pH 5.3–5.9, (b) pH 6.1–6.4, (c) pH 6.7–6.9, and (d) pH 7.3–7.7 with citrate-phosphate buffers (pH 5.0, 5.8, 6.6, and 7.6). The resultant samples were incubated with human serum and analyzed for potential protein aggregates. (a) At pH 5.3–5.9, all 11 mAbs formed insoluble aggregates, displaying similar band patterns on SDS-PAGE, with lower levels of aggregates being seen for mAbs in ionic formulations (mAbs 2, 4, 6, 9, and 11). (b-d) The levels of protein aggregates decreased by raising pH in the D5W-buffer-mAb mixtures. (e) Lanes 1–6, the indicated mAbs in acidic formulations formed massive aggregates after incubation with dextrose and serum at 25°C for 15 minutes. Lanes 7–12, duplicate samples were incubated for an additional 15 minutes after adjusting to pH 7.3–7.6. The preformed aggregates were no longer detectable in the resultant samples.

Figure 4. Ionic strength-dependence of aggregation of therapeutic mAbs in mixtures with dextrose and human serum

(a) The indicated mAbs were mixed with a NaCl-containing dextrose solution (D5W + 0.45% NaCl (77 mM)) and human serum. At such ionic strength, little or no aggregates were detected across all the samples. (b) Aliquots of mAb-1 were diluted into D5W solutions with varying amounts of NaCl. The mixed solutions were adjusted to pH 5.0–5.3 with citrate-phosphate buffer (pH 5.0). When incubated with serum, the presence of NaCl suppressed aggregate formation in a dose-dependent manner. (c) Aliquots of 10% NaCl were added to the six D5W-mAb-serum solutions that contained the most aggregates (lanes 1–6) to mimic physiological NaCl concentration (final concentration 0.9% or 154 mM). After an additional 15 minutes of incubation, little or no aggregates were detected across the samples (lanes 7–12).

Table 3. Comparison of protein aggregates in the mAb-dextrose mixtures with human serum and plasma

					Total Protein Spectral Intensity (×10^Citation7)***
					Aggregates Formed in Serum				Aggregates Formed in Plasma
#	Protein Name*	pI**	MW (kDa)	Uniprot #	mAb1	mAb5	mAb7	mAb10	mAb1	mAb5	mAb7	mAb10
Common Proteins Identified in All Eight Insoluble Aggregates
1	Apolipoprotein B-100	6.6	517	P04114	16	19	7	7	17	13	10	22
2	Fibronectin	5.5	266	P02751	3	19	7	29	1	5	5	55
3	Complement C4-A/C4-B	6.7/6.9	194	P0C0L4/P0C0L5	56	44	61	79	50	48	56	62
4	Complement C5	6.1	190	P01031	6	5	8	12	5	7	8	12
5	Complement C3	6.0	189	P01024	29	31	31	30	93	111	118	122
6	Complement factor H	6.2	144	P08603	72	52	63	55	90	88	84	91
7	Plasminogen	7.1	93	P00747	2	2	3	26	4	3	3	13
8	Prothrombin	5.6	72	P00734	2	4	2	4	8	24	15	15
9	Serum albumin	5.9	71	P02768	12	6	6	2	2	4	6	4
10	C4b-binding protein alpha chain	7.3	69	P04003	55	22	45	52	50	45	55	51
11	Ig mu chain C region	–	–	P01871	119	77	67	64	102	65	90	90
12	Ig gamma-1 chain C region	–	–	P01857	134	46	77	95	55	142	59	38
13	Ig kappa chain C region	–	–	P01834	122	55	157	67	38	68	48	27
14	Ig lambda-2 chain C regions	–	–	P0CG05	24	11	19	20	12	13	7	19
Unique Proteins Identified only in Aggregates Formed in Plasma****
1	Fibrinogen alpha chain	5.7	96	P02671	none	none	none	none	3	3	5	46
2	Fibrinogen beta chain	9.0	57	P02675	none	none	none	none	4	4	8	50
3	Fibrinogen gamma chain	5.4	52	P02679	none	none	none	none	2	2	6	38

*Proteins were identified by the presence of at least three distinct peptides in the replicated samples from two independent experiments.

**Theoretical protein isoelectric point (pI), molecular weight (MW), and database accession number were obtained from UniProtKB/Swiss-Prot database using the Agilent Spectrum Mill mass spectrometry data analysis software. The proteins were sorted by their MWs.

***Total protein spectral intensity is the sum of all peptides that were assigned to the specific protein. Peptide intensity was determined on Spectrum Mill using extracted ion chromatograms. Listed proteins are those with an intensity value greater than 10 × 10⁷ in at least one sample.

****Proteins that were found in the aggregates from using plasma but were absent in those of serum.

Figure 5. Comparison of protein aggregates in the mAb-dextrose mixtures with serum and plasma

The indicated mAbs were diluted into dextrose at pH 4.7–6.2 and incubated with human plasma and serum, respectively, as in Figures 2a and 3a. (a) Serum mediated protein aggregates (left panel) showed similar band patterns as those of plasma (right panel) on SDS-PAGE. Duplicate aggregate samples were subjected to in-solution trypsin digestion followed by LC-MS/MS analysis revealing 14 of the most abundant proteins (Table 3). (b) Insoluble protein aggregates were prepared using mAb-7 and mAb-10 after mixing with dextrose at indicated pH and incubation with human serum and plasma, respectively. After resolving by SDS-PAGE, a set of 12 major protein bands (labeled 1 to 12) were retrieved for in-gel trypsin digestion followed by protein identification. (c) Shown are the most abundant protein(s) identified in the corresponding bands. * Proteins that were found in the aggregates from using plasma, but were absent in those of serum.

Figure 6. Potential biochemical pathways driving protein aggregation at the IV infusion interface

Therapeutic mAbs, especially those formulated in acidic buffers (pH ≤ 6.5) with low ionic strength, could form insoluble protein aggregates at the interface of IV infusion with blood components when diluted into a nonionic dextrose for intravenous infusion (Figure 2). The underlying biochemical pathways may involve isoelectric precipitation of abundant plasma proteins (e.g., complement proteins) whose pI values are proximate to the formulation pH of the mAb (Table 3). This hypothesis is supported by the evidence that protein aggregation was effectively prevented by increasing ionic strength and/or pH in the mAb-diluent solution (Figures 3 and 4). By contrast, no protein aggregation was detected for mAbs that are formulated at physiological pH (mAb-10 and mAb-11) even when mixed with nonionic dextrose and human serum (Figure 2).

Remicade prescribing information (1998) [cited 2019 Sept 22]: https://www.accessdata.fda.gov/drugsatfda_docs/label/1998/inflcen082498lb.pdf.

 Google Scholar